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Sample records for anandamide transport inhibitor

  1. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity.

    PubMed

    De Petrocellis, L; Bisogno, T; Davis, J B; Pertwee, R G; Di Marzo, V

    2000-10-13

    Some synthetic agonists of the VR1 vanilloid (capsaicin) receptor also inhibit the facilitated transport into cells of the endogenous cannabinoid anandamide (arachidonoylethanolamide, AEA). Here we tested several AEA derivatives containing various derivatized phenyl groups or different alkyl chains as either inhibitors of the AEA membrane transporter (AMT) in intact cells or functional agonists of the VR1 vanilloid receptor in HEK cells transfected with the human VR1. We found that four known AMT inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400-10000-fold lower than those necessary to inhibit the AMT. However, we also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the AMT as potently as AM404 but exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB(1)/CB(2) receptor ligands. We show for the first time that, despite the overlap between the chemical moieties of AMT inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed.

  2. Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study

    PubMed Central

    Ruiz-Llorente, Lidia; Ortega-Gutiérrez, Silvia; Viso, Alma; Sánchez, María G; Sánchez, Ana M; Fernández, Carlos; Ramos, José A; Hillard, Cecilia; Lasunción, Miguel A; López-Rodríguez, María L; Díaz-Laviada, Inés

    2004-01-01

    The response of anandamide is terminated by a carrier-mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC-3 cells by a saturable and temperature-dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7±0.2 μM and Vmax=3.3±0.3 pmol min−1 (106 cells)−1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC-3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1–8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC-3 cells, with (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3±0.5 μM, respectively. In conclusion, PC-3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator. PMID:14718261

  3. Role of FAAH-like anandamide transporter in anandamide inactivation.

    PubMed

    Leung, Kwannok; Elmes, Matthew W; Glaser, Sherrye T; Deutsch, Dale G; Kaczocha, Martin

    2013-01-01

    The endocannabinoid system modulates numerous physiological processes including nociception and reproduction. Anandamide (AEA) is an endocannabinoid that is inactivated by cellular uptake followed by intracellular hydrolysis by fatty acid amide hydrolase (FAAH). Recently, FAAH-like anandamide transporter (FLAT), a truncated and catalytically-inactive variant of FAAH, was proposed to function as an intracellular AEA carrier and mediate its delivery to FAAH for hydrolysis. Pharmacological inhibition of FLAT potentiated AEA signaling and produced antinociceptive effects. Given that endocannabinoids produce analgesia through central and peripheral mechanisms, the goal of the current work was to examine the expression of FLAT in the central and peripheral nervous systems. In contrast to the original report characterizing FLAT, expression of FLAT was not observed in any of the tissues examined. To investigate the role of FLAT as a putative AEA binding protein, FLAT was generated from FAAH using polymerase chain reaction and further analyzed. Despite its low cellular expression, FLAT displayed residual catalytic activity that was sensitive to FAAH inhibitors and abolished following mutation of its catalytic serine. Overexpression of FLAT potentiated AEA cellular uptake and this appeared to be dependent upon its catalytic activity. Immunofluorescence revealed that FLAT localizes primarily to intracellular membranes and does not contact the plasma membrane, suggesting that its capability to potentiate AEA uptake may stem from its enzymatic rather than transport activity. Collectively, our data demonstrate that FLAT does not serve as a global intracellular AEA carrier, although a role in mediating localized AEA inactivation in mammalian tissues cannot be ruled out.

  4. The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism.

    PubMed

    De Petrocellis, L; Bisogno, T; Maccarrone, M; Davis, J B; Finazzi-Agro, A; Di Marzo, V

    2001-04-20

    The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhanced anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A inhibited also the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism.

  5. The endogenous cannabinoid anandamide produces delta-9-tetrahydrocannabinol-like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of anandamide transport.

    PubMed

    Solinas, Marcello; Tanda, Gianluigi; Justinova, Zuzana; Wertheim, Carrie E; Yasar, Sevil; Piomelli, Daniele; Vadivel, Subramanian K; Makriyannis, Alexandros; Goldberg, Steven R

    2007-04-01

    Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methanandamide (AM-356), a metabolically stable anandamide analog, produced clear dose-related THC-like discriminative effects, but anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of anandamide, with 3 mg/kg anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of anandamide after URB-597 and methanandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of anandamide or its dopamine-elevating effects. Thus, anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting

  6. Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

    PubMed

    Nicolussi, Simon; Chicca, Andrea; Rau, Mark; Rihs, Sabine; Soeberdt, Michael; Abels, Christoph; Gertsch, Jürg

    2014-12-15

    Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

  7. New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis.

    PubMed

    Ligresti, Alessia; Cascio, Maria Grazia; Pryce, Gareth; Kulasegram, Sanjitha; Beletskaya, Irina; De Petrocellis, Luciano; Saha, Bijali; Mahadevan, Anu; Visintin, Cristina; Wiley, Jenny L; Baker, David; Martin, Billy R; Razdan, Raj K; Di Marzo, Vincenzo

    2006-01-01

    We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB(1) and CB(2) receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca(2+)](i)); (c) [(14)C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse 'tetrad' tests (analgesia on a hot plate, immobility on a 'ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the 'tetrad' up to a 20 mg kg(-1) dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB(1) (K(i) from 1.3 to >10 microM) and CB(2) binding assays (1.310 microM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC(50)>25 microM) and were inactive in the 'tetrad' up to a 30 mg kg(-1) dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [(14)C]AEA cellular uptake (IC(50)>40 microM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC(50)=1.4 microM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective

  8. New potent and selective inhibitors of anandamide reuptake with antispastic activity in a mouse model of multiple sclerosis

    PubMed Central

    Ligresti, Alessia; Cascio, Maria Grazia; Pryce, Gareth; Kulasegram, Sanjitha; Beletskaya, Irina; De Petrocellis, Luciano; Saha, Bijali; Mahadevan, Anu; Visintin, Cristina; Wiley, Jenny L; Baker, David; Martin, Billy R; Razdan, Raj K; Di Marzo, Vincenzo

    2005-01-01

    We previously reported that the compound O-2093 is a selective inhibitor of the reuptake of the endocannabinoid anandamide (AEA). We have now re-examined the activity of O-2093 in vivo and synthesized four structural analogs (O-2247, O-2248, O-3246, and O-3262), whose activity was assessed in: (a) binding assays carried out with membranes from cells overexpressing the human CB1 and CB2 receptors; (b) assays of transient receptor potential of the vanilloid type-1 (TRPV1) channel functional activity (measurement of [Ca2+]i); (c) [14C]AEA cellular uptake and hydrolysis assays in rat basophilic leukaemia (RBL-2H3) cells; (d) the mouse ‘tetrad' tests (analgesia on a hot plate, immobility on a ‘ring', rectal hypothermia and hypolocomotion in an open field); and (e) the limb spasticity test in chronic relapsing experimental allergic encephalomyelitis (CREAE) mice, a model of multiple sclerosis (MS). O-2093, either synthesized by us or commercially available, was inactive in the ‘tetrad' up to a 20 mg kg−1 dose (i.v.). Like O-2093, the other four compounds exhibited low affinity in CB1 (Ki from 1.3 to >10 μM) and CB2 binding assays (1.310 μM), very low potency as fatty acid amide hydrolase (FAAH) inhibitors (IC50>25 μM) and were inactive in the ‘tetrad' up to a 30 mg kg−1 dose (i.v.). While O-2247 and O-2248 were poor inhibitors of [14C]AEA cellular uptake (IC50>40 μM), O-3246 and O-3262 were quite potent in this assay. O-3246, which exhibits only a very subtle structural difference with O-2093, is the most potent inhibitor of AEA uptake reported in vitro under our experimental conditions (IC50=1.4 μM) and is 12-fold more potent than O-2093. When injected intravenously O-3246 and O-3262, again like O-2093 and unlike O-2247 and O-2248, significantly inhibited limb spasticity in mice with CREAE. These data confirm the potential utility of selective AEA uptake

  9. N-acyl-dopamines: novel synthetic CB(1) cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo.

    PubMed Central

    Bisogno, T; Melck, D; Bobrov MYu; Gretskaya, N M; Bezuglov, V V; De Petrocellis, L; Di Marzo, V

    2000-01-01

    We reported previously that synthetic amides of polyunsaturated fatty acids with bioactive amines can result in substances that interact with proteins of the endogenous cannabinoid system (ECS). Here we synthesized a series of N-acyl-dopamines (NADAs) and studied their effects on the anandamide membrane transporter, the anandamide amidohydrolase (fatty acid amide hydrolase, FAAH) and the two cannabinoid receptor subtypes, CB(1) and CB(2). NADAs competitively inhibited FAAH from N18TG2 cells (IC(50)=19-100 microM), as well as the binding of the selective CB(1) receptor ligand, [(3)H]SR141716A, to rat brain membranes (K(i)=250-3900 nM). The arachidonoyl (20:4 omega 6), eicosapentaenoyl (20:5 omega 3), docosapentaenoyl (22:5 omega 3), alpha-linolenoyl (18:3 omega 3) and pinolenoyl (5c,9c,12c 18:3 omega 6) homologues were also found to inhibit the anandamide membrane transporter in RBL-2H3 basophilic leukaemia and C6 glioma cells (IC(50)=17.5-33 microM). NADAs did not inhibit the binding of the CB(1)/CB(2) receptor ligand, [(3)H]WIN55,212-2, to rat spleen membranes (K(i)>10 microM). N-arachidonyl-dopamine (AA-DA) exhibited 40-fold selectivity for CB(1) (K(i)=250 nM) over CB(2) receptors, and N-docosapentaenoyl-dopamine exhibited 4-fold selectivity for the anandamide transporter over FAAH. AA-DA (0.1-10 microM) did not displace D1 and D2 dopamine-receptor high-affinity ligands from rat brain membranes, thus suggesting that this compound has little affinity for these receptors. AA-DA was more potent and efficacious than anandamide as a CB(1) agonist, as assessed by measuring the stimulatory effect on intracellular Ca(2+) mobilization in undifferentiated N18TG2 neuroblastoma cells. This effect of AA-DA was counteracted by the CB(1) antagonist SR141716A. AA-DA behaved as a CB(1) agonist in vivo by inducing hypothermia, hypo-locomotion, catalepsy and analgesia in mice (1-10 mg/kg). Finally, AA-DA potently inhibited (IC(50)=0.25 microM) the proliferation of human breast MCF

  10. The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

    PubMed

    Oz, Murat; Ravindran, Arippa; Diaz-Ruiz, Oscar; Zhang, Li; Morales, Marisela

    2003-09-01

    The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and

  11. Inhibition of fatty acid amide hydrolase and monoacylglycerol lipase by the anandamide uptake inhibitor VDM11: evidence that VDM11 acts as an FAAH substrate.

    PubMed

    Vandevoorde, Séverine; Fowler, Christopher J

    2005-08-01

    There is some dispute concerning the extent to which the uptake inhibitor VDM11 (N-(4-hydroxy-2-methylphenyl) arachidonoyl amide) is capable of inhibiting the metabolism of the endocannabinoid anandamide (AEA) by fatty acid amide hydrolase (FAAH). In view of a recent study demonstrating that the closely related compound AM404 (N-(4-hydroxyphenyl)arachidonylamide) is a substrate for FAAH, we re-examined the interaction of VDM11 with FAAH. In the presence of fatty acid-free bovine serum albumin (BSA, 0.125% w v(-1)), both AM404 and VDM11 inhibited the metabolism of AEA by rat brain FAAH with similar potencies (IC(50) values of 2.1 and 2.6 microM, respectively). The compounds were about 10-fold less potent as inhibitors of the metabolism of 2-oleoylglycerol (2-OG) by cytosolic monoacylglycerol lipase (MAGL). The potency of VDM11 towards FAAH was dependent upon the assay concentration of fatty acid-free bovine serum albumin (BSA). Thus, in the absence of fatty acid-free BSA, the IC(50) value for inhibition of FAAH was reduced by a factor of about two (from 2.9 to 1.6 microM). A similar reduction in the IC(50) value for the inhibition of membrane bound MAGL by both this compound (from 14 to 6 microM) and by arachidonoyl serinol (from 24 to 13 microM) was seen. An HPLC assay was set up to measure 4-amino-m-cresol, the hypothesised product of FAAH-catalysed VDM11 hydrolysis. 4-Amino-m-cresol was eluted with a retention time of approximately 2.4 min, but showed a time-dependent degradation to compounds eluting at peaks of approximately 5.6 and approximately 8 min. Peaks with the same retention times were also found following incubation of the membranes with VDM11, but were not seen when the membranes were preincubated with the FAAH inhibitors URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) and CAY10401 (1-oxazolo[4,5-b]pyridin-2-yl-9-octadecyn-1-one) prior to addition of VDM11. The rate of metabolism of VDM11 was estimated to be roughly 15-20% of that for

  12. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs)

    PubMed Central

    Deutsch, Dale G.

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that “solubilize” anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992

  13. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    PubMed

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  14. Glycine Transporters and Their Inhibitors

    NASA Astrophysics Data System (ADS)

    Gilfillan, Robert; Kerr, Jennifer; Walker, Glenn; Wishart, Grant

    Glycine plays a ubiquitous role in many biological processes. In the central nervous system it serves as an important neurotransmitter acting as an agonist at strychnine-sensitive glycine receptors and as an essential co-agonist with glutamate at the NMDA receptor complex. Control of glycine concentrations in the vicinity of these receptors is mediated by the specific glycine transporters, GlyT1 and GlyT2. Inhibition of these transporters has been postulated to be of potential benefit in several therapeutic indications including schizophrenia and pain. In this review we discuss our current knowledge of glycine transporters and focus on recent advances in the medicinal chemistry of GlyT1 and GlyT2 inhibitors.

  15. A biosynthetic pathway for anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C.; Zhou, Zhifeng; Huang, Bill X.; Kim, Hee-Yong; Kunos, George

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE–PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target. PMID:16938887

  16. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  17. Arachidonic acid release and prostaglandin F(2alpha) formation induced by anandamide and capsaicin in PC12 cells.

    PubMed

    Someya, Akiyoshi; Horie, Syunji; Murayama, Toshihiko

    2002-08-23

    Anandamide, an endogenous agonist of cannabinoid receptors, activates various signal transduction pathways. Anandamide also activates vanilloid VR(1) receptor, which was a nonselective cation channel with high Ca(2+) permeability and had sensitivity to capsaicin, a pungent principle in hot pepper. The effects of anandamide and capsaicin on arachidonic acid metabolism in neuronal cells have not been well established. We examined the effects of anandamide and capsaicin on arachidonic acid release in rat pheochromocytoma PC12 cells. Both agents stimulated [3H]arachidonic acid release in a concentration-dependent manner from the prelabeled PC12 cells even in the absence of extracellular CaCl(2). The effect of anandamide was neither mimicked by an agonist nor inhibited by an antagonist for cannabinoid receptors. The effects of anandamide and capsaicin were inhibited by phospholipase A(2) inhibitors, but not by an antagonist for vanilloid VR(1) receptor. In PC12 cells preincubated with anandamide or capsaicin, [3H]arachidonic acid release was marked and both agents were no more effective. Co-addition of anandamide or capsaicin synergistically enhanced [3H]arachidonic acid release by mastoparan in the absence of CaCl(2). Anandamide stimulated prostaglandin F(2alpha) formation. These findings suggest that anandamide and capsaicin stimulated arachidonic acid metabolism in cannabinoid receptors- and vanilloid VR(1) receptor-independent manner in PC12 cells. The possible mechanisms are also discussed.

  18. Anandamide acts as an intracellular messenger amplifying Ca2+ influx via TRPV1 channels

    PubMed Central

    van der Stelt, Mario; Trevisani, Marcello; Vellani, Vittorio; De Petrocellis, Luciano; Schiano Moriello, Aniello; Campi, Barbara; McNaughton, Peter; Geppetti, Piero; Di Marzo, Vincenzo

    2005-01-01

    The endocannabinoid anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca2+-ATPase inhibitor thapsigargin leads to formation of anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of anandamide cellular efflux. Thus, endogenous anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release. PMID:16107881

  19. Reduced Endothelium-Dependent Relaxation to Anandamide in Mesenteric Arteries from Young Obese Zucker Rats

    PubMed Central

    Lobato, Nubia S.; Filgueira, Fernando P.; Prakash, Roshini; Giachini, Fernanda R.; Ergul, Adviye; Carvalho, Maria Helena C.; Webb, R. Clinton; Tostes, Rita C.; Fortes, Zuleica B.

    2013-01-01

    Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity. PMID:23667622

  20. Auxin transport: a new synthetic inhibitor.

    PubMed

    Beyer, E M

    1972-09-01

    The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-(14)C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-(14)C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-(14)C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.DPX1840 also inhibited the lateral displacement of auxin. In horizontally placed stem sections of Helianthus annuus L. pretreated with DPX1840, the ratio of radioactivity from indoleacetic acid-1-(14)C in the upper versus the lower halves of the sections following basipetal indoleacetic acid-1-(14)C transport was approximately 50:50, whereas in the corresponding controls it was approximately 40:60.The data indicate that many of the characteristic effects of DPX1840 on plants, especially those which are known to involve auxin (e.g., epinasty, abscission, apical dominance, tropism), are due, at least in part, to its effects on auxin transport.

  1. Molecular modeling of auxin transport inhibitors

    SciTech Connect

    Gardner, G.; Black-Schaefer, C.; Bures, M.G. )

    1990-05-01

    Molecular modeling techniques have been used to study the chemical and steric properties of auxin transport inhibitors. These bind to a specific site on the plant plasma membrane characterized by its affinity for N-1-naphthylphthalamic acid (NPA). A three-dimensional model was derived from critical features of ligands for the NPA receptor, and a suggested binding conformation is proposed. This model, along with three-dimensional structural searching techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the search criteria, 77 representative molecules were evaluated for their ability to compete for ({sup 3}H)NPA binding to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, from chemical classes not included in the original compound set, also inhibited polar auxin transport through corn coleoptile sections.

  2. Involvement of central beta2-adrenergic, NMDA and thromboxane A2 receptors in the pressor effect of anandamide in rats.

    PubMed

    Malinowska, B; Zakrzeska, A; Kurz, C M; Göthert, M; Kwolek, G; Wielgat, P; Braszko, J J; Schlicker, E

    2010-04-01

    Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A(2) TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and beta(2)-adrenergic receptors (ICI 118551) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5-3 micromol/kg, i.v.) or its stable analogue methanandamide (0.75 micromol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 mumol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB(1) and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 mumol/kg each), and/or SQ 29548 (1 mumol/kg). The effect of anandamide i.v. was reduced by SQ 29548 (0.02 mumol per animal i.c.v.) and by the thromboxane A(2) synthesis inhibitor furegrelate i.c.v. (1.8 micromol per animal). ICI 118551, MK-801 (1 micromol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central beta(2)-adrenergic, NMDA and thromboxane A(2) receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats.

  3. Screening For Inhibitors Of Essential Leishmania Glucose Transporters

    DTIC Science & Technology

    2011-07-01

    parasitic protozoa that cause devastating diseases throughout much of the tropical and subtropical world (4), and infections of military personnel in the...o T t c p t s t t f e t lucose transporter igh-throughput screening porter inhibitors was dem inhibitor of PfHT. . Introduction Parasitic protozoa

  4. Truffles contain endocannabinoid metabolic enzymes and anandamide.

    PubMed

    Pacioni, Giovanni; Rapino, Cinzia; Zarivi, Osvaldo; Falconi, Anastasia; Leonardi, Marco; Battista, Natalia; Colafarina, Sabrina; Sergi, Manuel; Bonfigli, Antonella; Miranda, Michele; Barsacchi, Daniela; Maccarrone, Mauro

    2015-02-01

    Truffles are the fruiting body of fungi, members of the Ascomycota phylum endowed with major gastronomic and commercial value. The development and maturation of their reproductive structure are dependent on melanin synthesis. Since anandamide, a prominent member of the endocannabinoid system (ECS), is responsible for melanin synthesis in normal human epidermal melanocytes, we thought that ECS might be present also in truffles. Here, we show the expression, at the transcriptional and translational levels, of most ECS components in the black truffle Tuber melanosporum Vittad. at maturation stage VI. Indeed, by means of molecular biology and immunochemical techniques, we found that truffles contain the major metabolic enzymes of the ECS, while they do not express the most relevant endocannabinoid-binding receptors. In addition, we measured anandamide content in truffles, at different maturation stages (from III to VI), through liquid chromatography-mass spectrometric analysis, whereas the other relevant endocannabinoid 2-arachidonoylglycerol was below the detection limit. Overall, our unprecedented results suggest that anandamide and ECS metabolic enzymes have evolved earlier than endocannabinoid-binding receptors, and that anandamide might be an ancient attractant to truffle eaters, that are well-equipped with endocannabinoid-binding receptors.

  5. Cannabinoid receptors and their endogenous agonist, anandamide.

    PubMed

    Axelrod, J; Felder, C C

    1998-05-01

    Cannabinoids are a class of compound found in marijuana which have been known for their therapeutic and psychoactive properties for at least 4000 years. Isolation of the active principle in marijuana, delta9-THC, provided the lead structure in the development of highly potent congeners which were used to probe for the mechanism of marijuana action. Cannabinoids were shown to bind to selective binding sites in brain tissue thereby regulating second messenger formation. Such studies led to the cloning of three cannabinoid receptor subtypes, CB1, CB2, and CB1A all of which belong to the superfamily of G protein-coupled plasma membrane receptors. Analogous to the discovery of endogenous opiates, isolation of cannabinoid receptors provided the appropriate tool to isolate an endogenous cannabimimetic eicosanoid, anandamide, from porcine brain. Recent studies indicate that anandamide is a member of a family of fatty acid ethanolamides that may represent a novel class of lipid neurotransmitters. This review discusses recent progress in cannabinoid research with a focus on the receptors for delta9-THC, their coupling to second messenger responses, and the endogenous lipid cannabimimetic, anandamide.

  6. Comparisons of Δ9-Tetrahydrocannabinol and Anandamide on a Battery of Cognition-Related Behavior in Nonhuman Primates

    PubMed Central

    Leonard, Michael Z.; Shukla, Vidyanand G.; Alapafuja, Shakiru O.; Nikas, Spyros P.; Makriyannis, Alexandros; Bergman, Jack

    2016-01-01

    The primary psychoactive ingredient of marijuana, Δ9-tetrahydrocannabinol (Δ9-THC), has medicinal value but also produces unwanted deleterious effects on cognitive function, promoting the search for improved cannabinergic therapeutics. The present studies used a battery of touchscreen procedures in squirrel monkeys to compare the effects of different types of cannabinergic drugs on several measures of performance including learning (repeated acquisition), cognitive flexibility (discrimination reversal), short-term memory (delayed matching-to-sample), attention (psychomotor vigilance), and motivation (progressive ratio). Drugs studied included the cannabinoid agonist Δ9-THC, fatty acid amide hydrolase (FAAH) inhibitor cyclohexylcarbamic acid 3-carbamoylbiphenyl-3-yl ester (URB597), and endocannabinoid anandamide and its stable synthetic analog methanandamide [(R)-(+)-arachidonyl-1′-hydroxy-2′-propylamide]. The effects of Δ9-THC and anandamide after treatment with the cannabinoid receptor type 1 inverse agonist/antagonist rimonabant [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1Hpyrazole-3-carboxamide] and the FAAH inhibitor URB597, respectively, also were examined. The results showed the following: 1) Δ9-THC produced dose-related impairments of discrimination-based cognitive behavior with potency that varied across tasks (discriminative capability < learning < flexibility < short-term memory); 2) anandamide alone and URB597 alone were without effect on all endpoints; 3) anandamide following URB597 pretreatment and methanandamide had negligible effects on discriminative capability, learning, and reversal, but following large doses affected delayed matching-to-sample performance in some subjects; 4) all drugs, except anandamide and URB597, disrupted attention; and 5) progressive ratio breakpoints were generally unaffected by all drugs tested, suggesting little to no effect on motivation. Taken together, these data indicate that

  7. Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

    NASA Astrophysics Data System (ADS)

    Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

    2014-07-01

    Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

  8. Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

    PubMed Central

    Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

    2014-01-01

    Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors. PMID:25047372

  9. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    SciTech Connect

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.

    1988-01-01

    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 ..mu..M). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 ..mu..M). Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  10. Monoamine Transporter Inhibitors and Substrates as Treatments for Stimulant Abuse

    PubMed Central

    Howell, Leonard L.; Negus, S. Stevens

    2015-01-01

    The acute and chronic effects of abused psychostimulants on monoamine transporters and associated neurobiology have encouraged development of candidate medications that target these transporters. Monoamine transporters in general, and dopamine transporters in particular, are critical molecular targets that mediate abuse-related effects of psychostimulants such as cocaine and amphetamine. Moreover, chronic administration of psychostimulants can cause enduring changes in neurobiology reflected in dysregulation of monoamine neurochemistry and behavior. The current review will evaluate evidence for the efficacy of monoamine transporter inhibitors and substrates to reduce abuse-related effects of stimulants in preclinical assays of stimulant self-administration, drug discrimination and reinstatement. In considering deployment of monoamine transport inhibitors and substrates as agonist-type medications to treat stimulant abuse, the safety and abuse liability of the medications are an obvious concern, and this will also be addressed. Future directions in drug discovery should identify novel medications that retain efficacy to decrease stimulant use but possess lower abuse liability, and evaluate the degree to which efficacious medications can attenuate or reverse neurobiological effects of chronic stimulant use. PMID:24484977

  11. Monoamine transporter inhibitors and substrates as treatments for stimulant abuse.

    PubMed

    Howell, Leonard L; Negus, S Stevens

    2014-01-01

    The acute and chronic effects of abused psychostimulants on monoamine transporters and associated neurobiology have encouraged development of candidate medications that target these transporters. Monoamine transporters, in general, and dopamine transporters, in particular, are critical molecular targets that mediate abuse-related effects of psychostimulants such as cocaine and amphetamine. Moreover, chronic administration of psychostimulants can cause enduring changes in neurobiology reflected in dysregulation of monoamine neurochemistry and behavior. The current review will evaluate evidence for the efficacy of monoamine transporter inhibitors and substrates to reduce abuse-related effects of stimulants in preclinical assays of stimulant self-administration, drug discrimination, and reinstatement. In considering deployment of monoamine transport inhibitors and substrates as agonist-type medications to treat stimulant abuse, the safety and abuse liability of the medications are an obvious concern, and this will also be addressed. Future directions in drug discovery should identify novel medications that retain efficacy to decrease stimulant use but possess lower abuse liability and evaluate the degree to which efficacious medications can attenuate or reverse neurobiological effects of chronic stimulant use.

  12. Cannabidiol enhances anandamide signaling and alleviates psychotic symptoms of schizophrenia.

    PubMed

    Leweke, F M; Piomelli, D; Pahlisch, F; Muhl, D; Gerth, C W; Hoyer, C; Klosterkötter, J; Hellmich, M; Koethe, D

    2012-03-20

    Cannabidiol is a component of marijuana that does not activate cannabinoid receptors, but moderately inhibits the degradation of the endocannabinoid anandamide. We previously reported that an elevation of anandamide levels in cerebrospinal fluid inversely correlated to psychotic symptoms. Furthermore, enhanced anandamide signaling let to a lower transition rate from initial prodromal states into frank psychosis as well as postponed transition. In our translational approach, we performed a double-blind, randomized clinical trial of cannabidiol vs amisulpride, a potent antipsychotic, in acute schizophrenia to evaluate the clinical relevance of our initial findings. Either treatment was safe and led to significant clinical improvement, but cannabidiol displayed a markedly superior side-effect profile. Moreover, cannabidiol treatment was accompanied by a significant increase in serum anandamide levels, which was significantly associated with clinical improvement. The results suggest that inhibition of anandamide deactivation may contribute to the antipsychotic effects of cannabidiol potentially representing a completely new mechanism in the treatment of schizophrenia.

  13. The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation

    PubMed Central

    De Petrocellis, Luciano; Melck, Dominique; Palmisano, Antonella; Bisogno, Tiziana; Laezza, Chiara; Bifulco, Maurizio; Di Marzo, Vincenzo

    1998-01-01

    Anandamide was the first brain metabolite shown to act as a ligand of “central” CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells in vitro. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC50 values between 0.5 and 1.5 μM and 83–92% maximal inhibition at 5–10 μM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 μM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (R)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [3H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1–0.5 μM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene brca1. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor. PMID:9653194

  14. Glycine transport inhibitors for the treatment of schizophrenia.

    PubMed

    Hashimoto, Kenji

    2010-05-27

    Multiple lines of evidence indicate that hypofunction of glutamatergic neurotransmission via N-methyl-D-aspartate (NMDA) receptors might be implicated in the pathophysiology of schizophrenia, suggesting that increasing NMDA receptor function via pharmacological manipulation could provide a new strategy for the management of schizophrenia. Currently, the glycine modulatory sites on NMDA receptors present the most attractive therapeutic targets for the treatment of schizophrenia. One means of enhancing NMDA receptor neurotransmission is to increase the availability of the obligatory co-agonist glycine at modulatory sites on the NMDA receptors through the inhibition of glycine transporter-1 (GlyT-1) on glial cells. Clinical studies have demonstrated that the GlyT-1 inhibitor sarcosine (N-methyl glycine) shows antipsychotic activity in patients with schizophrenia. Accordingly, a number of pharmaceutical companies have developed novel and selective GlyT-1 inhibitors for the treatment of schizophrenia. This paper provides an overview of the various GlyT-1 inhibitors and their therapeutic potential.

  15. Glycine Transport Inhibitors for the Treatment of Schizophrenia

    PubMed Central

    Hashimoto, Kenji

    2010-01-01

    Multiple lines of evidence indicate that hypofunction of glutamatergic neurotransmission via N-methyl-D-aspartate (NMDA) receptors might be implicated in the pathophysiology of schizophrenia, suggesting that increasing NMDA receptor function via pharmacological manipulation could provide a new strategy for the management of schizophrenia. Currently, the glycine modulatory sites on NMDA receptors present the most attractive therapeutic targets for the treatment of schizophrenia. One means of enhancing NMDA receptor neurotransmission is to increase the availability of the obligatory co-agonist glycine at modulatory sites on the NMDA receptors through the inhibition of glycine transporter-1 (GlyT-1) on glial cells. Clinical studies have demonstrated that the GlyT-1 inhibitor sarcosine (N-methyl glycine) shows antipsychotic activity in patients with schizophrenia. Accordingly, a number of pharmaceutical companies have developed novel and selective GlyT-1 inhibitors for the treatment of schizophrenia. This paper provides an overview of the various GlyT-1 inhibitors and their therapeutic potential. PMID:21253021

  16. Convergent translational evidence of a role for anandamide in amygdala-mediated fear extinction, threat processing and stress-reactivity

    PubMed Central

    Gunduz-Cinar, O; MacPherson, KP; Cinar, R; Gamble-George, J; Sugden, K; Williams, B; Godlewski, G; Ramikie, TS; Gorka, AX; Alapafuja, SO; Nikas, SP; Makriyannis, A; Poulton, R; Patel, S; Hariri, AR; Caspi, A; Moffitt, TE; Kunos, G; Holmes, A

    2012-01-01

    Endocannabinoids are released ‘on-demand’ on the basis of physiological need, and can be pharmacologically augmented by inhibiting their catabolic degradation. The endocannabinoid anandamide is degraded by the catabolic enzyme fatty acid amide hydrolase (FAAH). Anandamide is implicated in the mediation of fear behaviors, including fear extinction, suggesting that selectively elevating brain anandamide could modulate plastic changes in fear. Here we first tested this hypothesis with preclinical experiments employing a novel, potent and selective FAAH inhibitor, AM3506 (5-(4-hydroxyphenyl)pentanesulfonyl fluoride). Systemic AM3506 administration before extinction decreased fear during a retrieval test in a mouse model of impaired extinction. AM3506 had no effects on fear in the absence of extinction training, or on various non-fear-related measures. Anandamide levels in the basolateral amygdala were increased by extinction training and augmented by systemic AM3506, whereas application of AM3506 to amygdala slices promoted long-term depression of inhibitory transmission, a form of synaptic plasticity linked to extinction. Further supporting the amygdala as effect-locus, the fear-reducing effects of systemic AM3506 were blocked by intra-amygdala infusion of a CB1 receptor antagonist and were fully recapitulated by intra-amygdala infusion of AM3506. On the basis of these preclinical findings, we hypothesized that variation in the human FAAH gene would predict individual differences in amygdala threat-processing and stress-coping traits. Consistent with this, carriers of a low-expressing FAAH variant (385A allele; rs324420) exhibited quicker habituation of amygdala reactivity to threat, and had lower scores on the personality trait of stress-reactivity. Our findings show that augmenting amygdala anandamide enables extinction-driven reductions in fear in mouse and may promote stress-coping in humans. PMID:22688188

  17. Glycine transporter inhibitors as therapeutic agents for schizophrenia.

    PubMed

    Hashimoto, Kenji

    2006-01-01

    Multiple lines of evidence suggest that a dysfunction in the glutamatergic neurotransmission via the N-methyl-D-aspartate (NMDA) receptors contributes to the pathophysiology of psychiatric diseases including schizophrenia. The potentiation of NMDA receptor function may be a useful approach for the treatment of diseases associated with NMDA receptor hypofunction. One possible strategy is to increase synaptic levels of glycine by blocking the glycine transporter-1 (GlyT-1) in glia cells, since glycine acts as a co-agonist site on the NMDA receptor. In this article, the author reviews the recent important patents on GlyT-1 inhibitors for treatment of schizophrenia and other psychiatric diseases associated with the NMDA receptor hypofunction.

  18. Screening for Inhibitors of Essential Leishmania Glucose Transporters

    DTIC Science & Technology

    2010-07-01

    4 Introduction: Leishmania are parasitic protozoa that cause devastating diseases throughout much of the tropical and subtropical...inhibitors was dem inhibitor of PfHT. . Introduction Parasitic protozoa such as Leishmania species, Trypanosoma rucei, and Plasmodium falciparum, the

  19. Production and physiological actions of anandamide in the vasculature of the rat kidney.

    PubMed Central

    Deutsch, D G; Goligorsky, M S; Schmid, P C; Krebsbach, R J; Schmid, H H; Das, S K; Dey, S K; Arreaza, G; Thorup, C; Stefano, G; Moore, L C

    1997-01-01

    The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects. PMID:9294122

  20. Adipocyte glucose transport regulation by eicosanoid precursors and inhibitors

    SciTech Connect

    Lee, H.C.C.

    1987-01-01

    Glucose uptake and free fatty acid release by adipocytes are increased by catecholamines. The mechanism of the stimulatory action of catecholamines on glucose uptake may be via eicosanoid production from release fatty acids. Rats were fed iso-nutrient diets with high or low safflower oil. After one month, 5 rats per diet group were fed diets with aspirin or without aspirin for 2 days. Isolated adipocytes from epididymal fat pads were incubated at 37/sup 0/C, gassed with 95% O/sub 2/-5% CO/sub 2/ in KRB buffer with 3% bovine serum albumin and with or without eicosanoid modifiers; a stimulator (10/sup -5/ M norepinephrine, N), or inhibitors (167 ..mu..l of antiserum to prostaglandin E (AntiE) per 1600 ..mu..l or 23mM Asp), or combinations of these. At 2-, 5-, and 10-min incubation, samples of incubation mixtures were taken to measure 2-deoxy glucose transport using /sup 3/H-2-deoxy glucose, /sup 14/C-inulin, and liquid scintillation counter.

  1. Anandamide and analogous endocannabinoids: a lipid self-assembly study

    SciTech Connect

    Sagnella, Sharon M.; Conn, Charlotte E.; Krodkiewska, Irena; Mulet, Xavier; Drummond, Calum J.

    2014-09-24

    Anandamide, the endogenous agonist of the cannabinoid receptors, has been widely studied for its interesting biological and medicinal properties and is recognized as a highly significant lipid signaling molecule within the nervous system. Few studies have, however, examined the effect of the physical conformation of anandamide on its function. The study presented herein has focused on characterizing the self-assembly behaviour of anandamide and four other endocannabinoid analogues of anandamide, viz., 2-arachidonyl glycerol, arachidonyl dopamine, 2-arachidonyl glycerol ether (noladin ether), and o-arachidonyl ethanolamide (virodhamine). Molecular modeling of the five endocannabinoid lipids indicates that the highly unsaturated arachidonyl chain has a preference for a U or J shaped conformation. Thermal phase studies of the neat amphiphiles showed that a glass transition was observed for all of the endocannabinoids at {approx} -110 C with the exception of anandamide, with a second glass transition occurring for 2-arachidonyl glycerol, 2-arachidonyl glycerol ether, and virodhamine (-86 C, -95 C, -46 C respectively). Both anandamide and arachidonyl dopamine displayed a crystal-isotropic melting point (-4.8 and -20.4 C respectively), while a liquid crystal-isotropic melting transition was seen for 2-arachidonyl glycerol (-40.7 C) and 2-arachidonyl glycerol ether (-71.2 C). No additional transitions were observed for virodhamine. Small angle X-ray scattering and cross polarized optical microscopy studies as a function of temperature indicated that in the presence of excess water, both 2-arachidonyl glycerol and anandamide form co-existing Q{sub II}{sup G} (gyroid) and Q{sub II}{sup D} (diamond) bicontinuous cubic phases from 0 C to 20 C, which are kinetically stable over a period of weeks but may not represent true thermodynamic equilibrium. Similarly, 2-arachidonyl glycerol ether acquired an inverse hexagonal (HII) phase in excess water from 0 C to 40 C, while

  2. Flavonoids are inhibitors of human organic anion transporter 1 (OAT1)-mediated transport.

    PubMed

    An, Guohua; Wang, Xiaodong; Morris, Marilyn E

    2014-09-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of <0.3 and 0.47 μM, respectively. In contrast to the tested flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway.

  3. Differential effects of glutamate transporter inhibitors on the global electrophysiological response of astrocytes to neuronal stimulation.

    PubMed

    Bernardinelli, Yann; Chatton, Jean-Yves

    2008-11-13

    Astrocytes are responsible for regulating extracellular levels of glutamate and potassium during neuronal activity. Glutamate clearance is handled by glutamate transporter subtypes glutamate transporter 1 and glutamate-aspartate transporter in astrocytes. DL-threo-beta-benzyloxyaspartate (TBOA) and dihydrokainate (DHK) are extensively used as inhibitors of glial glutamate transport activity. Using whole-cell recordings, we characterized the effects of both transporter inhibitors on afferent-evoked astrocyte currents in acute cortical slices of 3-week-old rats. When neuronal afferents were stimulated, passive astrocytes responded by a rapid inward current followed by a persistent tail current. The first current corresponded to a glutamate transporter current. This current was inhibited by both inhibitors and by tetrodotoxin. The tail current is an inward potassium current as it was blocked by barium. Besides inhibiting transporter currents, TBOA strongly enhanced the tail current. This effect was barium-sensitive and might be due to a rise in extracellular potassium level and increased glial potassium uptake. Unlike TBOA, DHK did not enhance the tail current but rather inhibited it. This result suggests that, in addition to inhibiting glutamate transport, DHK prevents astrocyte potassium uptake, possibly by blockade of inward-rectifier channels. This study revealed that, in brain slices, glutamate transporter inhibitors exert complex effects that cannot be attributed solely to glutamate transport inhibition.

  4. Interactions between glycine transporter type 1 (GlyT-1) and some inhibitor molecules - glycine transporter type 1 and its inhibitors (review).

    PubMed

    Harsing, Laszlo G; Zsilla, G; Matyus, P; Nagy, K M; Marko, B; Gyarmati, Zs; Timar, J

    2012-03-01

    Glycine is a mandatory positive allosteric modulator of N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors in the central nervous system. Elevation of glycine concentrations by inhibition of its reuptake in the vicinity of NMDA receptors may positively influence receptor functions as glycine B binding site on NR1 receptor subunit is not saturated in physiological conditions. Synaptic and extrasynaptic concentrations of glycine are regulated by its type-1 glycine transporter, which is primarily expressed in astroglial and glutamatergic cell membranes. Alteration of synaptic glycine levels may have importance in the treatment of various forms of endogenous psychosis characterized by hypofunctional NMDA receptors. Several lines of evidence indicate that impaired NMDA receptor-mediated glutamatergic neurotransmission is involved in development of the negative (and partly the positive) symptoms and the cognitive deficit in schizophrenia. Inhibitors of glycine transporter type-1 may represent a newly developed therapeutic intervention in treatment of this mental illness. We have synthesized a novel series of N-substituted sarcosines, analogues of the glycine transporter-1 inhibitor NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine). Of the pyridazinone-containing compounds, SzV-1997 was found to be a potent glycine transporter-1 inhibitor in rat brain synaptosomes and it markedly increased extracellular glycine concentrations in conscious rat striatum. SzV-1997 did not exhibit toxic symptoms such as hyperlocomotion, restless movements, respiratory depression, and lethality, characteristic for NFPS. Besides pyridazinone-based, sarcosine-containing glycine transporter-1 inhibitors, a series of substrate-type amino acid inhibitors was investigated in order to obtain better insight into the ligand-binding characteristics of the substrate binding cavity of the transporter.

  5. Is there a temperature-dependent uptake of anandamide into cells?

    PubMed Central

    Thors, L; Fowler, C J

    2006-01-01

    Background and purpose: The temperature dependency of anandamide uptake into cells implies an active mechanism but this is still a matter of considerable debate. We have therefore re-examined the temperature-sensitive uptake of anandamide in ND7/23 mouse neuroblastoma × rat dorsal root ganglion neurone hybrid cells and RBL2H3 rat basophilic leukaemia cells. Experimental approach: Cellular uptake of [3H] anandamide was measured in the presence of bovine serum albumin at different incubation temperatures and times. Rates of uptake were also measured in wells alone. Free anandamide concentrations were calculated by published methods. Key results: Anandamide showed a time-dependent saturable uptake into ND7/23 cells. The uptake was greater at 37°C than at 4°C for a given added anandamide concentration following a 5 min incubation. However, this temperature-dependency reflected temperature-dependent effects on the concentration of anandamide available for uptake, rather than the uptake process itself. A similar conclusion could be drawn for the rapid (∼1 min) uptake of anandamide into RBL2H3 cells. In contrast, re-analysis of published data for P19 cells indicated a clear temperature-dependency of the uptake at long (15 min) incubation times. The level of anandamide retained by wells alone provided a better measure of free anandamide concentrations than calculated values. Conclusions and implications: ND7/23 cells may be a useful model system for the study of anandamide uptake. The temperature-dependent uptake of anandamide may reflect effects on free anandamide concentrations rather than on the uptake process itself. PMID:16865094

  6. Solute transport and oxygen consumption along the nephrons: effects of Na+ transport inhibitors.

    PubMed

    Layton, Anita T; Laghmani, Kamel; Vallon, Volker; Edwards, Aurélie

    2016-12-01

    Sodium and its associated anions are the major determinant of extracellular fluid volume, and the reabsorption of Na(+) by the kidney plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial Na(+) transport (TNa) along the nephron alter urinary solute excretion and TNa efficiency and how those effects may vary along different nephron segments. To accomplish that goal, we used the multinephron model developed in the companion study (28). That model represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na(+)/H(+) exchanger (NHE3), the bumetanide-sensitive Na(+)-K(+)-2Cl(-) transporter (NKCC2), the Na(+)-Cl(-) cotransporter (NCC), and the amiloride-sensitive Na(+) channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC, and ENaC reabsorb 36, 22, 4, and 7%, respectively, of filtered Na(+) The model predicted that inhibition of NHE3 substantially reduced proximal tubule TNa and oxygen consumption (QO2 ). Whole-kidney TNa efficiency, as reflected by the number of moles of Na(+) reabsorbed per moles of O2 consumed (denoted by the ratio TNa/QO2 ), decreased by ∼20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb TNa and QO2 ; however, the effect on whole-kidney TNa/QO2 was minor. Tubular K(+) transport was also substantially impaired, resulting in elevated urinary K(+) excretion. The most notable effect of NCC inhibition was to increase the excretion of Na(+), K(+), and Cl(-); its impact on whole-kidney TNa and its efficiency was minor. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na(+) (increased) and K(+) (decreased) and to have only a minor impact on whole-kidney TNa and TNa/QO2 Overall, model predictions agree well with measured changes in Na(+) and K(+) excretion in response to

  7. Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters.

    PubMed

    Tsuda, Etsuko; Yang, Haibing; Nishimura, Takeshi; Uehara, Yukiko; Sakai, Tatsuya; Furutani, Masahiko; Koshiba, Tomokazu; Hirose, Masakazu; Nozaki, Hiroshi; Murphy, Angus S; Hayashi, Ken-ichiro

    2011-01-21

    Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

  8. Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters*

    PubMed Central

    Tsuda, Etsuko; Yang, Haibing; Nishimura, Takeshi; Uehara, Yukiko; Sakai, Tatsuya; Furutani, Masahiko; Koshiba, Tomokazu; Hirose, Masakazu; Nozaki, Hiroshi; Murphy, Angus S.; Hayashi, Ken-ichiro

    2011-01-01

    Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCFTIR1 auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development. PMID:21084292

  9. Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration.

    PubMed

    Hansen, H H; Schmid, P C; Bittigau, P; Lastres-Becker, I; Berrendero, F; Manzanares, J; Ikonomidou, C; Schmid, H H; Fernández-Ruiz, J J; Hansen, H S

    2001-09-01

    Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.

  10. A novel strategy for the treatment of diabetes mellitus - sodium glucose co-transport inhibitors

    PubMed Central

    Niazi, Asfandyar Khan; Niazi, Saad Hameed

    2010-01-01

    Background: Diabetes is one of the most common chronic diseases, affecting almost 3 million in Canada alone and is characterized by increased blood glucose levels. Treatment varies from lifestyle changes to oral anti-diabetics and/or insulin. Sodium glucose co-transport inhibitors may offer promising treatment for patients suffering from diabetes. The inhibitors act by increasing the loss of glucose in urine by decreasing the reabsorption of glucose from the proximal tubules of nephrons. Aims: The aim of this review was to assess the efficacy of sodium glucose co-transport inhibitors in the treatment of diabetes as well as any adverse effects. Materials and Methods: Databases such as MEDLINE, COCHRANE and EMBASE were systematically searched for literature on the efficacy of sodium glucose co-transport inhibitors in improving the glycemic control of patients with diabetes. Results: Research showed that sodium glucose co-transport inhibitors significantly decreased blood glucose levels by increasing glucosuria. Due to the diuretic effects of these inhibitors, diabetic patients who were suffering from hypertension showed a decrease in blood pressure. The caloric loss associated with these inhibitors resulted in weight loss as well. The most common adverse effect seen in patients on these medications was mycotic infection of the urinary or genital tract. Conclusion: Sodium glucose co-transport inhibitors may be an effective line of treatment for diabetes. Although short-term research has shown these drugs to be safe and well-tolerated, studies should be conducted to assess the long-term effects of these drugs. PMID:22558567

  11. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter.

    PubMed

    Andersen, Jacob; Ringsted, Kristoffer B; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S

    2015-10-27

    The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs.

  12. Binding site residues control inhibitor selectivity in the human norepinephrine transporter but not in the human dopamine transporter

    PubMed Central

    Andersen, Jacob; Ringsted, Kristoffer B.; Bang-Andersen, Benny; Strømgaard, Kristian; Kristensen, Anders S.

    2015-01-01

    The transporters for norepinephrine and dopamine (NET and DAT, respectively) constitute the molecular targets for recreational drugs and therapeutics used in the treatment of psychiatric disorders. Despite a strikingly similar amino acid sequence and predicted topology between these transporters, some inhibitors display a high degree of selectivity between NET and DAT. Here, a systematic mutational analysis of non-conserved residues within the extracellular entry pathway and the high affinity binding site in NET and DAT was performed to examine their role for selective inhibitor recognition. Changing the six diverging residues in the central binding site of NET to the complementary residues in DAT transferred a DAT-like pharmacology to NET, showing that non-conserved binding site residues in NET are critical determinants for inhibitor selectivity. In contrast, changing the equivalent residues in the central site of DAT to the corresponding residues in NET had modest effects on the same inhibitors, suggesting that non-conserved binding site residues in DAT play a minor role for selective inhibitor recognition. Our data points towards distinct structural determinants governing inhibitor selectivity in NET and DAT, and provide important new insight into the molecular basis for NET/DAT selectivity of therapeutic and recreational drugs. PMID:26503701

  13. Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors

    PubMed Central

    Duan, Haichuan; Hu, Tao; Foti, Robert S.; Pan, Yongmei; Swaan, Peter W.

    2015-01-01

    Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1–3 (OCT1–3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1–3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC50 = 1.4 ± 0.2 µM) over OCT1 (IC50 = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1–3. PMID:26285765

  14. Inhibition of anandamide hydrolysis attenuates nociceptor sensitization in a murine model of chemotherapy-induced peripheral neuropathy

    PubMed Central

    Uhelski, Megan L.; Khasabova, Iryna A.

    2014-01-01

    Painful neuropathy frequently develops as a consequence of commonly used chemotherapy agents for cancer treatment and is often a dose-limiting side effect. Currently available analgesic treatments are often ineffective on pain induced by neurotoxicity. Although peripheral administration of cannabinoids, endocannabinoids, and inhibitors of endocannabinoid hydrolysis has been effective in reducing hyperalgesia in models of peripheral neuropathy, including chemotherapy-induced peripheral neuropathy (CIPN), few studies have examined cannabinoid effects on responses of nociceptors in vivo. In this study we determined whether inhibition of fatty acid amide hydrolase (FAAH), which slows the breakdown of the endocannabinoid anandamide (AEA), reduced sensitization of nociceptors produced by chemotherapy. Over the course of a week of daily treatments, mice treated with the platinum-based chemotherapy agent cisplatin developed robust mechanical allodynia that coincided with sensitization of cutaneous C-fiber nociceptors as indicated by the development of spontaneous activity and increased responses to mechanical stimulation. Administration of the FAAH inhibitor URB597 into the receptive field of sensitized C-fiber nociceptors decreased spontaneous activity, increased mechanical response thresholds, and decreased evoked responses to mechanical stimuli. Cotreatment with CB1 (AM281) or CB2 (AM630) receptor antagonists showed that the effect of URB597 was mediated primarily by CB1 receptors. These changes following URB597 were associated with an increase in the endocannabinoid anandamide in the skin. Our results suggest that enhanced signaling in the peripheral endocannabinoid system could be utilized to reduce nociceptor sensitization and pain associated with CIPN. PMID:25505113

  15. Substituted quinolines as inhibitors of L-glutamate transport into synaptic vesicles.

    PubMed

    Bartlett, R D; Esslinger, C S; Thompson, C M; Bridges, R J

    1998-07-01

    This study investigated the structure-activity relationships and kinetic properties of a library of kynurenate analogues as inhibitors of 3H-L-glutamate transport into rat forebrain synaptic vesicles. The lack of inhibitory activity observed with the majority of the monocyclic pyridine derivatives suggested that the second aromatic ring of the quinoline-based compounds played a significant role in binding to the transporter. A total of two kynurenate derivatives, xanthurenate and 7-chloro-kynurenate, differing only in the carbocyclic ring substituents, were identified as potent competitive inhibitors, exhibiting Ki values of 0.19 and 0.59 mM, respectively. The Km value for L-glutamate was found to be 2.46 mM. Parallel experiments demonstrated that while none of the kynurenate analogues tested effectively inhibited the synaptosomal transport of 3H-D-aspartate, some cross-reactivity was observed with the EAA ionotropic receptors. Molecular modeling studies were carried out with the identified inhibitors and glutamate in an attempt to preliminarily define the pharmacophore of the vesicular transporter. It is hypothesized that the ability of the kynurenate analogues to bind to the transporter may be tied to the capacity of the quinoline carbocyclic ring to mimic the negative charge of the gamma-carboxylate of glutamate. A total of two low energy solution conformers of glutamate were identified that exhibited marked functional group overlap with the most potent inhibitor, xanthurenate. These results help to further refine the pharmacological specificity of the glutamate binding site on the vesicular transporter and identify a series of inhibitors with which to investigate transporter function.

  16. Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

    PubMed Central

    Kelley, G G; Aassar, O S; Forrest, J N

    1991-01-01

    The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport. Images PMID:1752953

  17. Enzyme- and transporter-mediated drug interactions with small molecule tyrosine kinase inhibitors.

    PubMed

    Shao, Jie; Markowitz, John S; Bei, Di; An, Guohua

    2014-12-01

    Among the novel and target-specific classes of anticancer drugs, small molecule tyrosine kinase inhibitors (TKIs) represent an extremely promising and rapidly expanding group. TKIs attack cancer-specific targets and therefore have a favorable safety profile. However, as TKIs are taken orally along with other medications on a daily basis, there is an elevated risk of potentially significant drug-drug interactions. Most TKIs are metabolized primarily through CYP3A4. In addition, many TKIs are also CYP3A4 inhibitors at the same time. In addition to drug metabolizing enzymes (DMEs), another determinant of TKI disposition are drug transporters. There is accumulating evidence showing that the majority of currently marketed TKIs interact with ATP-binding cassette transporters, particularly P-glycoprotein as well as Breast Cancer Resistance Protein and serve as both substrates and inhibitors. Considering the dual roles of TKIs on both DMEs and drug transporters, and the importance of these enzyme and transporters in drug disposition, the potential for enzyme- and transporter-mediated TKI-drug interactions in patients with cancer is an important consideration. This review provides a comprehensive overview of drug interactions with small molecule TKIs mediated by DMEs and drug transporters. The TKI-drug interactions with TKIs being victims and/or perpetrators are summarized.

  18. Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2.

    PubMed

    Singh, Kurnvir; Tanui, Rose; Gameiro, Armanda; Eisenberg, Gilad; Colas, Claire; Schlessinger, Avner; Grewer, Christof

    2017-02-01

    The glutamine transporter ASCT2 has been identified as a promising target to inhibit rapid growth of cancer cells. However, ASCT2 pharmacology is not well established. In this report, we performed a systematic structure activity analysis of a series of substituted benzylproline derivatives. Substitutions on the phenyl ring resulted in compounds with characteristics of ASCT2 inhibitors. Apparent binding affinity increased with increasing hydrophobicity of the side chain. In contrast, interaction of the ASCT2 binding site with specific positions on the phenyl ring was not observed. The most potent compound inhibits the ASCT2 anion conductance with a Ki of 3μM, which is in the same range as that of more bulky and higher molecular weight inhibitors recently reported by others. The experimental results are consistent with computational analysis based on docking of the inhibitors against an ASCT2 homology model. The benzylproline scaffold provides a valuable tool for further improving binding potency of future ASCT2 inhibitors.

  19. The organic anion transport inhibitor probenecid increases brain concentrations of the NKCC1 inhibitor bumetanide.

    PubMed

    Töllner, Kathrin; Brandt, Claudia; Römermann, Kerstin; Löscher, Wolfgang

    2015-01-05

    Bumetanide is increasingly being used for experimental treatment of brain disorders, including neonatal seizures, epilepsy, and autism, because the neuronal Na-K-Cl cotransporter NKCC1, which is inhibited by bumetanide, is implicated in the pathophysiology of such disorders. However, use of bumetanide for treatment of brain disorders is associated with problems, including poor brain penetration and systemic adverse effects such as diuresis, hypokalemic alkalosis, and hearing loss. The poor brain penetration is thought to be related to its high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, but more recently brain efflux transporters have been involved, too. Multidrug resistance protein 4 (MRP4), organic anion transporter 3 (OAT3) and organic anion transporting polypeptide 2 (OATP2) were suggested to mediate bumetanide brain efflux, but direct proof is lacking. Because MRP4, OAT3, and OATP2 can be inhibited by probenecid, we studied whether this drug alters brain levels of bumetanide in mice. Probenecid (50 mg/kg) significantly increased brain levels of bumetanide up to 3-fold; however, it also increased its plasma levels, so that the brain:plasma ratio (~0.015-0.02) was not altered. Probenecid markedly increased the plasma half-life of bumetanide, indicating reduced elimination of bumetanide most likely by inhibition of OAT-mediated transport of bumetanide in the kidney. However, the diuretic activity of bumetanide was not reduced by probenecid. In conclusion, our study demonstrates that the clinically available drug probenecid can be used to increase brain levels of bumetanide and decrease its elimination, which could have therapeutic potential in the treatment of brain disorders.

  20. Uptake inhibitors but not substrates induce protease resistance in extracellular loop two of the dopamine transporter.

    PubMed

    Gaffaney, Jon D; Vaughan, Roxanne A

    2004-03-01

    Changes in protease sensitivity of extracellular loop two (EL2) of the dopamine transporter (DAT) during inhibitor and substrate binding were examined using trypsin proteolysis and epitope-specific immunoblotting. In control rat striatal membranes, proteolysis of DAT in a restricted region of EL2 was produced by 0.001 to 10 microg/ml trypsin. However, in the presence of the dopamine uptake blockers [2-(diphenylmethoxyl) ethyl]-4-(3phenylpropyl) piperazine (GBR 12909), mazindol, 2beta-carbomethoxy-3beta-(4-flourophenyl)tropane (beta-CFT), nomifensine, benztropine, or (-)-cocaine, 100- to 1000-fold higher concentrations of trypsin were required to produce comparable levels of proteolysis. Protease resistance induced by ligands was correlated with their affinity for DAT binding, was not observed with Zn2+, (+)-cocaine, or inhibitors of norepinephrine or serotonin transporters, and was not caused by altered catalytic activity of trypsin. Together, these results support the hypothesis that the interaction of uptake inhibitors with DAT induces a protease-resistant conformation in EL2. In contrast, binding of substrates did not induce protease resistance in EL2, suggesting that substrates and inhibitors interact with DAT differently during binding. To assess the effects of EL2 proteolysis on DAT function, the binding and transport properties of trypsin-digested DAT were assayed with [3H]CFT and [3H]dopamine. Digestion decreased the Bmax for binding and the Vmax for uptake in amounts that were proportional to the extent of proteolysis, indicating that the structural integrity of EL2 is required for maintenance of both DAT binding and transport functions. Together this data provides novel information about inhibitor and substrate interactions at EL2, possibly relating the protease resistant DAT conformation to a mechanism of transport inhibition.

  1. Anandamide inhibits adhesion and migration of breast cancer cells

    SciTech Connect

    Grimaldi, Claudia; Pisanti, Simona; Laezza, Chiara; Malfitano, Anna Maria; Santoro, Antonietta; Vitale, Mario; Caruso, Maria Gabriella; Notarnicola, Maria; Iacuzzo, Irma; Portella, Giuseppe; Di Marzo, Vincenzo . E-mail: vdimarzo@icmib.na.cnr.it; Bifulco, Maurizio . E-mail: maubiful@unina.it

    2006-02-15

    The endocannabinoid system regulates cell proliferation in human breast cancer cells. We reasoned that stimulation of cannabinoid CB{sub 1} receptors could induce a non-invasive phenotype in breast mtastatic cells. In a model of metastatic spreading in vivo, the metabolically stable anandamide analogue, 2-methyl-2'-F-anandamide (Met-F-AEA), significantly reduced the number and dimension of metastatic nodes, this effect being antagonized by the selective CB{sub 1} antagonist SR141716A. In MDA-MB-231 cells, a highly invasive human breast cancer cell line, and in TSA-E1 cells, a murine breast cancer cell line, Met-F-AEA inhibited adhesion and migration on type IV collagen in vitro without modifying integrin expression: both these effects were antagonized by SR141716A. In order to understand the molecular mechanism involved in these processes, we analyzed the phosphorylation of FAK and Src, two tyrosine kinases involved in migration and adhesion. In Met-F-AEA-treated cells, we observed a decreased tyrosine phosphorylation of both FAK and Src, this effect being attenuated by SR141716A. We propose that CB{sub 1} receptor agonists inhibit tumor cell invasion and metastasis by modulating FAK phosphorylation, and that CB{sub 1} receptor activation might represent a novel therapeutic strategy to slow down the growth of breast carcinoma and to inhibit its metastatic diffusion in vivo.

  2. Does the Neuroprotective Role of Anandamide Display Diurnal Variations?

    PubMed Central

    Martinez-Vargas, Marina; Morales-Gomez, Julio; Gonzalez-Rivera, Ruben; Hernandez-Enriquez, Carla; Perez-Arredondo, Adan; Estrada-Rojo, Francisco; Navarro, Luz

    2013-01-01

    The endocannabinoid system is a component of the neuroprotective mechanisms that an organism displays after traumatic brain injury (TBI). A diurnal variation in several components of this system has been reported. This variation may influence the recovery and survival rate after TBI. We have previously reported that the recovery and survival rate of rats is higher if TBI occurs at 1:00 than at 13:00. This could be explained by a diurnal variation of the endocannabinoid system. Here, we describe the effects of anandamide administration in rats prior to the induction of TBI at two different times of the day: 1:00 and 13:00. We found that anandamide reduced the neurological damage at both times. Nevertheless, its effects on bleeding, survival, food intake, and body weight were dependent on the time of TBI. In addition, we analyzed the diurnal variation of the expression of the cannabinoid receptors CB1R and CB2R in the cerebral cortex of both control rats and rats subjected to TBI. We found that CB1R protein was expressed more during the day, whereas its mRNA level was higher during the night. We did not find a diurnal variation for the CB2R. In addition, we also found that TBI increased CB1R and CB2R in the contralateral hemisphere and disrupted the CB1R diurnal cycle. PMID:24287910

  3. Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters

    PubMed Central

    Shimokawa, Yoshihiko; Shibata, Masakazu; Hashizume, Kenta; Hamasako, Yusuke; Ohzone, Yoshihiro; Kashiyama, Eiji; Umehara, Ken

    2016-01-01

    Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro. Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 μmol/liter, respectively; M2, 7.80 and 6.02 μmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters. PMID:27021329

  4. Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1.

    PubMed

    Parry, G; Delbarre, A; Marchant, A; Swarup, R; Napier, R; Perrot-Rechenmann, C; Bennett, M J

    2001-02-01

    The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.

  5. Down-regulation of the rat serotonin transporter upon exposure to a selective serotonin reuptake inhibitor.

    PubMed

    Horschitz, S; Hummerich, R; Schloss, P

    2001-07-20

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) into the nerve terminal or axonal varicosities. SERT represents the target of various antidepressants which inhibit 5-HT transport and are widely used for the pharmacotherapy of depression. Here, we have analyzed the function of SERT stably expressed in HEK 293 cells upon exposure to citalopram, a selective serotonin reuptake inhibitor (SSRI), with respect to 5-HT transport activity and protein expression as estimated by ligand binding experiments. Our results show that long-term exposure to an SSRI causes a down-regulation of transport activity as revealed by a reduction of the maximal transport rate, without affecting substrate affinity, accompanied by a decrease in ligand binding sites.

  6. A specific pharmacophore model of sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors.

    PubMed

    Tang, Chunlei; Zhu, Xiaoyun; Huang, Dandan; Zan, Xin; Yang, Baowei; Li, Ying; Du, Xiaoyong; Qian, Hai; Huang, Wenlong

    2012-06-01

    Sodium-dependent glucose co-transporter 2 (SGLT2) plays a pivotal role in maintaining glucose equilibrium in the human body, emerging as one of the most promising targets for the treatment of diabetes mellitus type 2. Pharmacophore models of SGLT2 inhibitors have been generated with a training set of 25 SGLT2 inhibitors using Discovery Studio V2.1. The best hypothesis (Hypo1(SGLT2)) contains one hydrogen bond donor, five excluded volumes, one ring aromatic and three hydrophobic features, and has a correlation coefficient of 0.955, cost difference of 68.76, RMSD of 0.85. This model was validated by test set, Fischer randomization test and decoy set methods. The specificity of Hypo1(SGLT2) was evaluated. The pharmacophore features of Hypo1(SGLT2) were different from the best pharmacophore model (Hypo1(SGLT1)) of SGLT1 inhibitors we developed. Moreover, Hypo1(SGLT2) could effectively distinguish selective inhibitors of SGLT2 from those of SGLT1. These results indicate that a highly predictive and specific pharmacophore model of SGLT2 inhibitors has been successfully obtained. Then Hypo1(SGLT2) was used as a 3D query to screen databases including NCI and Maybridge for identifying new inhibitors of SGLT2. The hit compounds were subsequently subjected to filtering by Lipinski's rule of five. And several compounds selected from the top ranked hits have been suggested for further experimental assay studies.

  7. Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

    PubMed

    Domin, B A; Mahony, W B; Zimmerman, T P

    1988-07-05

    A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

  8. Identification and Characterization of ML352: A Novel, Noncompetitive Inhibitor of the Presynaptic Choline Transporter

    PubMed Central

    2015-01-01

    The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents. PMID:25560927

  9. Nonclassical pharmacology of the dopamine transporter: atypical inhibitors, allosteric modulators, and partial substrates.

    PubMed

    Schmitt, Kyle C; Rothman, Richard B; Reith, Maarten E A

    2013-07-01

    The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic, and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. Because both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands possess an addictive liability equivalent to that of cocaine. However, certain recently developed ligands, such as atypical benztropine-like DAT inhibitors with reduced or even a complete lack of cocaine-like rewarding effects, suggest that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classic preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein that can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychological effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.

  10. Activity of anandamide (AEA) metabolic enzymes in rat placental bed.

    PubMed

    Fonseca, B M; Battista, N; Correia-da-Silva, G; Rapino, C; Maccarrone, M; Teixeira, N A

    2014-11-01

    Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.

  11. Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2.

    PubMed

    Xu, Chenghao; Zhu, Ling; Chan, Ting; Lu, Xiaoxi; Shen, Weiyong; Madigan, Michele C; Gillies, Mark C; Zhou, Fanfan

    2016-02-01

    Chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used to treat malaria and inflammatory diseases, long-term usage of which often causes severe side effects, especially retinopathy. Solute carrier transporters (SLCs) are important proteins responsible for the cellular uptake of endogenous and exogenous substances. Inhibitors competing with transporter substrates for SLCs often results in unfavorable toxicities and unsatisfactory therapeutic outcomes. We investigated the inhibitory effect of CQ and HCQ on substrate uptake mediated through a range of important SLC transporters in overexpressing human embryonic kidney (HEK293) cells. Our data revealed that both CQ and HCQ potently inhibit the uptake activity of organic anion transporting polypeptide 1A2 (OATP1A2). We recently reported OATP1A2 to be expressed in human retinal pigment epithelium (RPE), where it mediates cellular uptake of all-trans-retinol (atROL), a key step in the classical visual cycle. In this study, we demonstrate that CQ and HCQ could markedly impair atROL uptake in OATP1A2-expressing HEK293 cells and more importantly, in primary human RPE cells. Our study shows that CQ and HCQ are novel inhibitors of OATP1A2 and significantly impair OATP1A2-mediated substrate uptake, particularly transport of atROL into the RPE. This effect may compromise the function of the classic visual cycle leading to vision impairment and contribute to the retinopathy observed clinically in patients using CQ or HCQ.

  12. Anandamide and 2-arachidonoylglycerol: pharmacological properties, functional features, and emerging specificities of the two major endocannabinoids.

    PubMed

    Luchicchi, Antonio; Pistis, Marco

    2012-10-01

    Since the discovery of endocannabinoids and their receptors, two major members of the endocannabinoid family, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), have been regarded almost as twin brothers. Pharmacological properties were initially considered to be similar, as these molecules were believed mutually exchangeable and almost indistinguishable in the regulation of synaptic functions, such as long- and short-term synaptic plasticity, and in behavioral aspects, such as learning and memory, reward and addiction, antinociception, and anxiety. In recent years, however, endocannabinoid signaling specificity began to emerge, in particular, due to the production of genetically engineered mice lacking key enzymes in endocannabinoid synthesis or degradation, together with the development of selective inhibitors of AEA or 2-AG catabolic enzymes. Evidence now suggests that AEA and 2-AG possess specific pharmacological properties, are engaged in different forms of synaptic plasticity, and take part in different behavioral functions. In this review, we provide an overview on similarities and specificities of the two endocannabinoids in the CNS and on the unresolved questions concerning their role in synaptic signaling.

  13. Small molecule peptidomimetic inhibitors of importin α/β mediated nuclear transport

    PubMed Central

    Ambrus, Géza; Whitby, Landon R.; Singer, Eric L.; Trott, Oleg; Choi, Euna; Olson, Arthur J.; Boger, Dale L.; Gerace, Larry

    2010-01-01

    Nucleocytoplasmic transport of macromolecules is a fundamental process of eukaryotic cells. Translocation of proteins and many RNAs between the nucleus and cytoplasm is carried out by shuttling receptors of the β-karyopherin family, also called importins and exportins. Leptomycin B, a small molecule inhibitor of the exportin CRM1, has proved to be an invaluable tool for cell biologists, but up to now no small molecule inhibitors of nuclear import have been described. We devised a microtiter plate based permeabilized cell screen for small molecule inhibitors of the importin α/β pathway. By analyzing peptidomimetic libraries, we identified β-turn and α-helix peptidomimetic compounds that selectively inhibit nuclear import by importin α/β but not by transportin. Structure-activity relationship analysis showed that large aromatic residues and/or a histidine side chain are required for effective import inhibition by these compounds. Our validated inhibitors can be useful for in vitro studies of nuclear import, and can also provide a framework for synthesis of higher potency nuclear import inhibitors. PMID:20869252

  14. Combinatorial pharmacophore modeling of organic cation transporter 2 (OCT2) inhibitors: insights into multiple inhibitory mechanisms.

    PubMed

    Xu, Yuan; Liu, Xian; Li, Shanshan; Zhou, Nannan; Gong, Likun; Luo, Cheng; Luo, Xiaomin; Zheng, Mingyue; Jiang, Hualiang; Chen, Kaixian

    2013-12-02

    Organic cation transporter 2 (OCT2) is responsible for the entry step of many drugs in renal elimination, of which the changing activity may cause unwanted drug-drug interactions (DDIs). To develop drugs with favorable safety profile and provide instruction for rational clinical drug administration, it is of great interest to investigate the multiple mechanisms of OCT2 inhibition. In this study, we designed a combinatorial scheme to screen the optimum combination of pharmacophores from a pool of hypotheses established based on 162 OCT2 inhibitors. Among them, one single pharmacophore hypothesis represents a potential binding mode that may account for one unique inhibitory mechanism, and the obtained pharmacophore combination describes the multimechanisms of OCT2 inhibition. The final model consists of four individual pharmacophores, i.e., DHPR18, APR2, PRR5 and HHR4. Given a query ligand, it is considered as an inhibitor if it matches at least one of the hypotheses, or a noninhibitor if it fails to match any of four hypotheses. Our combinatorial pharmacophore model performs reasonably well to discriminate inhibitors and noninhibitors, yielding an overall accuracy around 0.70 for a test set containing 81 OCT2 inhibitors and 218 noninhibitors. Intriguingly, we found that the number of matched hypotheses was positively correlated with inhibition rate, which coincides with the pharmacophore modeling result of P-gp substrate binding. Further analysis suggested that the hypothesis PRR5 was responsible for competitive inhibition of OCT2, and other hypotheses were important for interaction between the inhibitor and OCT2. In light of the results, a hypothetical model for inhibiting transporting mediated by OCT2 was proposed.

  15. Tyrosine kinase inhibitors as modulators of ABC transporter-mediated drug resistance

    PubMed Central

    Shukla, Suneet; Chen, Zhe-Sheng; Ambudkar, Suresh V.

    2012-01-01

    Tyrosine kinases (TKs) are involved in key signaling events/pathways that regulate cancer cell proliferation, apoptosis, angiogenesis and metastasis. Deregulated activity of TKs has been implicated in several types of cancers. In recent years, tyrosine kinase inhibitors (TKIs) have been developed to inhibit specific kinases whose constitutive activity results in specific cancer types. These TKIs have been found to demonstrate effective anticancer activity and some of them have been approved by the Food and Drug Administration for clinical use or are in clinical trials. However, these targeted therapeutic agents are also transported by ATP-binding cassette (ABC) transporters, resulting in altered pharmacokinetics or development of resistance to these drugs in cancer patients. This review covers the recent findings on the interactions of clinically important TKIs with ABC drug transporters. Future research efforts in the development of novel TKIs with specific targets, seeking improved activity, should consider these underlying causes of resistance to TKIs in cancer cells. PMID:22325423

  16. Spinal anandamide produces analgesia in neuropathic rats: possible CB(1)- and TRPV1-mediated mechanisms.

    PubMed

    Starowicz, K; Makuch, W; Osikowicz, M; Piscitelli, F; Petrosino, S; Di Marzo, V; Przewlocka, B

    2012-03-01

    The endocannabinoid anandamide (AEA) activates also transient receptor potential vanilloid-1 (TRPV1) channels. However, no data exist on the potential role of spinal TRPV1 activation by AEA in neuropathic pain. We tested the effect of: 1) AEA (5-100 μg), alone or in the presence of an inhibitor of its hydrolysis, and 2) elevated levels of endogenous AEA (following inhibition of AEA hydrolysis), in CCI rats, and the involvement of TRPV1 or cannabinoid CB(1) receptors in the observed effects. Levels of AEA in the spinal cord of CCI rats were measured following all treatments. AEA (50 μg) displayed anti-allodynic and anti-hyperalgesic effects which were abolished by previous antagonism of TRPV1, but not CB(1), receptors. Depending on the administered dose, the selective inhibitor of AEA enzymatic hydrolysis, URB597 (10-100 μg), reduced thermal and tactile nociception via CB(1) or CB(1)/TRPV1 receptors. The anti-nociceptive effects of co-administered per se ineffective doses of AEA (5 μg) and URB597 (5 μg) was abolished by antagonism of CB(1), but not TRPV1, receptors. Spinal AEA levels were increased after CCI, slightly increased further by URB597, 10 μg i.t., and strongly elevated by URB597, 100 μg. Injection of AEA (50 μg) into the lumbar spinal cord led to its dramatic elevation in this tissue, whereas, when a lower dose was used (5 μg) AEA endogenous levels were elevated only in the presence of URB597 (5 μg). We suggest that spinal AEA reduces neuropathic pain via CB(1) or TRPV1, depending on its local concentration.

  17. Sodium-glucose co-transporter 2 (SGLT2) inhibitors: a growing class of antidiabetic agents.

    PubMed

    Vivian, Eva M

    2014-01-01

    Although several treatment options are available to reduce hyperglycemia, only about half of individuals with diagnosed diabetes mellitus (DM) achieve recommended glycemic targets. New agents that reduce blood glucose concentrations by novel mechanisms and have acceptable safety profiles are needed to improve glycemic control and reduce the complications associated with type 2 diabetes mellitus (T2DM). The renal sodium-glucose co-transporter 2 (SGLT2) is responsible for reabsorption of most of the glucose filtered by the kidney. Inhibitors of SGLT2 lower blood glucose independent of the secretion and action of insulin by inhibiting renal reabsorption of glucose, thereby promoting the increased urinary excretion of excess glucose. Canagliflozin, dapagliflozin, and empagliflozin are SGLT2 inhibitors approved as treatments for T2DM in the United States, Europe, and other countries. Canagliflozin, dapagliflozin, and empagliflozin increase renal excretion of glucose and improve glycemic parameters in patients with T2DM when used as monotherapy or in combination with other antihyperglycemic agents. Treatment with SGLT2 inhibitors is associated with weight reduction, lowered blood pressure, and a low intrinsic propensity to cause hypoglycemia. Overall, canagliflozin, dapagliflozin, and empagliflozin are well tolerated. Cases of genital infections and, in some studies, urinary tract infections have been more frequent in canagliflozin-, dapagliflozin-, and empagliflozin-treated patients compared with those receiving placebo. Evidence from clinical trials suggests that SGLT2 inhibitors are a promising new treatment option for T2DM.

  18. Sodium glucose CoTransporter 2 (SGLT2) inhibitors: Current status and future perspective.

    PubMed

    Madaan, Tushar; Akhtar, Mohd; Najmi, Abul Kalam

    2016-10-10

    Diabetes mellitus is a disease that affects millions of people worldwide and its prevalence is estimated to rise in the future. Billions of dollars are spent each year around the world in health expenditure related to diabetes. There are several anti-diabetic drugs in the market for the treatment of non-insulin dependent diabetes mellitus. In this article, we will be talking about a relatively new class of anti-diabetic drugs called sodium glucose co-transporter 2 (SGLT2) inhibitors. This class of drugs has a unique mechanism of action focusing on inhibition of glucose reabsorption that separates it from other classes. This article covers the mechanism of glucose reabsorption in the kidneys, the mechanism of action of SGLT2 inhibitors, several SGLT2 inhibitors currently available in the market as well as those in various phases of development, their individual pharmacokinetics as well as the discussion about the future role of SGLT2 inhibitors, not only for the treatment of diabetes, but also for various other diseases like obesity, hepatic steatosis, and cardiovascular disorders.

  19. Structure activity relationships of benzylproline-derived inhibitors of the glutamine transporter ASCT2

    PubMed Central

    Singh, Kurnvir; Tanui, Rose; Gameiro, Armanda; Eisenberg, Gilad; Colas, Claire; Schlessinger, Avner; Grewer, Christof

    2017-01-01

    The glutamine transporter ASCT2 has been identified as a promising target to inhibit rapid growth of cancer cells. However, ASCT2 pharmacology is not well established. In this report, we performed a systematic structure activity analysis of a series of substituted benzylproline derivatives. Substitutions on the phenyl ring resulted in compounds with characteristics of ASCT2 inhibitors. Apparent binding affinity increased with increasing hydrophobicity of the side chain. In contrast, interaction of the ASCT2 binding site with specific positions on the phenyl ring was not observed. The most potent compound inhibits the ASCT2 anion conductance with a Ki of 3 μM, which is in the same range as that of more bulky and higher molecular weight inhibitors recently reported by others. The experimental results are consistent with computational analysis based on docking of the inhibitors against an ASCT2 homology model. The benzylproline scaffold provides a valuable tool for further improving binding potency of future ASCT2 inhibitors. PMID:28057420

  20. Sodium-glucose co-transporter 2 (SGLT2) inhibitors: a growing class of antidiabetic agents

    PubMed Central

    Vivian, Eva M

    2014-01-01

    Although several treatment options are available to reduce hyperglycemia, only about half of individuals with diagnosed diabetes mellitus (DM) achieve recommended glycemic targets. New agents that reduce blood glucose concentrations by novel mechanisms and have acceptable safety profiles are needed to improve glycemic control and reduce the complications associated with type 2 diabetes mellitus (T2DM). The renal sodium-glucose co-transporter 2 (SGLT2) is responsible for reabsorption of most of the glucose filtered by the kidney. Inhibitors of SGLT2 lower blood glucose independent of the secretion and action of insulin by inhibiting renal reabsorption of glucose, thereby promoting the increased urinary excretion of excess glucose. Canagliflozin, dapagliflozin, and empagliflozin are SGLT2 inhibitors approved as treatments for T2DM in the United States, Europe, and other countries. Canagliflozin, dapagliflozin, and empagliflozin increase renal excretion of glucose and improve glycemic parameters in patients with T2DM when used as monotherapy or in combination with other antihyperglycemic agents. Treatment with SGLT2 inhibitors is associated with weight reduction, lowered blood pressure, and a low intrinsic propensity to cause hypoglycemia. Overall, canagliflozin, dapagliflozin, and empagliflozin are well tolerated. Cases of genital infections and, in some studies, urinary tract infections have been more frequent in canagliflozin-, dapagliflozin-, and empagliflozin-treated patients compared with those receiving placebo. Evidence from clinical trials suggests that SGLT2 inhibitors are a promising new treatment option for T2DM. PMID:25598831

  1. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

    PubMed Central

    Dhonukshe, Pankaj; Grigoriev, Ilya; Fischer, Rainer; Tominaga, Motoki; Robinson, David G.; Hašek, Jiří; Paciorek, Tomasz; Petrášek, Jan; Seifertová, Daniela; Tejos, Ricardo; Meisel, Lee A.; Zažímalová, Eva; Gadella, Theodorus W. J.; Stierhof, York-Dieter; Ueda, Takashi; Oiwa, Kazuhiro; Akhmanova, Anna; Brock, Roland; Spang, Anne; Friml, Jiří

    2008-01-01

    Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role. PMID:18337510

  2. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279

    PubMed Central

    2015-01-01

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio. PMID:26713105

  3. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279.

    PubMed

    Keith, John M; Jones, William M; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A; Webb, Michael; Karbarz, Mark; Scott, Brian P; Wilson, Sandy J; Luo, Lin; Wennerholm, Michelle L; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R; Breitenbucher, J Guy

    2015-12-10

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio.

  4. Both P-gp and MRP2 mediate transport of Lopinavir, a protease inhibitor

    PubMed Central

    Agarwal, Sheetal; Pal, Dhananjay; Mitra, Ashim K.

    2011-01-01

    Polarized epithelial non-human (canine) cell lines stably transfected with human or murine complementary DNA (cDNA) encoding for various efflux transporters (P-gp/MDR1, MRP1, MRP2, and Bcrp1) were used to study transepithelial transport of Lopinavir (LVR) and compare results with the MDCKII-Wild type cells. These transmembrane proteins cause multidrug resistance by decreasing the total intracellular accumulation of drugs. Lopinavir efflux was directional and was completely inhibited by MK-571, a selective MRP family inhibitor in the MDCKII-MRP2 cell line. Similarly, LVR efflux was also inhibited by P-gp inhibitors P-gp 4008 and GF120918 in the MDCKII-MDR1 cell line. The efflux ratios (Efflux rate/ Influx rate) of LVR in the absence of any efflux inhibitors in the MDCK-Wild type, MDCKII-MDR1, MDCKII-MRP1 and MDCKII-MRP2 cell monolayers were 1.32, 4.91, 1.26 and 2.89 respectively. The MDCKII-MDR1 and MDCKII-MRP2 cells have significantly increased LVR efflux ratio relative to the parental cells due to the apically directed transport by MDR1 and MRP2 respectively. The efflux ratios in MRP2 and MDR1 transfected cell lines were close to unity in the presence of MK-571 and P-gp 4008 respectively; indicating that LVR efflux by MRP2 and P-gp was completely inhibited by their selective inhibitors. MDCKII-MRP1 cells did not exhibit a significant reduction in the LVR efflux relative to the parental cells, indicating that LVR is not a good substrate for MRP1. Transport studies across MDCKII-Bcrp1 cells indicated that LVR is not transported by Bcrp1 and is not a substrate for this efflux protein. In conclusion, this study presents direct evidence that LVR is effluxed by both P-gp and MRP2 which may contribute to its poor oral bioavailability and limited penetration into the CNS. PMID:17451894

  5. Biaryls as potent, tunable dual neurokinin 1 receptor antagonists and serotonin transporter inhibitors.

    PubMed

    Degnan, Andrew P; Tora, George O; Han, Ying; Rajamani, Ramkumar; Bertekap, Robert; Krause, Rudolph; Davis, Carl D; Hu, Joanna; Morgan, Daniel; Taylor, Sarah J; Krause, Kelly; Li, Yu-Wen; Mattson, Gail; Cunningham, Melissa A; Taber, Matthew T; Lodge, Nicholas J; Bronson, Joanne J; Gillman, Kevin W; Macor, John E

    2015-08-01

    Depression is a serious illness that affects millions of patients. Current treatments are associated with a number of undesirable side effects. Neurokinin 1 receptor (NK1R) antagonists have recently been shown to potentiate the antidepressant effects of serotonin-selective reuptake inhibitors (SSRIs) in a number of animal models. Herein we describe the optimization of a biaryl chemotype to provide a series of potent dual NK1R antagonists/serotonin transporter (SERT) inhibitors. Through the choice of appropriate substituents, the SERT/NK1R ratio could be tuned to afford a range of target selectivity profiles. This effort culminated in the identification of an analog that demonstrated oral bioavailability, favorable brain uptake, and efficacy in the gerbil foot tap model. Ex vivo occupancy studies with compound 58 demonstrated the ability to maintain NK1 receptor saturation (>88% occupancy) while titrating the desired level of SERT occupancy (11-84%) via dose selection.

  6. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

    PubMed Central

    Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

    2001-01-01

    The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium

  7. Effects of glial glutamate transporter inhibitors on intracellular Na+ in mouse astrocytes.

    PubMed

    Chatton, J Y; Shimamoto, K; Magistretti, P J

    2001-03-02

    The effects of inhibitors of the glial Na+/glutamate co-transporter on the intracellular Na+ concentration ([Na+](i)) were investigated in mouse cortical astrocytes. [Na+](i) was monitored by fluorescence microscopy on single astrocytes using the Na+-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-beta-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in robust and reversible increases in [Na+](i) that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the [Na+](i) response to these compounds was concentration-dependent with EC(50) values of 11.1 microM (THA) and 7.6 microM (t-PDC), as was the initial rate of [Na+](i) rise (EC(50) values of 14.8 microM for THA and 11.5 microM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-beta-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na+](i) up to a concentration of 500 microM . TBOA inhibited the [Na+](i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) values of 114 and 63 microM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na+](i) increase by TBOA was approximately 70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters.

  8. Diphenyleneiodonium, an inhibitor of NOXes and DUOXes, is also an iodide-specific transporter.

    PubMed

    Massart, C; Giusti, N; Beauwens, R; Dumont, J E; Miot, F; Sande, J Van

    2013-01-01

    NADPH oxidases (NOXes) and dual oxidases (DUOXes) generate O2 (.-) and H2O2. Diphenyleneiodonium (DPI) inhibits the activity of these enzymes and is often used as a specific inhibitor. It is shown here that DPI, at concentrations similar to those which inhibit the generation of O2 derivatives, activated the efflux of radioiodide but not of its analog (99m)TcO4 (-) nor of the K(+) cation mimic (86)Rb(+) in thyroid cells, in the PCCl3 rat thyroid cell line and in COS cell lines expressing the iodide transporter NIS. Effects obtained with DPI, especially in thyroid cells, should therefore be interpreted with caution.

  9. Amygdala FAAH and anandamide: mediating protection and recovery from stress.

    PubMed

    Gunduz-Cinar, Ozge; Hill, Matthew N; McEwen, Bruce S; Holmes, Andrew

    2013-11-01

    A long-standing literature linking endocannabinoids (ECBs) to stress, fear, and anxiety has led to growing interest in developing novel anxiolytics targeting the ECB system. Following rapid on-demand biosynthesis and degradation upon neuronal activation, the ECB N-arachidonoylethanolamide (anandamide, AEA) is actively degraded by the serine hydrolase enzyme, fatty acid amide hydrolase (FAAH). Exposure to stress rapidly mobilizes FAAH to deplete the signaling pool of AEA and increase neuronal excitability in a key anxiety-mediating region--the basolateral amygdala (BLA). Gene deletion or pharmacological inhibition of FAAH prevents stress-induced reductions in AEA and associated increases in BLA dendritic hypertrophy and anxiety-like behavior. Additionally, inhibition of FAAH facilitates long-term fear extinction and rescues deficient fear extinction in rodent models by enhancing AEA-CB1 (cannabinoid type 1) receptor signaling and synaptic plasticity in the BLA. These preclinical findings propose restoring deficient BLA AEA levels by pharmacologically inhibiting FAAH as a mechanism to therapeutically mitigate the effects of traumatic stress.

  10. The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls.

    PubMed

    Amijima, Makoto; Iwata, Yuji; Koizumi, Nozomu; Mishiba, Kei-Ichiro

    2014-08-01

    We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

  11. Anandamide inhibits nuclear factor-kappaB activation through a cannabinoid receptor-independent pathway.

    PubMed

    Sancho, Rocío; Calzado, Marco A; Di Marzo, Vincenzo; Appendino, Giovanni; Muñoz, Eduardo

    2003-02-01

    Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB(1) receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factor-kappaB (NF-kappaB) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NF-kappaB. This study shows that AEA inhibits tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation by direct inhibition of the IkappaB kinase (IKK)beta and, to a lesser extent, the IKKalpha subunits of kappaB inhibitor (IkappaB) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of IkappaBalpha degradation, NF-kappaB binding to DNA, and NF-kappaB-dependent transcription in TNFalpha-stimulated cells. AEA also prevents NF-kappaB-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NF-kappaB-inducing kinase. The NF-kappaB inhibitory activity of AEA was independent of CB(1) and CB(2) activation in TNFalpha-stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NF-kappaB, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine "head" with a vanillyl group led to potent inhibition of TNFalpha-induced NF-kappaB-dependent transcription. These findings provide new mechanistic insights into the anti-inflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents.

  12. Corticotropin-Releasing Hormone Drives Anandamide Hydrolysis in the Amygdala to Promote Anxiety

    PubMed Central

    Gray, J. Megan; Vecchiarelli, Haley A.; Morena, Maria; Lee, Tiffany T.Y.; Hermanson, Daniel J.; Kim, Alexander B.; McLaughlin, Ryan J.; Hassan, Kowther I.; Kühne, Claudia; Wotjak, Carsten T.; Deussing, Jan M.; Patel, Sachin

    2015-01-01

    Corticotropin-releasing hormone (CRH) is a central integrator in the brain of endocrine and behavioral stress responses, whereas activation of the endocannabinoid CB1 receptor suppresses these responses. Although these systems regulate overlapping functions, few studies have investigated whether these systems interact. Here we demonstrate a novel mechanism of CRH-induced anxiety that relies on modulation of endocannabinoids. Specifically, we found that CRH, through activation of the CRH receptor type 1 (CRHR1), evokes a rapid induction of the enzyme fatty acid amide hydrolase (FAAH), which causes a reduction in the endocannabinoid anandamide (AEA), within the amygdala. Similarly, the ability of acute stress to modulate amygdala FAAH and AEA in both rats and mice is also mediated through CRHR1 activation. This interaction occurs specifically in amygdala pyramidal neurons and represents a novel mechanism of endocannabinoid–CRH interactions in regulating amygdala output. Functionally, we found that CRH signaling in the amygdala promotes an anxious phenotype that is prevented by FAAH inhibition. Together, this work suggests that rapid reductions in amygdala AEA signaling following stress may prime the amygdala and facilitate the generation of downstream stress-linked behaviors. Given that endocannabinoid signaling is thought to exert “tonic” regulation on stress and anxiety responses, these data suggest that CRH signaling coordinates a disruption of tonic AEA activity to promote a state of anxiety, which in turn may represent an endogenous mechanism by which stress enhances anxiety. These data suggest that FAAH inhibitors may represent a novel class of anxiolytics that specifically target stress-induced anxiety. PMID:25740517

  13. Further structure-activity relationship studies of piperidine-based monoamine transporter inhibitors: effects of piperidine ring stereochemistry on potency. Identification of norepinephrine transporter selective ligands and broad-spectrum transporter inhibitors.

    PubMed

    He, Rong; Kurome, Toru; Giberson, Kelly M; Johnson, Kenneth M; Kozikowski, Alan P

    2005-12-15

    4-(4-Chlorophenyl)piperidine analogues each bearing a thioacetamide side chain appendage similar to that found in the wake-promoting drug modafinil have been synthesized. The transporter inhibitory activity of both the cis and trans isomers of these 3,4-disubstituted piperidines in both their (+)- and (-)-enantiomeric forms was determined. These studies reveal that the (-)-cis analogues exhibit dopamine transporter/norepinephrine transporter (DAT/NET) selectivity as was previously reported for the (+)-trans analogues. On the other hand, the (-)-trans and the (+)-cis isomers show serotonin transporter (SERT) or SERT/NET selectivity. Among them, (+)-cis-5b shows a low nanomolar Ki for the NET with 39-fold and 321-fold lower potency at the DAT and SERT, respectively, thus making it a useful pharmacological research tool for exploring NET-associated behavioral signatures. On the other hand, several of the compounds described herein, such as (+)-trans-5c, show comparable activity at all three transporters. Because broad-spectrum transporter inhibitors have been hypothesized to exhibit a more rapid onset of action and/or a greater efficacy as antidepressant agents than those selective for SERT or SERT + NET, some of the present compounds will be valuable to study in animal models of depression.

  14. Glycine transporters type 1 inhibitor promotes brain preconditioning against NMDA-induced excitotoxicity.

    PubMed

    Pinto, Mauro Cunha Xavier; Lima, Isabel Vieira de Assis; da Costa, Flávia Lage Pessoa; Rosa, Daniela Valadão; Mendes-Goulart, Vânia Aparecida; Resende, Rodrigo Ribeiro; Romano-Silva, Marco Aurélio; de Oliveira, Antônio Carlos Pinheiro; Gomez, Marcus Vinícius; Gomez, Renato Santiago

    2015-02-01

    Brain preconditioning is a protective mechanism, which can be activated by sub-lethal stimulation of the NMDA receptors (NMDAR) and be used to achieve neuroprotection against stroke and neurodegenerative diseases models. Inhibitors of glycine transporters type 1 modulate glutamatergic neurotransmission through NMDAR, suggesting an alternative therapeutic strategy of brain preconditioning. The aim of this work was to evaluate the effects of brain preconditioning induced by NFPS, a GlyT1 inhibitor, against NMDA-induced excitotoxicity in mice hippocampus, as well as to study its neurochemical mechanisms. C57BL/6 mice (male, 10-weeks-old) were preconditioned by intraperitoneal injection of NFPS at doses of 1.25, 2.5 or 5.0 mg/kg, 24 h before intrahippocampal injection of NMDA. Neuronal death was evaluated by fluoro jade C staining and neurochemical parameters were evaluated by gas chromatography-mass spectrometry, scintillation spectrometry and western blot. We observed that NFPS preconditioning reduced neuronal death in CA1 region of hippocampus submitted to NMDA-induced excitotoxicity. The amino acids (glycine and glutamate) uptake and content were increased in hippocampus of animals treated with NFPS 5.0 mg/kg, which were associated to an increased expression of type-2 glycine transporter (GlyT2) and glutamate transporters (EAAT1, EAAT2 and EAAT3). The expression of GlyT1 was reduced in animals treated with NFPS. Interestingly, the preconditioning reduced expression of GluN2B subunits of NMDAR, whereas did not change the expression of GluN1 or GluN2A in all tested doses. Our study suggests that NFPS preconditioning induces resistance against excitotoxicity, which is associated with neurochemical changes and reduction of GluN2B-containing NMDAR expression.

  15. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

    PubMed

    Vuille-dit-Bille, Raphael N; Camargo, Simone M; Emmenegger, Luca; Sasse, Tom; Kummer, Eva; Jando, Julia; Hamie, Qeumars M; Meier, Chantal F; Hunziker, Schirin; Forras-Kaufmann, Zsofia; Kuyumcu, Sena; Fox, Mark; Schwizer, Werner; Fried, Michael; Lindenmeyer, Maja; Götze, Oliver; Verrey, François

    2015-04-01

    Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

  16. Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors.

    PubMed Central

    Sanglard, D; Ischer, F; Monod, M; Bille, J

    1996-01-01

    Some Candida albicans isolates from AIDS patients with oropharyngeal candidiasis are becoming resistant to the azole antifungal agent fluconazole after prolonged treatment with this compound. Most of the C. albicans isolates resistant to fluconazole fail to accumulate this antifungal agent, and this has been considered a cause of resistance. This phenomenon was shown to be linked to an increase in the amounts of mRNA of a C. albicans ABC (ATP-binding cassette) transporter gene called CDR1 and of a gene conferring benomyl resistance (BENr), the product of which belongs to the class of major facilitator multidrug efflux transporters (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). To analyze the roles of these multidrug transporters in the efflux of azole antifungal agents, we constructed C. albicans mutants with single and double deletion mutations of the corresponding genes. The mutants were tested for their susceptibilities to these antifungal agents. Our results indicated that the delta cdr1 C. albicans mutant was hypersusceptible to the azole derivatives fluconazole, itraconazole, and ketoconazole, thus showing that the ABC transporter Cdr1 can use these compounds as substrates. The delta cdr1 mutant was also hypersusceptible to other antifungal agents (terbinafine and amorolfine) and to different metabolic inhibitors (cycloheximide, brefeldin A, and fluphenazine). The same mutant was slightly more susceptible than the wild type to nocodazole, cerulenin, and crystal violet but not to amphotericin B, nikkomycin Z, flucytosine, or pradimicin. In contrast, the delta ben mutant was rendered more susceptible only to the mutagen 4-nitroquinoline-N-oxide. However, this mutation increased the susceptibilities of the cells to cycloheximide and cerulenin when the mutation was constructed in a delta cdr1 background. The assay used in the present study could be implemented with new antifungal

  17. Combinatorial Pharmacophore Modeling of Multidrug and Toxin Extrusion Transporter 1 Inhibitors: a Theoretical Perspective for Understanding Multiple Inhibitory Mechanisms

    PubMed Central

    Xu, Yuan; Liu, Xian; Wang, Yulan; Zhou, Nannan; Peng, Jianlong; Gong, Likun; Ren, Jing; Luo, Cheng; Luo, Xiaomin; Jiang, Hualiang; Chen, Kaixian; Zheng, Mingyue

    2015-01-01

    A combinatorial pharmacophore (CP) model for Multidrug and toxin extrusion 1 (MATE1/SLC47A1) inhibitors was developed based on a data set including 881 compounds. The CP model comprises four individual pharmacophore hypotheses, HHR1, DRR, HHR2 and AAAP, which can successfully identify the MATE1 inhibitors with an overall accuracy around 75%. The model emphasizes the importance of aromatic ring and hydrophobicity as two important structural determinants for MATE1 inhibition. Compared with the pharmacophore model of Organic Cation Transporter 2 (OCT2/ SLC22A2), a functional related transporter of MATE1, the hypotheses of AAAP and PRR5 are suggested to be responsible for their ligand selectivity, while HHR a common recognition pattern for their dual inhibition. A series of analysis including molecular sizes of inhibitors matching different hypotheses, matching of representative MATE1 inhibitors and molecular docking indicated that the small inhibitors matching HHR1 and DRR involve in competitive inhibition, while the relatively large inhibitors matching AAAP are responsible for the noncompetitive inhibition by locking the conformation changing of MATE1. In light of the results, a hypothetical model for inhibiting transporting mediated by MATE1 was proposed. PMID:26330298

  18. Inhibitors of Glycine Transporter-1: Potential Therapeutics for the Treatment of CNS Disorders.

    PubMed

    Cioffi, Christopher L; Guzzo, Peter R

    2016-01-01

    Glycine acts as an inhibitory neurotransmitter at glycine receptor (GlyR)-enriched synapses and as an obligatory co-agonist at the N-methyl-D-aspartate (NMDA) receptor, where it facilitates neuronal excitation. Two high-affinity and substrate selective transporters, glycine transporter-1 and glycine transporter-2 (GlyT-1 and GlyT-2), regulate extracellular glycine concentrations within the CNS and as such, play critical roles in maintaining a balance between inhibitory and excitatory neurotransmission. GlyT-1 inhibition has been extensively examined as a potential means by which to treat several CNS disorders that include schizophrenia, depression, anxiety, obsessive compulsive disorder (OCD), and addiction. More recently, preclinical studies have emerged that indicate the approach may also promote neuroprotection, provide a pharmacotherapeutic strategy for autism spectrum disorders (ASDs), and treat symptomology associated with pain, Parkinson's disease, and epilepsy. This review examines the pharmacological aspects of GlyT-1 inhibition and describes drug discovery and development efforts toward the identification of novel inhibitors.

  19. Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model.

    PubMed

    Sostelly, Alexandre; Payen, Léa; Guitton, Jérôme; Di Pietro, Attilio; Falson, Pierre; Honorat, Mylène; Boumendjel, Ahcène; Gèze, Annabelle; Freyer, Gilles; Tod, Michel

    2014-04-01

    ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

  20. The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase.

    PubMed

    De Petrocellis, L; Harrison, S; Bisogno, T; Tognetto, M; Brandi, I; Smith, G D; Creminon, C; Davis, J B; Geppetti, P; Di Marzo, V

    2001-06-01

    The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect was decreased five-fold in the presence of forskolin (FRSK, 1-5 microM) or the cAMP analogue, 8-Br-cAMP (10-100 microM). The effects of 8-Br-cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhanced the sensory neurone- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induced release of substance P, an effect mediated by VR1 activation, was enhanced three-fold by FRSK (10 nM). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.

  1. Glycine Transporter Inhibitor Attenuates the Psychotomimetic Effects of Ketamine in Healthy Males: Preliminary Evidence

    PubMed Central

    D'Souza, Deepak Cyril; Singh, Nagendra; Elander, Jacqueline; Carbuto, Michelle; Pittman, Brian; de Haes, Joanna Udo; Sjogren, Magnus; Peeters, Pierre; Ranganathan, Mohini; Schipper, Jacques

    2012-01-01

    Enhancing glutamate function by stimulating the glycine site of the NMDA receptor with glycine, -serine, or with drugs that inhibit glycine reuptake may have therapeutic potential in schizophrenia. The effects of a single oral dose of cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-ylmethyl) amino-methylcarboxylic acid hydrochloride (Org 25935), a glycine transporter-1 (GlyT1) inhibitor, and placebo pretreatment on ketamine-induced schizophrenia-like psychotic symptoms, perceptual alterations, and subjective effects were evaluated in 12 healthy male subjects in a randomized, counter-balanced, within-subjects, crossover design. At 2.5 h after administration of the Org 25935 or placebo, subjects received a ketamine bolus and constant infusion lasting 100 min. Psychotic symptoms, perceptual, and a number of subjective effects were assessed repeatedly before, several times during, and after completion of ketamine administration. A cognitive battery was administered once per test day. Ketamine produced behavioral, subjective, and cognitive effects consistent with its known effects. Org 25935 reduced the ketamine-induced increases in measures of psychosis (Positive and Negative Syndrome Scale (PANSS)) and perceptual alterations (Clinician Administered Dissociative Symptoms Scale (CADSS)). The magnitude of the effect of Org 25935 on ketamine-induced increases in Total PANSS and CADSS Clinician-rated scores was 0.71 and 0.98 (SD units), respectively. None of the behavioral effects of ketamine were increased by Org 25935 pretreatment. Org 25935 worsened some aspects of learning and delayed recall, and trended to improve choice reaction time. This study demonstrates for the first time in humans that a GlyT1 inhibitor reduces the effects induced by NMDA receptor antagonism. These findings provide preliminary support for further study of the antipsychotic potential of GlyT1 inhibitors. PMID:22113087

  2. The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

    PubMed

    Firth, J A; Marland, B Y

    1975-08-01

    The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

  3. Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

    NASA Technical Reports Server (NTRS)

    Lee, J. S.; Mulkey, T. J.; Evans, M. L.

    1984-01-01

    Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.

  4. Atypical Dopamine Uptake Inhibitors that Provide Clues About Cocaine's Mechanism at the Dopamine Transporter

    NASA Astrophysics Data System (ADS)

    Hauck Newman, Amy; Katz, Jonathan L.

    The dopamine transporter (DAT) has been a primary target for cocaine abuse/addiction medication discovery. However predicted addiction liability and limited clinical evaluation has provided a formidable challenge for development of these agents for human use. The unique and atypical pharmacological profile of the benztropine (BZT) class of dopamine uptake inhibitors, in preclinical models of cocaine effects and abuse, has encouraged further development of these agents. Moreover, in vivo studies have challenged the original DAT hypothesis and demonstrated that DAT occupancy and subsequent increases in dopamine produced by BZT analogues are significantly delayed and long lasting, as compared to cocaine. These important and distinctive elements are critical to the lack of abuse liability among BZT analogues, and improve their potential for development as treatments for cocaine abuse and possibly other neuropsychiatric disorders.

  5. The Glycine Transport Inhibitor Sarcosine Is an Inhibitory Glycine Receptor Agonist

    PubMed Central

    Zhang, Hai Xia; Lyons-Warren, Ariel; Thio, Liu Lin

    2009-01-01

    Summary Sarcosine is an endogenous amino acid that is a competitive inhibitor of the type I glycine transporter (GlyT1), an N-methyl-D-aspartate receptor (NMDAR) co-agonist, and an important intermediate in one-carbon metabolism. Its therapeutic potential for schizophrenia further underscores its clinical importance. The structural similarity between sarcosine and glycine and sarcosine's ability to serve as an NMDAR co-agonist led us to examine whether sarcosine is also an agonist at the inhibitory glycine receptor (GlyR). We examined this possibility using whole-cell recordings from cultured embryonic mouse hippocampal neurons and found that sarcosine evoked a dose-dependent, strychnine sensitive, Cl- current that cross-inhibited glycine currents. Sarcosine evoked this current with Li+ in the extracellular solution to block GlyT1, in neurons treated with the essentially irreversible GlyT1 inhibitor N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS), and in neurons plated in the absence of glia. These results indicate that the sarcosine currents did not result from GlyT1 inhibition or heteroexchange. We conclude that sarcosine is a GlyR agonist. PMID:19619564

  6. Sodium-glucose co-transporter-2 inhibitors and euglycemic ketoacidosis: Wisdom of hindsight

    PubMed Central

    Singh, Awadhesh Kumar

    2015-01-01

    Sodium-glucose co-transporter-2 inhibitors (SGLT-2i) are newly approved class of oral anti-diabetic drugs, in the treatment of type 2 diabetes, which reduces blood glucose through glucouresis via the kidney, independent, and irrespective of available pancreatic beta-cells. Studies conducted across their clinical development program found, a modest reduction in glycated hemoglobin ranging from −0.5 to −0.8%, without any significant hypoglycemia. Moreover, head-to-head studies versus active comparators yielded comparable efficacy. Interestingly, weight and blood pressure reduction were additionally observed, which was not only consistent but significantly superior to active comparators, including metformin, sulfonylureas, and dipeptydylpeptide-4 inhibitors. Indeed, these additional properties makes this class a promising oral anti-diabetic drug. Surprisingly, a potentially fatal unwanted side effect of diabetic ketoacidosis has been noted with its widespread use, albeit rarely. Nevertheless, this has created a passé among the clinicians. This review is an attempt to pool those ketosis data emerging with SGLT-2i, and put a perspective on its implicated mechanism. PMID:26693421

  7. Molecular Docking Analysis of Steroid-based Copper Transporter 1 Inhibitors.

    PubMed

    Kadioglu, Onat; Serly, Julianna; Seo, Ean-Jeong; Vincze, Irén; Somlai, Csaba; Saeed, Mohamed E M; Molnár, József; Efferth, Thomas

    2015-12-01

    Copper transporter 1 (CTR1) represents an important determinant of cisplatin resistance. A series of 35 semi-substituted steroids were recently investigated on cisplatin-resistant CTR1-expressing A2780cis ovarian carcinoma cells as well as their parental sensitive counterparts regarding their cytotoxic and resistance-reversing features. In the present investigation, three compounds ( 4: , 5: , 25: ) were selected for molecular docking analysis on the homology-modelled human CTR1 transmembrane domain. Steroids 4: , 5: and 25: interacted with CTR1 at a similar docking pose and with even higher binding affinities than the known CTR1 inhibitor, cimetidine. Applying the defined docking mode, the binding energies were found to be -7.15±<0.001 kcal/mol (compound 4: ), -8.71±0.06 kcal/mol (compound 5: ), -7.63±0.01 kcal/mol (compound 25: ), and -5.05±0.02 kcal/mol (for cimetidine). These steroids have the potential for further development as CTR1 inhibitors overcoming cisplatin resistance.

  8. Simultaneous modulation of transport and metabolism of acyclovir prodrugs across rabbit cornea: An approach involving enzyme inhibitors.

    PubMed

    Katragadda, Suresh; Talluri, Ravi S; Mitra, Ashim K

    2006-08-31

    The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir (ACV) and to modulate transport and metabolism of amino acid and dipeptide prodrugs of acyclovir by enzyme inhibitors across rabbit cornea. l-Valine ester of acyclovir, valacyclovir (VACV) and l-glycine-valine ester of acyclovir, gly-val-acyclovir (GVACV) were used as model compounds. Hydrolysis studies of VACV and GVACV in corneal homogenate were conducted in presence of various enzyme inhibitors. IC(50) values were determined for the enzyme inhibitors. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Complete inhibition of VACV hydrolysis was observed in the presence of Pefabloc SC (4-(2-aminoethyl)-benzenesulfonyl-fluoride) and PCMB (p-chloromercuribenzoic acid). Similar trend was also observed with GVACV in the presence of bestatin. IC(50) values of PCMB and bestatin for VACV and GVACV were found to be 3.81+/-0.94 and 0.34+/-0.08muM respectively. Eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) also produced significant inhibition of VACV hydrolysis. Transport of VACV and GVACV across cornea showed decreased metabolic rate and modulation of transport in presence of PCMB and bestain respectively. The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were carboxylesterases and aminopeptidases respectively. Enzyme inhibitors modulated the transport and metabolism of prodrugs simultaneously even though their affinity towards prodrugs was distinct. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs appears to be promising strategy for enhancing drug transport across cornea.

  9. Potent inhibitors of human organic anion transporters 1 and 3 from clinical drug libraries: discovery and molecular characterization.

    PubMed

    Duan, Peng; Li, Shanshan; Ai, Ni; Hu, Longqin; Welsh, William J; You, Guofeng

    2012-11-05

    Transporter-mediated drug-drug interactions in the kidney dramatically influence the pharmacokinetics and other clinical effects of drugs. Human organic anion transporters 1 (hOAT1) and 3 (hOAT3) are the major transporters in the basolateral membrane of kidney proximal tubules, mediating the rate-limiting step in the elimination of a broad spectrum of drugs. In the present study, we screened two clinical drug libraries against hOAT1 and hOAT3. Of the 727 compounds screened, 92 compounds inhibited hOAT1 and 262 compounds inhibited hOAT3. When prioritized based on the peak unbound plasma concentrations of these compounds, three inhibitors for hOAT1 and seven inhibitors for hOAT3 were subsequently identified with high inhibitory potency (>95%). Computational analyses revealed that inhibitors and noninhibitors can be differentiated from each other on the basis of several physicochemical features, including number of hydrogen-bond donors, number of rotatable bonds, and topological polar surface area (TPSA) for hOAT1; and molecular weight, number of hydrogen-bond donors and acceptors, TPSA, partition coefficient (log P(7.4)), and polarizability for hOAT3. Pharmacophore modeling identified two common structural features associated with inhibitors for hOAT1 and hOAT3, viz., an anionic hydrogen-bond acceptor atom, and an aromatic center separated by ∼5.7 Å. Such model provides mechanistic insights for predicting new OAT inhibitors.

  10. Drug interactions with HMG-CoA reductase inhibitors (statins): the importance of CYP enzymes, transporters and pharmacogenetics.

    PubMed

    Neuvonen, Pertti J

    2010-03-01

    HMG-CoA reductase inhibitors (statins) can cause skeletal muscle toxicity; the risk of toxicity is elevated by drug interactions and pharmacogenetic factors that increase the concentration of statins in the plasma. Statins are substrates for several membrane transporters that may mediate drug interactions. Inhibitors of the organic anion transporting polypeptide 1B1 can decrease the hepatic uptake of many statins, as well as the therapeutic index of these agents. Potent inhibitors of cytochrome P450 (CYP)3A4 can significantly increase the plasma concentrations of the active forms of simvastatin, lovastatin and atorvastatin. Fluvastatin, which is metabolized by CYP2C9, is less prone to pharmacokinetic interactions, while pravastatin, rosuvastatin and pitavastatin are not susceptible to any CYP inhibition. An understanding of the mechanisms of statin interactions will help to minimize drug interactions and to develop statins that are less prone to adverse interactions.

  11. Suppression of asymmetric acid efflux and gravitropism in maize roots treated with auxin transport inhibitors of sodium orthovanadate

    NASA Technical Reports Server (NTRS)

    Mulkey, T. J.; Evans, M. L.

    1982-01-01

    In gravitropically stimulated roots of maize (Zea mays L., hybrid WF9 x 38MS), there is more acid efflux on the rapidly growing upper side than on the slowly growing lower side. In light of the Cholodny/Went hypothesis of gravitropism which states that gravitropic curvature results from lateral redistribution of auxin, the effects of auxin transport inhibitors on the development of acid efflux asymmetry and curvature in gravistimulated roots were examined. All the transport inhibitors tested prevented both gravitropism and the development of asymmetric acid efflux in gravistimulated roots. The results indicate that auxin redistribution may cause the asymmetry of acid efflux, a finding consistent with the Cholodny/Went hypothesis of gravitropism. As further evidence that auxin-induced acid efflux asymmetry may mediate gravitropic curvature, sodium orthovanadate, an inhibitor of auxin-induced H+ efflux was found to prevent both gravitropism and the development of asymmetric acid efflux in gravistimulated roots.

  12. Anandamide, an endogenous cannabinoid, inhibits Shaker-related voltage-gated K+ channels.

    PubMed

    Poling, J S; Rogawski, M A; Salem, N; Vicini, S

    1996-01-01

    Anandamide has been identified in porcine brain as an endogenous cannabinoid receptor ligand and is believed to be a counterpart to the psychoactive component of marijuana, delta 9-tetrahydrocannabinol (delta 9-THC). Here we report that anandamide directly inhibits (IC50, 2.7 muM) Shaker-related Kv1.2 K+ channels that are found ubiquitously in the mammalian brain. Delta 9-THC also inhibited Kv1.2 channels with comparable potency (IC50, 2.4 muM), as did several N-acyl-ethanolamides with cannabinoid receptor binding activity. Potassium current inhibition occurred through a pertussis toxin-insensitive mechanism and was not prevented by the cannabinoid receptor antagonist SR141716A. Utilizing excised patches of Kv1.2 channel-rich membrane as a rapid and sensitive bioassay, we found that phospholipase D stimulated the release of an endogenous anandamide-like K+ channel blocker from rat brain slices. Structure-activity studies were consistent with the possibility that the released blocker was either anandamide or another N-acyl-ethanolamide.

  13. Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures

    PubMed Central

    Malek, Natalia; Popiolek-Barczyk, Katarzyna; Mika, Joanna; Przewlocka, Barbara; Starowicz, Katarzyna

    2015-01-01

    Microglial activation is a polarized process divided into potentially neuroprotective phenotype M2 and neurotoxic phenotype M1, predominant during chronic neuroinflammation. Endocannabinoid system provides an attractive target to control the balance between microglial phenotypes. Anandamide as an immune modulator in the central nervous system acts via not only cannabinoid receptors (CB1 and CB2) but also other targets (e.g., GPR18/GPR55). We studied the effect of anandamide on lipopolysaccharide-induced changes in rat primary microglial cultures. Microglial activation was assessed based on nitric oxide (NO) production. Analysis of mRNA was conducted for M1 and M2 phenotype markers possibly affected by the treatment. Our results showed that lipopolysaccharide-induced NO release in microglia was significantly attenuated, with concomitant downregulation of M1 phenotypic markers, after pretreatment with anandamide. This effect was not sensitive to CB1 or GPR18/GPR55 antagonism. Administration of CB2 antagonist partially abolished the effects of anandamide on microglia. Interestingly, administration of a GPR18/GPR55 antagonist by itself suppressed NO release. In summary, we showed that the endocannabinoid system plays a crucial role in the management of neuroinflammation by dampening the activation of an M1 phenotype. This effect was primarily controlled by the CB2 receptor, although functional cross talk with GPR18/GPR55 may occur. PMID:26090232

  14. Design and synthesis of dual inhibitors of acetylcholinesterase and serotonin transporter targeting potential agents for Alzheimer's disease.

    PubMed

    Kogen, Hiroshi; Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio

    2002-10-03

    Highly efficient acetylcholinesterase (AChE) and serotonin transporter (SERT) dual inhibitors, (S)-4 and (R)-13 were designed and synthesized on the basis of the hypothetical model of AChE active site. Both compounds showed potent inhibitory activities against AChE and SERT. [structure: see text

  15. Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction.

    PubMed Central

    Schuel, H; Goldstein, E; Mechoulam, R; Zimmerman, A M; Zimmerman, S

    1994-01-01

    Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening. PMID:8052642

  16. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

    PubMed Central

    Stark, Katarina; Dostalek, Miroslav; Guengerich, F. Peter

    2016-01-01

    Summary Cytochrome P450 (P450) 4X1 is one of the so-called “orphan” P450s without assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5α cells (<100 nmol P450/L). The highest level of expression (300-450 nmol P450/L culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b5. Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala, and basal ganglia), in addition to expression in human heart, liver, prostate, and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain. PMID:18549450

  17. Increasing oral absorption of polar neuraminidase inhibitors: a prodrug transporter approach applied to oseltamivir analogue.

    PubMed

    Gupta, Deepak; Varghese Gupta, Sheeba; Dahan, Arik; Tsume, Yasuhiro; Hilfinger, John; Lee, Kyung-Dall; Amidon, Gordon L

    2013-02-04

    showed that the l-valyl prodrug (P(app) = 1.7 × 10(-6) cm/s) has the potential to be rapidly transported across the epithelial cell apical membrane. Significantly, only the parent drug (GOCarb) appeared in the basolateral compartment, indicating complete activation (hydrolysis) during transport. Intestinal rat jejunal permeability studies showed that l-valyl and l-isoleucyl prodrugs are highly permeable compared to the orally well absorbed metoprolol, while the parent drug had essentially zero permeability in the jejunum, consistent with its known poor low absorption. Prodrugs were rapidly converted to parent in cell homogenates, suggesting their ability to be activated endogenously in the epithelial cell, consistent with the transport studies. Additionally, l-valyl prodrug was found to be a substrate for valacyclovirase (K(m) = 2.37 mM), suggesting a potential cell activation mechanism. Finally we determined the oral bioavailability of our most promising candidate, GOC-l-Val, in mice to be 23% under fed conditions and 48% under fasted conditions. In conclusion, GOC-l-Val prodrug was found to be a very promising antiviral agent for oral delivery. These findings indicate that the carrier-mediated prodrug approach is an excellent strategy for improving oral absorption of polar neuraminidase inhibitors. These promising results demonstrate that the oral peptide transporter-mediated prodrug strategy has enormous promise for improving the oral mucosal cell membrane permeability of polar, poorly absorbed antiviral agents and treating influenza via the oral route of administration.

  18. A novel monocarboxylate transporter inhibitor as a potential treatment strategy for γ-hydroxybutyric acid overdose

    PubMed Central

    Vijay, Nisha; Morse, Bridget L.; Morris, Marilyn E.

    2014-01-01

    Purpose Monocarboxylate transporter (MCT) inhibition represents a potential treatment strategy for γ-hydroxybutyric acid (GHB) overdose by blocking its renal reabsorption in the kidney. This study further evaluated the effects of a novel, highly potent MCT inhibitor, AR-C155858, on GHB toxicokinetics/toxicodynamics (TK/TD). Methods Rats were administered GHB (200, 600 or 1500 mg/kg i.v. or 1500 mg/kg po) with and without AR-C155858. Breathing frequency was continuously monitored using whole-body plethysmography. Plasma and urine samples were collected up to 8 hours. The effect of AR-C155858 on GHB brain/plasma partitioning was also assessed. Results AR-C155858 treatment significantly increased GHB renal and total clearance after intravenous GHB administration at all the GHB doses used in this study. GHB-induced respiratory depression was significantly improved by AR-C155858 as demonstrated by an improvement in the respiratory rate. AR-C155858 treatment also resulted in a significant reduction in brain/plasma partitioning of GHB (0.1 ± 0.03) when compared to GHB alone (0.25 ± 0.02). GHB CLR and CLoral (CL/F) following oral administration were also significantly increased following AR-C155858 treatment (from 1.82 ± 0.63 to 5.74 ± 0.86 and 6.52 ± 0.88 to 10.2 ± 0.75 ml/min/kg, respectively). Conclusion The novel and highly potent MCT inhibitor represents a potential treatment option for GHB overdose. PMID:25480120

  19. Sodium-glucose co-transporter 2 inhibitors: from apple tree to 'Sweet Pee'.

    PubMed

    Hardman, Timothy C; Rutherford, Peter; Dubrey, Simon W; Wierzbicki, Anthony S

    2010-01-01

    The sodium-glucose co-transporter 2 (SGLT2), located in the plasma membrane of cells lining the proximal tubule, facilitates the reabsorbtion of glucose in the kidney. Inhibition of SGLT2 has the potential to reduce blood glucose and represents an opportune target for managing blood glucose. By promoting the excretion of glucose, SGLT2 inhibitors are the first anti-diabetic treatment to target the removal rather than the metabolic redirection of glucose. Their mechanism of action is independent of that of endogenous insulin status and thus provides a means of managing plasma glucose irrespective of a patient's glycaemic status or treatments being used in combination. Several candidate SGLT2 inhibitors based on the core glucoside structure of phlorizin are currently being developed, of which, the metabolically more stable aromatic and heteroaromatic C-glucosides have demonstrated the most promising preclinical and clinical data. The inhibition of SGLT2 by messenger antisense technology is also being investigated. Current indications suggest that short-term benefits, in terms of HbA1(c) reductions, are modest and it remains to be seen whether encouraging exogenous glucose disposal will result in long term patient benefits in terms of returning metabolic balance or even weight loss. Indications are that clinical efficacy will be greater with molecules based on an O-glucoside structure. Concerns have been raised over the safety of these agents, particularly a possible predisposition to urinary tract infections, but these concerns have yet to be confirmed in clinical studies. Clinical development programs will need to establish those patients most likely to benefit from inhibition of SGLT2.

  20. Differing effects of transport inhibitor on glutamate uptake by nerve terminals before and after exposure of rats to artificial gravity.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    Glutamate is the major excitatory neurotransmitter in the brain. Subsequent to its release from glutamatergic neurons and activation of receptors, it is removed from extracellular space by high affinity Na^+-dependent glutamate transporters, which utilize the Na^+/K^+ electrochemical gradient as a driving force and located in nerve terminals and astrocytes. The glutamate transporters may modify the time course of synaptic events. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity (e.g. cerebral ischemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia). The present study assessed transporter inhibitor for the ability to inhibit glutamate uptake by synaptosomes at the normal and hypergravity conditions (rats were rotated in a long-arm centrifuge at ten-G during one-hour period). DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a newly developed competitive inhibitor of the high-affinity, Na^+-dependent glutamate transporters. As a potent, non- transported inhibitor of glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms. We demonstrated that DL-TBOA inhibited glutamate uptake ( 100 μM glutamate, 30 sec incubation period) in dose-dependent manner as in control as in hypergravity. The effect of this transport inhibitor on glutamate uptake by control synaptosomes and synaptosomes prepared of animals exposed to hypergravity was different. IC50 values calculated on the basis of curves of non-linear regression kinetic analysis was 18±2 μM and 11±2 μM ((P≤0,05) before and after exposure to artificial gravity, respectively. Inhibition caused by 10 μM DL-TBOA was significantly increased from 38,0±3,8 % in control group to 51,0±4,1 % in animals, exposed to hypergravity (P≤0,05). Thus, DL-TBOA had complex effect on glutamate uptake process and perhaps, became more potent under

  1. Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors

    PubMed Central

    Esteva-Font, Cristina; Cil, Onur; Phuan, Puay-Wah; Su, Tao; Lee, Sujin; Anderson, Marc O.; Verkman, A. S.

    2014-01-01

    Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 μM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3–5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-d-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.—Esteva-Font, C., Cil, O., Phuan, P.-W., Su, T., Lee, S., Anderson, M. O., Verkman, A. S. Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors. PMID:24843071

  2. Determinants of Anti-Cancer Effect of Mitochondrial Electron Transport Chain Inhibitors: Bioenergetic Profile and Metabolic Flexibility of Cancer Cells.

    PubMed

    Urra, Félix A; Weiss-López, Boris; Araya-Maturana, Ramiro

    2016-01-01

    Recent evidence highlights that energy requirements of cancer cells vary greatly from normal cells and they exhibit different metabolic phenotypes with variable participation of both glycolysis and oxidative phosphorylation (OXPHOS). Interestingly, mitochondrial electron transport chain (ETC) has been identified as an essential component in bioenergetics, biosynthesis and redox control during proliferation and metastasis of cancer cells. This dependence converts ETC of cancer cells in a promising target to design small molecules with anti-cancer actions. Several small molecules have been described as ETC inhibitors with different consequences on mitochondrial bioenergetics, viability and proliferation of cancer cells, when the substrate availability is controlled to favor either the glycolytic or OXPHOS pathway. These ETC inhibitors can be grouped as 1) inhibitors of a respiratory complex (e.g. rotenoids, vanilloids, alkaloids, biguanides and polyphenols), 2) inhibitors of several respiratory complexes (e.g. capsaicin, ME-344 and epigallocatechin-3 gallate) and 3) inhibitors of ETC activity (e.g. elesclomol and VLX600). Although pharmacological ETC inhibition may produce cell death and a decrease of proliferation of cancer cells, factors such as degree of inhibition of ETC activity by small molecules, bioenergetic profile and metabolic flexibility of different cancer types or subpopulations of cells in a particular cancer type, can affect the impact of the anti-cancer actions. Particularly interesting are the adaptive mechanisms induced by ETC inhibition, such as induction of glutamine-dependent reductive carboxylation, which may offer a strategy to sensitize cancer cells to inhibitors of glutamine metabolism.

  3. Diuresis and reduced urinary osmolality in rats produced by small-molecule UT-A-selective urea transport inhibitors.

    PubMed

    Esteva-Font, Cristina; Cil, Onur; Phuan, Puay-Wah; Su, Tao; Lee, Sujin; Anderson, Marc O; Verkman, A S

    2014-09-01

    Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 μM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.

  4. The endocannabinoid transport inhibitor AM404 differentially modulates recognition memory in rats depending on environmental aversiveness

    PubMed Central

    Campolongo, Patrizia; Ratano, Patrizia; Manduca, Antonia; Scattoni, Maria L.; Palmery, Maura; Trezza, Viviana; Cuomo, Vincenzo

    2012-01-01

    Cannabinoid compounds may influence both emotional and cognitive processes depending on the level of environmental aversiveness at the time of drug administration. However, the mechanisms responsible for these responses remain to be elucidated. The present experiments investigated the effects induced by the endocannabinoid transport inhibitor AM404 (0.5–5 mg/kg, i.p.) on both emotional and cognitive performances of rats tested in a Spatial Open Field task and subjected to different experimental settings, named High Arousal (HA) and Low Arousal (LA) conditions. The two different experimental conditions influenced emotional reactivity independently of drug administration. Indeed, vehicle-treated rats exposed to the LA condition spent more time in the center of the arena than vehicle-treated rats exposed to the HA context. Conversely, the different arousal conditions did not affect the cognitive performances of vehicle-treated animals such as the capability to discriminate a spatial displacement of the objects or an object substitution. AM404 administration did not alter locomotor activity or emotional behavior of animals exposed to both environmental conditions. Interestingly, AM404 administration influenced the cognitive parameters depending on the level of emotional arousal: it impaired the capability of rats exposed to the HA condition to recognize a novel object while it did not induce any impairing effect in rats exposed to the LA condition. These findings suggest that drugs enhancing endocannabinoid signaling induce different effects on recognition memory performance depending on the level of emotional arousal induced by the environmental conditions. PMID:22454620

  5. Sleep improvement in dogs after oral administration of mioflazine, a nucleoside transport inhibitor.

    PubMed

    Wauquier, A; Van Belle, H; Van den Broeck, W A; Janssen, P A

    1987-01-01

    Mioflazine, a nucleoside transport inhibitor, was given PO to dogs at doses of 0.04-10 mg/kg. Sixteen hour polygraphic sleep recordings were made and analysis and sleep stage classification was done by computer. Mioflazine decreased wakefulness and increased slow wave sleep, but did not affect the latencies of either REM sleep or slow wave sleep. This increased sleep was due to an increase in the number of light and deep slow wave sleep epochs. The effect lasted for about 8 h. The decreased wakefulness and increased slow wave sleep could be antagonized by the adenosine antagonist caffeine (2.5 and 10 mg/kg, PO); however, there was not a pure antagonistic effect. It might be that the enhancement of slow wave sleep is due to an activation of brain adenosine receptors. This is the first report of a drug acting on adenosine that given orally improves sleep. Mioflazine might be the prototype of substances worth considering for the treatment of a variety of sleep disorders.

  6. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

    PubMed Central

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-01-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

  7. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

    PubMed

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T; Hocart, Charles H; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-08-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

  8. Enhanced functional preservation of cold-stored rat heart by a nucleoside transport inhibitor.

    PubMed

    Yang, X; Zhu, Q; Claydon, M A; Hicks, G L; Wang, T

    1994-07-15

    This study investigates the hypothesis that inhibition of nucleoside transport during hypothermic storage elevates tissue adenosine (ADO) content and improves the function of the isolated rat heart. The hearts, flushed with a cardioplegic solution containing varying concentrations (0-100 nM) of a nucleoside transport inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI), were immersion-stored at 0 degrees C for 9 hr. Function was assessed after 30 min of working reperfusion. Function of unstored fresh hearts served as controls and poststorage recovery is reported as percentage of control function. Poststorage heart rate in all groups returned to control level after reperfusion. Recovery of other functional parameters in the no-NBTI group was as follows: aortic flow (AF), 56.2 +/- 4.6%; coronary flow (CF), 53.9 +/- 3.2%; cardiac output (CO), 55.5 +/- 4.0%; systolic pressure, 81.6 +/- 2.5%; work, 47.0 +/- 4.2%; and coronary vascular resistance (CVR), 157.1 +/- 7.8% of control. NBTI improved functional recovery in a dose-dependent fashion; the maximal improvement was seen at a dose of 5 nM, in which the recovery was: AF, 78.1 +/- 3.4%; CF, 73.5 +/- 4.4%; CO, 76.7 +/- 3.6%; work, 70.7 +/- 5.0%; and CVR, 127.5 +/- 4.5% of control (P < 0.05 vs. no-NBTI). The ADO A1-receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (0.1 microM) blocked the effects of 5 nM NBTI; the recovery of AF, CF, CO, work, and CVR decreased to 62.8 +/- 8.0%, 58.3 +/- 5.0%, 61.5 +/- 3.9%, 54.4 +/- 4.5%, and 163.8 +/- 12.7% of control, respectively (P < 0.05 vs. 5 nM NBTI). Tissue ADO content in 5 nM NBTI hearts at the end of storage was 0.075 +/- 0.025 mumol/g dry wt, which was significantly elevated from 0.016 +/- 0.004 mumol/g dry wt in no-NBTI hearts. Purine release during initial reperfusion was delayed in 5 nM NBTI hearts, indicating the inhibition of nucleoside transport by NBTI. But NBTI treatment did not improve end-storage or end-reperfusion myocardial ATP. In conclusion, the addition of

  9. Role of sensory nerves in gastroprotective effect of anandamide in rats.

    PubMed

    Warzecha, Z; Dembinski, A; Ceranowicz, P; Dembinski, M; Cieszkowski, J; Kownacki, P; Konturek, P C

    2011-04-01

    Previous studies have shown that stimulation of cannabinoid 1 (CB1) receptor protects the gastric mucosa against stress-induced lesion. Aim of the present study was to examine the influence of anandamide on lipid peroxidation and antioxidant defense system in gastric mucosa and the role of sensory nerves in gastroprotective effects of cannabinoids. Studies were performed on rats with intact or ablated sensory nerves (by neurotoxic doses of capsaicin). Gastric lesions were induced by water immersion and restrain stress (WRS). Anandamide was administered at the dose of 0.3, 1.5 or 3.0 μmol/kg, 30 min before exposure to WRS. CB1 receptor antagonist, AM251 (4.0 μmol/kg) was administered 40 min before WRS. WRS induced gastric lesions associated with the decrease in gastric blood flow, mucosal DNA synthesis and mucosal activity of superoxide dismutase (SOD). Serum level of interleukin-1β (IL-1β) and mucosal level of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were increased. Administration of anandamide reduced the ulcers area, generation of MDA+4-HNE and serum level of IL-1β, and this effect was associated with the reduction in the WRS-induced decrease in gastric mucosal blood flow, mucosal DNA synthesis and SOD activity. Ablation of sensory nerves increased the area of ulcers, serum level of IL-1β and mucosal content of MDA+4-HNE, whereas mucosal DNA synthesis, SOD activity and blood flow were additionally decreased. In rats with ablation of sensory nerves, administration of anandamide at the high doses (1.5 and 3.0 μmol/kg) partly reduced deleterious effect of WRS on gastric mucosa, but this effect was weaker than in animals with intact sensory nerves. Low dose of anandamide (0.3 μmol/kg) was ineffective in the protection of gastric mucosa against the WRS-induced lesions in rats with ablation of sensory nerves. In rats with intact sensory nerves and exposed to WRS, administration of AM251 exhibited deleterious effect. In rats with ablation of sensory

  10. FAAH inhibition enhances anandamide mediated anti-tumorigenic effects in non-small cell lung cancer by downregulating the EGF/EGFR pathway.

    PubMed

    Ravi, Janani; Sneh, Amita; Shilo, Konstantin; Nasser, Mohd W; Ganju, Ramesh K

    2014-05-15

    The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

  11. Crystallographic study of FABP5 as an intracellular endocannabinoid transporter

    SciTech Connect

    Sanson, Benoît; Wang, Tao; Sun, Jing; Wang, Liqun; Kaczocha, Martin; Ojima, Iwao; Deutsch, Dale; Li, Huilin

    2014-02-01

    FABP5 was recently found to intracellularly transport endocannabinoid signaling lipids. The structures of FABP5 complexed with two endocannabinoids and an inhibitor were solved. Human FABP5 was found to dimerize via a domain-swapping mechanism. This work will help in the development of inhibitors to raise endocannabinoid levels. In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5–endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

  12. [The effect of membrane-transport inhibitors on the vacuolization of skeletal muscle fibers induced by glycerin removal from them].

    PubMed

    Krolenko, S A; Adamian, S Ia

    1996-01-01

    Effects of inhibitors of volume regulation on reversible vacuolation of skeletal muscle transverse sarcotubules have been studied. The drugs include inhibitors of sodium pump (ouabain), electrodiffusion Cl- and K+ channels (SITS, DIDS, niflumic acid, Ba2+), anion-cation cotransport and antiport (bumetanide, furosimide, amiloride), calcium channels (verapamil, nifedipine), stretch-activated ion channels (Gd3+) and water channels (Hg2+). The drugs were used at concentrations usually inhibiting the regulatory volume response in nonmuscle cells. The vacuolation-devacuolation cycles of frog muscle fibers were observed under the light microscope during efflux and entry of glycerol (90 mM). Neither of used inhibitors produced any noticeable effect on development and disappearance of vacuolation. A conclusion is made that development of vacuoles from the T-system during glycerol removal is not associated with activation of ion transport pathways that leads to regulatory volume decrease or increase in other cell types. The mechanisms of T-tubule vacuolation are discussed.

  13. Differential effects of histone deacetylase inhibitors on cellular drug transporters and their implications for using epigenetic modifiers in combination chemotherapy

    PubMed Central

    Valdez, Benigno C.; Li, Yang; Murray, David; Brammer, Jonathan E.; Liu, Yan; Hosing, Chitra; Nieto, Yago; Champlin, Richard E.; Andersson, Borje S.

    2016-01-01

    HDAC inhibitors, DNA alkylators and nucleoside analogs are effective components of combination chemotherapy. To determine a possible mechanism of their synergism, we analyzed the effects of HDAC inhibitors on the expression of drug transporters which export DNA alkylators. Exposure of PEER lymphoma T-cells to 15 nM romidepsin (Rom) resulted in 40%-50% reduction in mRNA for the drug transporter MRP1 and up to ~500-fold increase in the MDR1 mRNA within 32-48 hrs. MRP1 protein levels concomitantly decreased while MDR1 increased. Other HDAC inhibitors − panobinostat, belinostat and suberoylanilide hydroxamic acid (SAHA) − had similar effects on these transporters. The protein level of MRP1 correlated with cellular resistance to busulfan and chlorambucil, and Rom exposure sensitized cells to these DNA alkylators. The decrease in MRP1 correlated with decreased cellular drug export activity, and increased level of MDR1 correlated with increased export of daunorubicin. A similar decrease in the level of MRP1 protein, and increase in MDR1, were observed when mononuclear cells derived from patients with T-cell malignancies were exposed to Rom. Decreased MRP1 and increased MDR1 expressions were also observed in blood mononuclear cells from lymphoma patients who received SAHA-containing chemotherapy in a clinical trial. This inhibitory effect of HDAC inhibitors on the expression of MRP1 suggests that their synergism with DNA alkylating agents is partly due to decreased efflux of these alkylators. Our results further imply the possibility of antagonistic effects when HDAC inhibitors are combined with anthracyclines and other MDR1 drug ligands in chemotherapy. PMID:27564097

  14. Preclinical to Clinical Translation of CNS Transporter Occupancy of TD-9855, a Novel Norepinephrine and Serotonin Reuptake Inhibitor

    PubMed Central

    Patil, DL; Daniels, OT; Ding, Y-S; Gallezot, J-D; Henry, S; Kim, KHS; Kshirsagar, S; Martin, WJ; Obedencio, GP; Stangeland, E; Tsuruda, PR; Williams, W; Carson, RE; Patil, ST

    2015-01-01

    Background: Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters. Methods: We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor. Results: TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC50 of 11.7ng/mL and 50.8ng/mL, respectively, consistent with modest selectivity for NET in vivo. Accounting for species differences in protein binding, the projected human NET and SERT plasma EC50 values were 5.5ng/mL and 23.9ng/mL, respectively. A single-dose, open-label PET study (4–20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [11C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [11C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30–40h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC50 for NET was estimated to be 1.21ng/mL, and at doses of greater than 4mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35ng/mL. Conclusions: These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation. PMID:25522383

  15. Effect of inhibitors of auxin transport and of calmodulin on a gravisensing-dependent current in maize roots

    NASA Technical Reports Server (NTRS)

    Bjorkman, T.; Leopold, A. C.

    1987-01-01

    Some characteristics of the gravity sensing mechanism in maize root caps were investigated using a bioelectric current as an indicator of gravity sensing. This technique involves the measurement of a change in the current density which arises at the columella region coincidently with the presentation time. Two inhibitors of auxin transport, triiodobenzoic acid and naphthylphthalamic acid, blocked gravitropic curvature but not the change in current density. Two inhibitors of calmodulin activity, compound 48/80 and calmidazolium, blocked both curvature and gravity-induced current. The results suggest that auxin transport is not a component of gravity sensing in the root cap. By contrast, the results suggest that calmodulin plays an intrinsic role in gravity sensing.

  16. Influence of an oil soluble inhibitor on microbiologically influenced corrosion in a diesel transporting pipeline.

    PubMed

    Muthukumar, N; Maruthamuthu, S; Mohanan, S; Palaniswamy, N

    2007-01-01

    Microbial degradation of the oil soluble corrosion inhibitor (OSCI) Baker NC 351 contributed to a decrease in inhibitor efficiency. Corrosion inhibition efficiency was studied by the rotating cage and flow loop methods. The nature of the biodegradation of the corrosion inhibitor was also analysed using Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. The influence of bacterial activity on the degradation of the corrosion inhibitor and its influence on corrosion of API 5LX were evaluated using a weight loss technique and impedance studies. Serratia marcescens ACE2 and Bacillus cereus ACE4 can degrade aromatic and aliphatic hydrocarbons present in the corrosion inhibitor. The present study also discusses the demerits of the oil soluble corrosion inhibitors used in petroleum product pipeline.

  17. The novel endogenous cannabinoid 2-arachidonoylglycerol is inactivated by neuronal- and basophil-like cells: connections with anandamide.

    PubMed Central

    Di Marzo, V; Bisogno, T; Sugiura, T; Melck, D; De Petrocellis, L

    1998-01-01

    The novel endogenous cannabinoid 2-arachidonoylglycerol (2-AG) was rapidly inactivated by intact rat basophilic leukaemia (RBL-2H3) and mouse neuroblastoma (N18TG2) cells through diffusion/hydrolysis/reacylation processes. The hydrolysis of 2-AG was inhibited by typical esterase inhibitors and by more specific blockers of 'fatty acid amide hydrolase' (FAAH), the enzyme catalysing the hydrolysis of the other 'endocannabinoid', anandamide (AEA). No evidence for a facilitated-diffusion process was found. A 2-AG-hydrolysing activity was detected in homogenates from both cell lines, with the highest levels in membrane fractions. It exhibited an optimal pH at 10, and recognized both 2- and 1(3)- isomers of monoarachidonoylglycerol with similar efficiencies. The apparent Km and Vmax values for -3H-2-AG hydrolysis were 91 microM and 29 microM and 2.4 and 1.8 nmol.min-1.mg of protein-1 respectively in N18TG2 and RBL-2H3 cells. [3H]2-AG hydrolysis was inhibited by Cu2+, Zn2+ and p-hydroxymercuribenzoate, and by 2- or 1(3)-monolinoleoyl- and -linolenoyl-glycerols, but not by the oleoyl, palmitoyl and myristoyl congeners. Purified fractions from solubilized membrane proteins catalysed, at pH 9.5, the hydrolysis of 2-AG as well as AEA. Accordingly, AEA as well as FAAH inhibitors, including arachidonoyltrifluoromethyl ketone (ATFMK), blocked [3H]2-AG hydrolysis by N18TG2 and RBL-2H3 membranes, whereas 2-AG inhibited [14C]AEA hydrolysis. FAAH blockade by ATFMK preserved from inactivation the 2-AG synthesized de novo by intact N18TG2 cells stimulated with ionomycin. These data suggest that FAAH may be one of the enzymes deputed to the physiological inactivation of 2-AG, and create intriguing possibilities for the cross-regulation of 2-AG and AEA levels. PMID:9512456

  18. Effects of Selective Serotonin Reuptake Inhibitors on Interregional Relation of Serotonin Transporter Availability in Major Depression

    PubMed Central

    James, Gregory M.; Baldinger-Melich, Pia; Philippe, Cecile; Kranz, Georg S.; Vanicek, Thomas; Hahn, Andreas; Gryglewski, Gregor; Hienert, Marius; Spies, Marie; Traub-Weidinger, Tatjana; Mitterhauser, Markus; Wadsak, Wolfgang; Hacker, Marcus; Kasper, Siegfried; Lanzenberger, Rupert

    2017-01-01

    Selective serotonin reuptake inhibitors (SSRIs) modulate serotonergic neurotransmission by blocking reuptake of serotonin from the extracellular space. Up to now, it remains unclear how SSRIs achieve their antidepressant effect. However, task-based and resting state functional magnetic resonance imaging studies, have demonstrated connectivity changes between brain regions. Here, we use positron emission tomography (PET) to quantify SSRI’s main target, the serotonin transporter (SERT), and assess treatment-induced molecular changes in the interregional relation of SERT binding potential (BPND). Nineteen out-patients with major depressive disorder (MDD) and 19 healthy controls (HC) were included in this study. Patients underwent three PET measurements with the radioligand [11C]DASB: (1) at baseline, (2) after a first SSRI dose; and (3) following at least 3 weeks of daily intake. Controls were measured once with PET. Correlation analyses were restricted to brain regions repeatedly implicated in MDD pathophysiology. After 3 weeks of daily SSRI administration a significant increase in SERT BPND correlations of anterior cingulate cortex and insula with the amygdala, midbrain, hippocampus, pallidum and putamen (p < 0.05; false discovery rate, FDR corrected) was revealed. No significant differences were found when comparing MDD patients and HC at baseline. These findings are in line with the clinical observation that treatment response to SSRIs is often achieved only after a latency of several weeks. The elevated associations in interregional SERT associations may be more closely connected to clinical outcomes than regional SERT occupancy measures and could reflect a change in the regional interaction of serotonergic neurotransmission during antidepressant treatment. PMID:28220069

  19. Serotonin transporter occupancy in rats exposed to serotonin reuptake inhibitors in utero or via breast milk.

    PubMed

    Capello, Catherine F; Bourke, Chase H; Ritchie, James C; Stowe, Zachary N; Newport, D Jeffrey; Nemeroff, Amanda; Owens, Michael J

    2011-10-01

    Rigorous data regarding fetal central nervous system (CNS) exposure after antidepressant exposure are sparse. The magnitude of serotonin reuptake inhibitor (SRI) CNS exposure was measured in three groups of rats using ex vivo autoradiography of the serotonin transporter (SERT): 1) in utero, 2) postnatal clearance after birth, and 3) exposure through lactation. Rats were exposed to one of five SRI-type antidepressants (escitalopram, fluoxetine, paroxetine, sertraline, and venlafaxine) administered continuously via osmotic minipumps to pregnant or nursing dams. Dam dosing was adjusted to reflect the 50th and 85th percentiles of serum concentrations observed in pregnant women. Embryonic day 21 rat pups exposed in utero exhibited >80% SERT occupancy in brain tissue, which is equivalent to that of the pregnant dam and similar to that reported for human pharmacotherapy. Venlafaxine was the exception with occupancies ranging from 61 to 92% across different litters. The magnitude of SERT occupancy is essentially equivalent between dams and fetuses. By postnatal day 4, high SERT occupancy was observed only in fluoxetine-exposed pups (41-92% occupancy). Significantly less, but measurable, exposure occurred via breast milk exposure even in the absence of detectable drug concentrations in nursing pup sera. Pups exposed to SRIs via breast milk for 3 or 7 days exhibited varying SERT occupancies (0-57% depending on the individual medication and dam dose). These data highlight the need for animal modeling of fetal and nursing infant drug exposure using clinically meaningful dosing strategies and appropriate CNS measures to develop rational treatment guidelines that systematically minimize fetal and neonatal medication exposure in humans.

  20. Salicylic Acid Is an Uncoupler and Inhibitor of Mitochondrial Electron Transport1

    PubMed Central

    Norman, Christel; Howell, Katharine A.; Millar, A. Harvey; Whelan, James M.; Day, David A.

    2004-01-01

    The effect of salicylic acid (SA) on respiration and mitochondrial function was examined in tobacco (Nicotiana tabacum) suspension cell cultures in the range of 0.01 to 5 mm. Cells rapidly accumulated SA up to 10-fold of the externally applied concentrations. At the lower concentrations, SA accumulation was transitory. When applied at 0.1 mm or less, SA stimulated respiration of whole cells and isolated mitochondria in the absence of added ADP, indicating uncoupling of respiration. However, at higher concentrations, respiration was severely inhibited. Measurements of ubiquinone redox poise in isolated mitochondria suggested that SA blocked electron flow from the substrate dehydrogenases to the ubiquinone pool. This inhibition could be at least partially reversed by re-isolating the mitochondria. Two active analogs of SA, benzoic acid and acetyl-SA, had the same effect as SA on isolated tobacco mitochondria, whereas the inactive p-hydroxybenzoic acid was without effect at the same concentration. SA induced an increase in Aox protein levels in cell suspensions, and this was correlated with an increase in Aox1 transcript abundance. However, when applied at 0.1 mm, this induction was transient and disappeared as SA levels in the cells declined. SA at 0.1 mm also increased the expression of other SA-responsive genes, and this induction was dependent on active mitochondria. The results indicate that SA is both an uncoupler and an inhibitor of mitochondrial electron transport and suggest that this underlies the induction of some genes by SA. The possible implications of this for the interpretation of SA action in plants are discussed. PMID:14684840

  1. Relative contributions of norepinephrine and serotonin transporters to antinociceptive synergy between monoamine reuptake inhibitors and morphine in the rat formalin model.

    PubMed

    Shen, Fei; Tsuruda, Pamela R; Smith, Jacqueline A M; Obedencio, Glenmar P; Martin, William J

    2013-01-01

    Multimodal analgesia is designed to optimize pain relief by coadministering drugs with distinct mechanisms of action or by combining multiple pharmacologies within a single molecule. In clinical settings, combinations of monoamine reuptake inhibitors and opioid receptor agonists have been explored and one currently available analgesic, tapentadol, functions as both a µ-opioid receptor agonist and a norepinephrine transporter inhibitor. However, it is unclear whether the combination of selective norepinephrine reuptake inhibition and µ-receptor agonism achieves an optimal antinociceptive synergy. In this study, we assessed the pharmacodynamic interactions between morphine and monoamine reuptake inhibitors that possess different affinities and selectivities for norepinephrine and serotonin transporters. Using the rat formalin model, in conjunction with measurements of ex vivo transporter occupancy, we show that neither the norepinephrine-selective inhibitor, esreboxetine, nor the serotonin-selective reuptake inhibitor, fluoxetine, produce antinociceptive synergy with morphine. Atomoxetine, a monoamine reuptake inhibitor that achieves higher levels of norepinephrine than serotonin transporter occupancy, exhibited robust antinociceptive synergy with morphine. Similarly, a fixed-dose combination of esreboxetine and fluoxetine which achieves comparable levels of transporter occupancy potentiated the antinociceptive response to morphine. By contrast, duloxetine, a monoamine reuptake inhibitor that achieves higher serotonin than norepinephrine transporter occupancy, failed to potentiate the antinociceptive response to morphine. However, when duloxetine was coadministered with the 5-HT3 receptor antagonist, ondansetron, potentiation of the antinociceptive response to morphine was revealed. These results support the notion that inhibition of both serotonin and norepinephrine transporters is required for monoamine reuptake inhibitor and opioid-mediated antinociceptive

  2. Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma

    PubMed Central

    Eskiocak, Ugur; Ramesh, Vijayashree; Gill, Jennifer G.; Zhao, Zhiyu; Yuan, Stacy W.; Wang, Meng; Vandergriff, Travis; Shackleton, Mark; Quintana, Elsa; Johnson, Timothy M.; DeBerardinis, Ralph J.; Morrison, Sean J.

    2016-01-01

    New therapies are required for melanoma. Here, we report that multiple cardiac glycosides, including digitoxin and digoxin, are significantly more toxic to human melanoma cells than normal human cells. This reflects on-target inhibition of the ATP1A1 Na+/K+ pump, which is highly expressed by melanoma. MEK inhibitor and/or BRAF inhibitor additively or synergistically combined with digitoxin to induce cell death, inhibiting growth of patient-derived melanomas in NSG mice and synergistically extending survival. MEK inhibitor and digitoxin do not induce cell death in human melanocytes or haematopoietic cells in NSG mice. In melanoma, MEK inhibitor reduces ERK phosphorylation, while digitoxin disrupts ion gradients, altering plasma membrane and mitochondrial membrane potentials. MEK inhibitor and digitoxin together cause intracellular acidification, mitochondrial calcium dysregulation and ATP depletion in melanoma cells but not in normal cells. The disruption of ion homoeostasis in cancer cells can thus synergize with targeted agents to promote tumour regression in vivo. PMID:27545456

  3. [Sodium Glucose Co-transporter Type 2 (SGLT2) Inhibitors in CKD].

    PubMed

    Insalaco, Monica; Zanoli, Luca; Rastelli, Stefania; Lentini, Paolo; Rapisarda, Francesco; Fatuzzo, Pasquale; Castellino, Pietro; Granata, Antonio

    2015-01-01

    Among the new drugs used for the treatment of Diabetes Mellitus type 2, sodium-glucose cotransporter 2 (SGLT2) inhibitors represent a promising therapeutic option. Since their ability to lower glucose is proportional to GFR, their effect is reduced in patients with chronic kidney disease (CKD). The antidiabetic mechanism of these drugs is insulin-independent and, therefore, complimentary to that of others antihyperglicaemic agents. Moreover, SGLT2 inhibitors are able to reduce glomerular hyperfiltration, systemic and intraglomerular pressure and uric acid levels, with consequent beneficial effects on the progression of kidney disease in non diabetic patients as well. Only few studies have been performed to evaluate the effects of SGLT2 inhibitors in patients with CKD. Therefore, safety and efficacy of SGLT2 inhibitors should be better clarified in the setting of CKD. In this paper, we will review the use of SGLT2 inhibitors in diabetic patients, including those with CKD.

  4. The role of human equilibrative nucleoside transporter 1 on the cellular transport of the DNA methyltransferase inhibitors 5-azacytidine and CP-4200 in human leukemia cells.

    PubMed

    Hummel-Eisenbeiss, Johanna; Hascher, Antje; Hals, Petter-Arnt; Sandvold, Marit Liland; Müller-Tidow, Carsten; Lyko, Frank; Rius, Maria

    2013-09-01

    The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹⁴C]5-azacytidine demonstrated Na⁺-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.

  5. In vivo neuronal synthesis and axonal transport of Kunitz protease inhibitor (KPI)-containing forms of the amyloid precursor protein.

    PubMed

    Moya, K L; Confaloni, A M; Allinquant, B

    1994-11-01

    We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

  6. In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

    PubMed

    Mishra, Rama K; Wei, Changyong; Hresko, Richard C; Bajpai, Richa; Heitmeier, Monique; Matulis, Shannon M; Nooka, Ajay K; Rosen, Steven T; Hruz, Paul W; Schiltz, Gary E; Shanmugam, Mala

    2015-06-05

    Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

  7. In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy*

    PubMed Central

    Mishra, Rama K.; Wei, Changyong; Hresko, Richard C.; Bajpai, Richa; Heitmeier, Monique; Matulis, Shannon M.; Nooka, Ajay K.; Rosen, Steven T.; Hruz, Paul W.; Schiltz, Gary E.; Shanmugam, Mala

    2015-01-01

    Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics. PMID:25847249

  8. Presumed LRP1-targeting transport peptide delivers β-secretase inhibitor to neurons in vitro with limited efficiency

    PubMed Central

    Kim, Jong Ah; Casalini, Tommaso; Brambilla, Davide; Leroux, Jean-Christophe

    2016-01-01

    Interfering with the activity of β-secretase to reduce the production of Aβ peptides is a conceivable therapeutic strategy for Alzheimer’s disease. However, the development of efficient yet safe inhibitors is hampered by secondary effects, usually linked to the indiscriminate inhibition of other substrates’ processing by the targeted enzyme. Based on the spatial compartmentalization of the cleavage of the amyloid precursor protein by β-secretase, we hypothesized that by exploiting the endocytosis receptor low-density lipoprotein receptor-related protein it would be possible to direct an otherwise cell-impermeable inhibitor to the endosomes of neurons, boosting the drug’s efficacy and importantly, sparing the off-target effects. We used the transport peptide Angiopep to build an endocytosis-competent conjugate and found that although the peptide facilitated the inhibitor’s internalization into neurons and delivered it to the endosomes, the delivery was not efficient enough to potently reduce β-secretase activity at the cellular level. This is likely connected to the finding that in the cell lines we used, Angiopep’s internalization was not mediated by its presumed receptor to a significant extent. Additionally, Angiopep exploited different internalization mechanisms when applied alone or when conjugated to the inhibitor, highlighting the impact that drug conjugation can have on transport peptides. PMID:27682851

  9. Place of sodium-glucose co-transporter type 2 inhibitors for treatment of type 2 diabetes

    PubMed Central

    Mikhail, Nasser

    2014-01-01

    Inhibitors of sodium-glucose co-transporter type 2 (SGLT2), such as canagliflozin and dapagliflozin, are recently approved for treatment of type 2 diabetes. These agents lower blood glucose mainly by increasing urinary glucose excretion. Compared with placebo, SGLT2 inhibitors reduce hemoglobin A1c (HbA1c) levels by an average of 0.5%-0.8% when used as monotherapy or add-on therapy. Advantages of this drug class include modest weight loss of approximately 2 kg, low risk of hypoglycemia, and decrease blood pressure of approximately 4 mmHg systolic and 2 mmHg diastolic. These characteristics make these agents potential add-on therapy in patients with HbA1c levels close to 7%-8.0%, particularly if these patients are obese, hypertensive, and/or prone for hypoglycemia. Meanwhile, these drugs are limited by high frequency of genital mycotic infections. Less common adverse effects include urinary tract infections, hypotension, dizziness, and worsening renal function. SGLT2 inhibitors should be used with caution in the elderly because of increased adverse effects, and should not be used in chronic kidney disease due to decreased or lack of efficacy and nephrotoxicity. Overall, SGLT2 inhibitors are useful addition for treatment of select groups of patients with type 2 diabetes, but their efficacy and safety need to be established in long-term clinical trials. PMID:25512787

  10. Sodium-glucose linked transporter-2 inhibitors in chronic kidney disease.

    PubMed

    Zanoli, L; Granata, A; Lentini, P; Rastelli, S; Fatuzzo, P; Rapisarda, F; Castellino, P

    2015-01-01

    SGLT2 inhibitors are new antihyperglycaemic agents whose ability to lower glucose is directly proportional to GFR. Therefore, in chronic kidney disease (CKD) the blood glucose lowering effect is reduced. Unlike many current therapies, the mechanism of action of SGLT2 inhibitors is independent of insulin action or beta-cell function. In addition, the mechanism of action of SGLT2 inhibitors is complementary and not alternative to other antidiabetic agents. SGLT2 inhibitors could be potentially effective in attenuating renal hyperfiltration and, consequently, the progression of CKD. Moreover, the reductions in intraglomerular pressure, systemic blood pressure, and uric acid levels induced by SGLT inhibition may potentially be of benefit in CKD subjects without diabetes. However, at present, only few clinical studies were designed to evaluate the effects of SGLT2 inhibitors in CKD. Consequently, safety and potential efficacy beyond blood glucose lowering should be better clarified in CKD. In this paper we provide an updated review of the use of SGLT2 inhibitors in clinical practice, with particular attention on subjects with CKD.

  11. The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors.

    PubMed

    Chen, Fenghua; Larsen, Mads Breum; Sánchez, Connie; Wiborg, Ove

    2005-03-01

    The interaction of the S- and R-enantiomers (escitalopram and R-citalopram) of citalopram, with high- and low-affinity binding sites in COS-1 cell membranes expressing human SERT (hSERT) were investigated. Escitalopram affinity for hSERT and its 5-HT uptake inhibitory potency was in the nanomolar range and approximately 40-fold more potent than R-citalopram. Escitalopram considerably stabilised the [3H]-escitalopram/SERT complex via an allosteric effect at a low-affinity binding site. The stereoselectivity between escitalopram and R-citalopram was approximately 3:1 for the [3H]-escitalopram/hSERT complex. The combined effect of escitalopram and R-citalopram was additive. Paroxetine and sertraline mainly stabilised the [3H]-paroxetine/hSERT complex. Fluoxetine, duloxetine and venlafaxine have only minor effects. 5-HT stabilised the [125I]-RTI-55, [3H]-MADAM, [3H]-paroxetine, [3H]-fluoxetine and [3H]-venlafaxine/SERT complex to some extent. Thus, escitalopram shows a unique interaction with the hSERT compared with other 5-HT reuptake inhibitors (SSRIs) and, in addition to its 5-HT reuptake inhibitory properties, displays a pronounced effect via an affinity-modulating allosteric site.

  12. Fatty Acid Transport Protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    PubMed Central

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC50 8–11μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC50 58μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13C-oleate demonstrating its potential as a therapeutic agent. PMID:26284975

  13. SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria.

    PubMed

    Chino, Yukihiro; Samukawa, Yoshishige; Sakai, Soichi; Nakai, Yasuhiro; Yamaguchi, Jun-ichi; Nakanishi, Takeo; Tamai, Ikumi

    2014-10-01

    Sodium glucose cotransporter 2 (SGLT2) inhibitors have been reported to lower the serum uric acid (SUA) level. To elucidate the mechanism responsible for this reduction, SUA and the urinary excretion rate of uric acid (UE(UA)) were analysed after the oral administration of luseogliflozin, a SGLT2 inhibitor, to healthy subjects. After dosing, SUA decreased, and a negative correlation was observed between the SUA level and the UE(UA), suggesting that SUA decreased as a result of the increase in the UE(UA). The increase in UE(UA) was correlated with an increase in urinary D-glucose excretion, but not with the plasma luseogliflozin concentration. Additionally, in vitro transport experiments showed that luseogliflozin had no direct effect on the transporters involved in renal UA reabsorption. To explain that the increase in UE(UA) is likely due to glycosuria, the study focused on the facilitative glucose transporter 9 isoform 2 (GLUT9ΔN, SLC2A9b), which is expressed at the apical membrane of the kidney tubular cells and transports both UA and D-glucose. It was observed that the efflux of [(14) C]UA in Xenopus oocytes expressing the GLUT9 isoform 2 was trans-stimulated by 10 mm D-glucose, a high concentration of glucose that existed under SGLT2 inhibition. On the other hand, the uptake of [(14) C]UA by oocytes was cis-inhibited by 100 mm D-glucose, a concentration assumed to exist in collecting ducts. In conclusion, it was demonstrated that the UE(UA) could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose.

  14. Inhibition of anandamide hydrolysis dampens the neuroendocrine response to stress in neonatal rats subjected to suboptimal rearing conditions.

    PubMed

    McLaughlin, Ryan Joseph; Verlezza, Silvanna; Gray, Jennifer Megan; Hill, Matthew Nicholas; Walker, Claire-Dominique

    2016-01-01

    Exposure to stress during early development can exert profound effects on the maturation of the neuroendocrine stress axis. The endocannabinoid (ECB) system has recently surfaced as a fundamental component of the neuroendocrine stress response; however, the effect of early-life stress on neonatal ECB signaling and the capacity to which ECB enhancement may modulate neonatal stress responses is relatively unknown. The present study assessed whether exposure to early-life stress in the form of limited access to nesting/bedding material (LB) from postnatal (PND) day 2 to 9 alters neuroendocrine activity and hypothalamic ECB content in neonatal rats challenged with a novel immobilization stressor. Furthermore, we examined whether inhibition of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of anandamide (AEA) affects neuroendocrine responses in PND10 pups as a function of rearing conditions. Neonatal rats showed a robust increase in corticosterone (CORT) and adrenocorticotropin hormone (ACTH) secretion in response to immobilization stress, which was significantly blunted in pups reared in LB conditions. Accordingly, LB pups exhibited reduced stress-induced Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, with no significant differences in hypothalamic ECB content. Administration of the FAAH inhibitor URB597 (0.3 mg/kg, ip) 90 min prior to immobilization stress significantly dampened stress-induced CORT release, but only in pups reared in LB conditions. These results suggest that rearing in restricted bedding conditions dampens the neuroendocrine response to stress, while augmenting AEA mitigates stress-induced alterations in glucocorticoid secretion preferentially in pups subjected to early-life stress.

  15. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    SciTech Connect

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  16. Pharmacological and Behavioral Characterization of D-473, an Orally Active Triple Reuptake Inhibitor Targeting Dopamine, Serotonin and Norepinephrine Transporters

    PubMed Central

    Dutta, Aloke K.; Santra, Soumava; Sharma, Horrick; Voshavar, Chandrashekhar; Xu, Liping; Mabrouk, Omar; Antonio, Tamara; Reith, Maarten E. A.

    2014-01-01

    Major depressive disorder (MDD) is a debilitating disease affecting a wide cross section of people around the world. The current therapy for depression is less than adequate and there is a considerable unmet need for more efficacious treatment. Dopamine has been shown to play a significant role in depression including production of anhedonia which has been one of the untreated symptoms in MDD. It has been hypothesized that drugs acting at all three monoamine transporters including dopamine transporter should provide more efficacious antidepressants activity. This has led to the development of triple reuptake inhibitor D-473 which is a novel pyran based molecule and interacts with all three monoamine transporters. The monoamine uptake inhibition activity in the cloned human transporters expressed in HEK-293 cells (70.4, 9.18 and 39.7 for DAT, SERT and NET, respectively) indicates a serotonin preferring triple reuptake inhibition profile for this drug. The drug D-473 exhibited good brain penetration and produced efficacious activity in rat forced swim test under oral administration. The optimal efficacy dose did not produce any locomotor activation. Microdialysis experiment demonstrated that systemic administration of D-473 elevated extracellular level of the three monoamines DA, 5-HT, and NE efficaciously in the dorsal lateral striatum (DLS) and the medial prefrontal cortex (mPFC) area, indicating in vivo blockade of all three monoamine transporters by D-473. Thus, the current biological data from D-473 indicate potent antidepressant activity of the molecule. PMID:25427177

  17. [Escitalopram: a selective inhibitor and allosteric modulator of the serotonin transporter].

    PubMed

    Mnie-Filali, O; El Mansari, M; Scarna, H; Zimmer, L; Sánchez, C; Haddjeri, N

    2007-12-01

    Citalopram (Séropram) is an antidepressant of the selective serotonin (5-HT) reuptake inhibitor (SSRI) class, composed of equal amounts of S-enantiomer, escitalopram, and R-enantiomer, R-citalopram. Both clinical and preclinical studies have reported that escitalopram is a potent SSRI that possesses a faster onset of antidepressant activity in comparison with citalopram. Conversely, R-citalopram, although devoid of 5-HT reuptake inhibition property, was reported to counteract the effect of the S-enantiomer in several in vitro and in vivo experiments. For instance, microdialysis studies have shown that escitalopram increased the extracellular 5-HT levels in the frontal cortex and the ventral hippocampus, and this effect was prevented by concomitant injection of R-citalopram. The in vivo relevance of the antagonistic effect of R-citalopram on escitalopram efficacy was confirmed in dorsal raphe nucleus, a brain region known to be a target for SSRIs. In the later region, escitalopram was four times more potent than citalopram in suppressing the firing activity of 5-HT neurons and this effect of escitalopram was significantly prevented by R-citalopram. The antagonizing effect of R-citalopram on escitalopram efficacy was also observed in behavioural tests predictive of anxiolytic or antidepressant properties. In adult rats, R-citalopram reduced the anxiolytic-like effect of escitalopram obtained in the footshock-induced ultrasonic vocalization model, the conditioned fear model or the Vogel conflict and elevated plus maze tests. In validated chronic models with high predictive value for antidepressant activity, when escitalopram was administered for five weeks, either alone or with twice as much R-citalopram, the effect of the treatment regimens on reversal of hedonic deficit was significantly different. Importantly, chronic treatment with escitalopram reversed the decrease in cytogenesis in the rat dentate gyrus, induced by chronic mild stress. However, in naïve rats

  18. Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of Excitatory Amino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c -

    NASA Astrophysics Data System (ADS)

    Bridges, Richard J.; Patel, Sarjubhai A.

    As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates not only in standard fast synaptic communication, but also contributes to higher order signal processing, as well as neuropathology. Given this variety of functional roles, interest has been growing as to how the extracellular concentrations of l-glutamate surrounding neurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, each of which appears capable of influencing both the signaling and pathological actions of l-glutamate within the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystine exchanger, system x c - (Sx c -). While the family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequestering l-glutamate in neurons and glia, Sxc - provides a route for the export of glutamate from cells into the extracellular environment. The primary intent of this work is to provide an overview of the inhibitors and substrates that have been developed to delineate the pharmacological specificity of these transport systems, as well as be exploited as probes with which to selectively investigate function. Particular attention is paid to the development of small molecule templates that mimic the structural properties of the endogenous substrates, l-glutamate, l-aspartate and l-cystine and how strategic control of functional group position and/or the introduction of lipophilic R-groups can impact multiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrate activity.

  19. New lipid modulating drugs: the role of microsomal transport protein inhibitors.

    PubMed

    Rizzo, Manfredi; Wierzbicki, Anthony S

    2011-01-01

    Microsomal triglyceride transfer protein (MTP) is involved in the synthesis of very low density lipoprotein in the liver. Its deficiency results in abetalipoproteinemia. MTP inhibitors target the assembly and secretion of apolipoprotein B-containing lipoproteins. These agents may potentially play a role, alone or in combination, in the treatment of hypercholesterolemia or hypertriglyceridaemia. Clinical applications of MTP inhibitors initially focused primarily on high-dose monotherapy in order to produce substantial reductions in LDL-cholesterol levels but these proved to induce significant hepatic steatosis and transaminase elevations. However, likely orphan indications for MTP inhibitors, where a different risk-benefit profile applies, include patients with homozygous familial hypercholesterolemia where statins often show a low response. Development of MTP inhibitors has continued to enter clinical trials at lower doses or in formulations aimed at utilizing their efficacy while avoiding their side effects. These have shown promising results in reducing cholesterol, triglycerides and apolipoprotein B with a far lower incidence of, often, transient side-effects. The clinical efficacy and safety of MTP inhibition in patients with hyperlipidaemia remains to be fully determined and to be proven in both surrogate and clinical endpoint trials but there may be a role for these agents in orphan indications for rarer severe hyperlipidaemias.

  20. Dipeptidyl peptidase-4 inhibitors or sodium glucose co-transporter-2 inhibitors as an add-on to insulin therapy: A comparative review.

    PubMed

    Singh, Awadhesh Kumar; Singh, Ritu

    2016-01-01

    The gradual decline in β-cell function is inevitable in type 2 diabetes mellitus and therefore, substantial proportions of patients require insulin subsequently, in order to achieve optimal glucose control. While weight gain, hypoglycemia, and fluid retention especially during dose intensification is a known limitation to insulin therapy, these adverse effects also reduce patient satisfaction and treatment adherence. It is also possible that the benefits of intensive control achieved by insulin therapy, perhaps get nullified by the weight gain and hypoglycemia. In addition, improvement in plasma glucose or glycated hemoglobin (HbA1c) itself is associated with weight gain. Notably, studies have already suggested that reduction in body weight by ~3-5%, may allow a significantly better glycemic control. Thus, a class of drugs, which can reduce HbA1c effectively, yet are weight neutral or preferably reduce body weight, could be the most sought out strategy as an add-on therapy to insulin. While sulfonylureas (SUs) are associated with weight gain and hypoglycemia, pioglitazone increases body weight and fluid retention. Moreover, SUs are not recommended once premix or prandial insulin is commenced. The addition of newer agents, such as glucagon-like peptide-1 receptor agonist to insulin certainly appears to be an effective tool in reducing both HbA1c and body weight as is evident across the studies; however, this approach incurs an additional injection as well as cost. Dipeptidyl peptidase-4 inhibitors (DPP-4I) and sodium-glucose co-transporter-2 inhibitors (SGLT-2I) are other exciting options, as an add-on to insulin therapy primarily because these are oral drugs and do not possess any intrinsic potential of hypoglycemia. Furthermore, these are either weight neutral or induce significant weight loss. This review article aims to comparatively analyze the safety and efficacy of DPP-4I and SGLT-2I, as an add-on therapy to insulin.

  1. Structure-activity analysis of thiourea analogs as inhibitors of UT-A and UT-B urea transporters

    PubMed Central

    Esteva-Font, Cristina; Phuan, Puay-Wah; Lee, Sujin; Su, Tao; Anderson, Marc O.; Verkman, A. S.

    2015-01-01

    Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ∼0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50 >15 vs. 2.8 mM). PMID:25613743

  2. Novel Inhibitors of Human Organic Cation/Carnitine Transporter (hOCTN2) via Computational Modeling and In Vitro Testing

    PubMed Central

    Diao, Lei; Ekins, Sean; Polli, James E.

    2010-01-01

    Purpose The objective was to elucidate the inhibition requirements of the human Organic Cation/Carnitine Transporter (hOCTN2). Methods Twenty-seven drugs were screened initially for their potential to inhibit uptake of L-carnitine into a stably transfected hOCTN2-MDCK cell monolayer. A HipHop common features pharmacophore was developed and used to search a drug database. Fifty-three drugs, including some not predicted to be inhibitors, were selected and screened in vitro. Results A common features pharmacophore was derived from initial screening data and consisted of three hydrophobic features and a positive ionizable feature. Among the 33 tested drugs that were predicted to map to the pharmacophore, 27 inhibited hOCTN2 in vitro (40% or less L-carnitine uptake from 2.5μM L-carnitine solution in presence of 500 μM drug, compared to L-carnitine uptake without drug present). Hence, the pharmacophore accurately prioritized compounds for testing. Ki measurements showed low micromolar inhibitors belonged to diverse therapeutic classes of drugs, including many not previously known to inhibit hOCTN2. Compounds were more likely to cause rhabdomyolysis if the Cmax/Ki ratio was higher than 0.0025. Conclusion A combined pharmacophore and in vitro approach found new, structurally diverse inhibitors for hOCTN2 that may possibly cause clinical significant toxicity such as rhabdomyolysis. PMID:19437106

  3. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).

    PubMed

    Elkind, N Barry; Szentpétery, Zsófia; Apáti, Agota; Ozvegy-Laczka, Csilla; Várady, György; Ujhelly, Olga; Szabó, Katalin; Homolya, László; Váradi, András; Buday, László; Kéri, György; Német, Katalin; Sarkadi, Balázs

    2005-03-01

    Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.

  4. Alpha adrenergic modulation on effects of norepinephrine transporter inhibitor reboxetine in five-choice serial reaction time task

    PubMed Central

    Liu, Yia-Ping; Lin, Yu-Lung; Chuang, Chia-Hsin; Kao, Yu-Cheng; Chang, Shang-Tang; Tung, Che-Se

    2009-01-01

    The study examined the effects of a norepinephrine transporter (NET) inhibitor reboxetine (RBX) on an attentional performance test. Adult SD rats trained with five-choice serial reaction time task (5-CSRTT) were administered with RBX (0, 3.0 and 10 mg/kg) in the testing day. Alpha-1 adrenergic receptor antagonist PRA and alpha-2 adrenergic receptor antagonist RX821002 were used to clarify the RBX effect. Results revealed that rat received RBX at 10 mg/kg had an increase in the percentage of the correct response and decreases in the numbers of premature response. Alpha-1 adrenergic receptor antagonist Prazosin (PRA) at 0.1 mg/kg reversed the RBX augmented correct responding rate. However, alpha-2 adrenergic receptor antagonist RX821002 at 0.05 and 0.1 mg/kg dose dependently reversed the RBX reduced impulsive responding. Our results suggested that RBX as a norepinephrine transporter inhibitor can be beneficial in both attentional accuracy and response control and alpha-1 and alpha-2 adrenergic receptors might be involved differently. PMID:19678962

  5. Silychristin, a Flavonolignan Derived From the Milk Thistle, Is a Potent Inhibitor of the Thyroid Hormone Transporter MCT8.

    PubMed

    Johannes, Jörg; Jayarama-Naidu, Roopa; Meyer, Franziska; Wirth, Eva Katrin; Schweizer, Ulrich; Schomburg, Lutz; Köhrle, Josef; Renko, Kostja

    2016-04-01

    Thyroid hormones (THs) are charged and iodinated amino acid derivatives that need to pass the cell membrane facilitated by thyroid hormone transmembrane transporters (THTT) to exert their biological function. The importance of functional THTT is affirmed by the devastating effects of mutations in the human monocarboxylate transporter (MCT) 8, leading to a severe form of psychomotor retardation. Modulation of THTT function by pharmacological or environmental compounds might disturb TH action on a tissue-specific level. Therefore, it is important to identify compounds with relevant environmental exposure and THTT-modulating activity. Based on a nonradioactive TH uptake assay, we performed a screening of 13 chemicals, suspicious for TH receptor interaction, to test their potential effects on THTT in MCT8-overexpressing MDCK1-cells. We identified silymarin, an extract of the milk thistle, to be a potent inhibitor of T3 uptake by MCT8. Because silymarin is a complex mixture of flavonolignan substances, we further tested its individual components and identified silychristin as the most effective one with an IC50 of approximately 100 nM. The measured IC50 value is at least 1 order of magnitude below those of other known THTT inhibitors. This finding was confirmed by T3 uptake in primary murine astrocytes expressing endogenous Mct8 but not in MCT10-overexpressing MDCK1-cells, indicating a remarkable specificity of the inhibitor toward MCT8. Because silymarin is a frequently used adjuvant therapeutic for hepatitis C infection and chronic liver disease, our observations raise questions regarding its safety with respect to unwanted effects on the TH axis.

  6. Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour.

    PubMed

    Maccarrone, Mauro; Valverde, Olga; Barbaccia, Maria L; Castañé, Anna; Maldonado, Rafael; Ledent, Catherine; Parmentier, Marc; Finazzi-Agrò, Alessandro

    2002-04-01

    Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.

  7. Cocaine- and amphetamine-related transcript is involved in the orexigenic effect of endogenous anandamide.

    PubMed

    Osei-Hyiaman, Douglas; Depetrillo, Michael; Harvey-White, Judith; Bannon, Anthony W; Cravatt, Benjamin F; Kuhar, Michael J; Mackie, Ken; Palkovits, Miklós; Kunos, George

    2005-01-01

    Endocannabinoids acting at CB1 cannabinoid receptors (CB1) increase appetite. In view of the predominant presynaptic localization of CB1 in the brain, we tested the hypothesis that the orexigenic effect of endocannabinoids involves inhibition of the release of a tonically active anorexigenic mediator, such as the peptide product of the cocaine- and amphetamine-related transcript (CART). The CB1 antagonist rimonabant inhibited food intake in food-restricted wild-type mice, but not in their CART-deficient littermates. Mice deficient in fatty acid amide hydrolase (FAAH), the enzyme responsible for the in vivo metabolism of the endocannabinoid anandamide, have reduced levels of CART-immunoreactive nerve fibers and terminals in several brain regions implicated in appetite control, including the arcuate, dorsomedial and periventricular nuclei of the hypothalamus, the amygdala, the bed nucleus of the stria terminalis and the nucleus accumbens, and treatment of FAAH(-/-) mice with rimonabant, 3 mg/kg/day for 7 days, increased CART levels toward those seen in FAAH(+/+) wild-type controls. In contrast, no difference in the density of CART-immunoreactive fibers was observed in the median eminence and the paraventricular nucleus of FAAH(+/+) and FAAH(-/-) mice. Acute treatment of wild-type mice with the cannabinoid agonist HU-210 resulted in elevated CART levels in the dorsomedial nucleus and the shell portion of the nucleus accumbens. These observations are compatible with CART being a downstream mediator of the CB1-mediated orexigenic effect of endogenous anandamide.

  8. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

    PubMed Central

    Bisogno, Tiziana; Hanuš, Lumír; De Petrocellis, Luciano; Tchilibon, Susanna; Ponde, Datta E; Brandi, Ines; Moriello, Aniello Schiano; Davis, John B; Mechoulam, Raphael; Di Marzo, Vincenzo

    2001-01-01

    (−)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA).CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5′ pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca2+ concentrations in cells over-expressing human VR1; (b) [14C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase.Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC50=3.2 – 3.5 μM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 – 70% of the effect obtained with ionomycin (4 μM). CBD (10 μM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive.(+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD inhibited [14C]-AEA uptake (IC50=10.0 and 7.0 μM); the (−)-enantiomers were slightly less active (IC50=14.0 and 12.5 μM). CBD and (+)-CBD were also active (IC50=22.0 and 17.0 μM).CBD (IC50=27.5 μM), (+)-CBD (IC50=63.5 μM) and (−)-7-hydroxy-CBD (IC50=34 μM), but not the other analogues (IC50>100 μM), weakly inhibited [14C]-AEA hydrolysis.Only the (+)-isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors.These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield

  9. Examination of the Addictive and Behavioral Properties of Fatty Acid-Binding Protein Inhibitor SBFI26

    PubMed Central

    Thanos, Panayotis K.; Clavin, Brendan H.; Hamilton, John; O’Rourke, Joseph R.; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested. PMID:27092087

  10. Examination of the Addictive and Behavioral Properties of Fatty Acid-Binding Protein Inhibitor SBFI26.

    PubMed

    Thanos, Panayotis K; Clavin, Brendan H; Hamilton, John; O'Rourke, Joseph R; Maher, Thomas; Koumas, Christopher; Miao, Erick; Lankop, Jessenia; Elhage, Aya; Haj-Dahmane, Samir; Deutsch, Dale; Kaczocha, Martin

    2016-01-01

    The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested.

  11. A conformational restriction approach to the development of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease.

    PubMed

    Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio; Kogen, Hiroshi

    2003-10-01

    Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).

  12. Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions.

    PubMed

    Byun, Sangwon; Tortorella, Micky D; Malfait, Anne-Marie; Fok, Kam; Frank, Eliot H; Grodzinsky, Alan J

    2010-07-01

    The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

  13. C1-transport in gastric micorsomes. An ATP-dependent influx sensitive to membrane potential and to protein kinase inhibitor.

    PubMed

    Soumarmon, A; Abastado, M; Bonfils, S; Lewin, M J

    1980-12-25

    Uptakes of radioactive C1- or 1- by gastric microsomal vesicles were stimulated 2- to 8-fold by AtP. The sensitivity of those uptakes to a C1- in equilibrium OH- ionophore and to osmotic swelling suggested they were due to transport rather than to binding. The ATP effect was labile, but dithiothreitol and methanol improved its stability. The stimulation of anion transport required magnesium; GTP and UTP were less potent than ATP whereas ADP and AMP had no effect. The apparent Km for ATP was estimated to be 2 X 10(-4) M at 22 degrees C. The rate of the ATP-dependent transport showed saturation-type kinetics, with half-maximal uptake at 10 mM for I- and 15 mM for C1-. Nonradioactive C1-, I-, and SCN- competed with 125I- uptake while SO42- did not. K+ valinomycin increased the ATP-dependent C1- uptake. The thermostable inhibitor of cAMP-dependent protein kinases inhibited the effect of ATP. These results suggest the existence of an anion conductance, permeant to C1-, I-, and SCN- and nonpermeant to SO42-, which could be linked to a cAMP-dependent protein kinase.

  14. Development of new brain imaging agents based upon nocaine-modafinil hybrid monoamine transporter inhibitors.

    PubMed

    Musachio, John L; Hong, Jinsoo; Ichise, Masanori; Seneca, Nicholas; Brown, Amira K; Liow, Jeih-San; Halldin, Christer; Innis, Robert B; Pike, Victor W; He, Rong; Zhou, Jia; Kozikowski, Alan P

    2006-06-15

    11C-labeled (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]ethanol ([11C]5) and (+)-trans-2-[[(3R,4S)-4-(4-chlorophenyl)-1-methylpiperidin-3-yl]methylsulfanyl]-1-(piperidin-1-yl)ethanone ([11C]6) were synthesized and evaluated as new imaging agents for the norepinephrine transporter (NET). [11C]5 and [11C]6 display high affinity for the NET in vitro (Ki = 0.94 and 0.68 nM, respectively) and significant selectivity over the dopamine (DAT) and serotonin transporters (SERT). Because of their high affinity and favorable transporter selectivities we speculated that these ligands might serve as useful PET agents for imaging NET in vivo. Contrary to our expectations, both of these ligands provided brain images that were more typical of those shown by agents binding to the DAT.

  15. Evidence for Bidirectional Endocannabinoid Transport across Cell Membranes*

    PubMed Central

    Chicca, Andrea; Marazzi, Janine; Nicolussi, Simon; Gertsch, Jürg

    2012-01-01

    Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as 2-arachidonoylglycerol (2-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and 2-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nm AEA and 1 μm 2-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and 2-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 μm) and OMDM-2 (5 μm), a functional synergism on cellular AEA and 2-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and 2-AG. We show, for the first time, that UCM707 and OMDM-2 inhibit the bidirectional movement of AEA and 2-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and 2-AG trafficking and metabolism. PMID:22879589

  16. Sodium-Glucose Cotransporter Inhibitors: Effects on Renal and Intestinal Glucose Transport: From Bench to Bedside.

    PubMed

    Mudaliar, Sunder; Polidori, David; Zambrowicz, Brian; Henry, Robert R

    2015-12-01

    Type 2 diabetes is a chronic disease with disabling micro- and macrovascular complications that lead to excessive morbidity and premature mortality. It affects hundreds of millions of people and imposes an undue economic burden on populations across the world. Although insulin resistance and insulin secretory defects play a major role in the pathogenesis of hyperglycemia, several other metabolic defects contribute to the initiation/worsening of the diabetic state. Prominent among these is increased renal glucose reabsorption, which is maladaptive in patients with diabetes. Instead of an increase in renal glucose excretion, which could ameliorate hyperglycemia, there is an increase in renal glucose reabsorption, which helps sustain hyperglycemia in patients with diabetes. The sodium-glucose cotransporter (SGLT) 2 inhibitors are novel antidiabetes agents that inhibit renal glucose reabsorption and promote glucosuria, thereby leading to reductions in plasma glucose concentrations. In this article, we review the long journey from the discovery of the glucosuric agent phlorizin in the bark of the apple tree through the animal and human studies that led to the development of the current generation of SGLT2 inhibitors.

  17. Intraplantar injection of anandamide inhibits mechanically-evoked responses of spinal neurones via activation of CB2 receptors in anaesthetised rats.

    PubMed

    Sokal, D M; Elmes, S J R; Kendall, D A; Chapman, V

    2003-09-01

    Anti-nociceptive effects of the endocannabinoid anandamide are well established. Anandamide has, however, also been shown to activate pro-nociceptive vanilloid 1 (VR1) receptors present on primary afferent nociceptors. The aim of the present study was to determine the effect of intraplantar injection of anandamide on dorsal spinal neuronal responses in control rats and rats with hindpaw carrageenan-induced inflammation. Effects of intraplantar administration of anandamide (50 microg in 50 microl) on peripheral mechanically-evoked responses of spinal neurones were studied in halothane-anaesthetised rats in vivo. Responses of spinal neurones to mechanical punctate stimulation (von Frey filaments, 8-80 g) of the peripheral receptive field were similar in non-inflamed rats and rats with hindpaw carrageenan-induced inflammation. Intraplantar injection of anandamide, but not vehicle, significantly (P<0.05) inhibited innocuous and noxious mechanically-evoked responses of spinal neurones in rats with hindpaw inflammation, but not in non-inflamed rats. Co-administration of the cannabinoid (2) (CB(2)) receptor antagonist, SR144528 (10 microg in 50 microl), but not the cannabinoid (1) (CB(1)) receptor antagonist, SR141716A (10 microg in 50 microl), significantly blocked inhibitory effects of anandamide on peripheral evoked neuronal responses in rats with hindpaw inflammation. This study demonstrates inhibitory effects of exogenous anandamide on mechanically-evoked responses under inflammatory conditions in vivo, which are mediated by peripheral CB(2) receptors.

  18. Antinociceptive effect of intracerebroventricular administration of glycine transporter-2 inhibitor ALX1393 in rat models of inflammatory and neuropathic pain.

    PubMed

    Takahashi, Yoshihiro; Hara, Koji; Haranishi, Yasunori; Terada, Tadanori; Obara, Goh; Sata, Takeyoshi

    2015-03-01

    Glycinergic transmission has an important role in regulating nociception in the spinal cord. The glycine transporter-2 (GlyT2) is localized at presynaptic terminals of glycinergic neurons and eliminates glycine from the synaptic cleft to terminate glycinergic transmission. Systemic and intrathecal administration of GlyT2 inhibitors alleviate various types of pain. Although the GlyT2s and glycine receptors are widely distributed in the central nervous system, little is known about the role of glycinergic transmission in pain perception at supraspinal regions. The present study examined the antinociceptive effect of intracerebroventricular (i.c.v.) administration of the selective GlyT2 inhibitor ALX1393 on inflammatory and neuropathic pain in experimental models. For i.c.v. administration, a guide cannula was implanted into the right lateral ventricle of male Sprague-Dawley rats. Normal rats were used to assess inflammatory nociception using the formalin test and motor function using the rotarod test. Chronic constriction injury (CCI) to the sciatic nerve was induced in the rats. The CCI rats were then used to assess mechanical, cold, and thermal hyperalgesia using the electronic von Frey test, cold plate test, and the plantar test, respectively. ALX1393 (25, 50, and 100 μg) was administered i.c.v. to examine its effects on supraspinal antinociception. Supraspinal ALX1393 in normal rats suppressed the late-phase response in the formalin test but did not affect motor performance. In the CCI rats, ALX1393 inhibited mechanical and cold hyperalgesia in a dose-dependent manner. The antihyperalgesic effects of ALX1393 (100 μg) were reversed completely by i.c.v. pretreatment with a glycine receptor antagonist strychnine (10 μg). These results suggest that GlyT2 contributes to nociceptive transmission at supraspinal level and that the selective GlyT2 inhibitor is a promising candidate for the treatment of inflammatory and neuropathic pain without causing motor dysfunction.

  19. [Role of the sympathetic nervous system in vasovagal syncope and rationale for beta-blockers and norepinephrine transporter inhibitors].

    PubMed

    Márquez, Manlio F; Gómez-Flores, Jorge Rafael; González-Hermosillo, Jesús A; Ruíz-Siller, Teresita de Jesús; Cárdenas, Manuel

    2016-12-29

    Vasovagal or neurocardiogenic syncope is a common clinical situation and, as with other entities associated with orthostatic intolerance, the underlying condition is a dysfunction of the autonomic nervous system. This article reviews various aspects of vasovagal syncope, including its relationship with orthostatic intolerance and the role of the autonomic nervous system in it. A brief history of the problem is given, as well as a description of how the names and associated concepts have evolved. The response of the sympathetic system to orthostatic stress, the physiology of the baroreflex system and the neurohumoral changes that occur with standing are analyzed. Evidence is presented of the involvement of the autonomic nervous system, including studies of heart rate variability, microneurography, cardiac innervation, and molecular genetic studies. Finally, we describe different studies on the use of beta-blockers and norepinephrine transporter inhibitors (sibutramine, reboxetine) and the rationality of their use to prevent this type of syncope.

  20. Renal Safety of Canagliflozin, a Sodium Glucose Co-transporter 2 Inhibitor, in Patients With Type 2 Diabetes Mellitus.

    PubMed

    Desai, Mehul; Yavin, Yshai; Balis, Dainius; Sun, Don; Xie, John; Canovatchel, William; Rosenthal, Norm

    2017-01-12

    The incidence of renal-related adverse events (AEs) with canagliflozin in patients with type 2 diabetes mellitus from a pooled population of patients in 7 active- and placebo-controlled trials (N = 5,598) and in a 104-week study versus glimepiride (N = 1,450) was low and similar in canagliflozin and non-canagliflozin groups. In the study versus glimepiride, canagliflozin was associated with an initial acute decrease in estimated glomerular filtration rate (eGFR) that attenuated over time, while eGFR declined progressively over 104 weeks with glimepiride; the incidence of renal-related AEs with canagliflozin was generally stable over time, while the incidence with glimepiride increased over 104 weeks. In the analysis reported in this manuscript based on postmarketing reports from the US Food and Drug Administration Adverse Event Reporting System, a potential signal was identified for acute kidney injury with all approved sodium glucose co-transporter 2 (SGLT2) inhibitors (ie, canagliflozin, dapagliflozin, empagliflozin). The early onset of acute kidney injury events with SGLT2 inhibitors in postmarketing reports likely reflects the acute changes in eGFR due to the known renal haemodynamic effects of SGLT2 inhibition.

  1. Chicago sky blue 6B, a vesicular glutamate transporters inhibitor, attenuates methamphetamine-induced hyperactivity and behavioral sensitization in mice.

    PubMed

    He, Zongsheng; Yan, Lingdi; Yong, Zheng; Dong, Zhaoqi; Dong, Huajin; Gong, Zehui

    2013-02-15

    Several lines of evidence demonstrate that glutamatergic system plays an important role in drug addiction. The present study was designed to investigate the effects of Chicago sky blue 6B (CSB6B), a vesicular glutamate transporters (VGLUTs) inhibitor, on methamphetamine (METH)-induced behaviors in mice. Mice were induced behavioral sensitization to METH by subcutaneous injection of 1mg/kg METH once daily for 7 days and then challenged with 1mg/kg METH in 14th day. Intracerebroventricular administration of CSB6B (7.5μg) 2.5h prior to METH was to observe its effects on METH -induced behavioral sensitization. Our results showed that the expressions of behavioral sensitization were significantly attenuated by intracerebroventricular administration of CSB6B 2.5h prior to METH either during the development period or before methamphetamine challenge in mice, while CSB6B itself had no effect on locomotor activity. Meanwhile, pretreatment of CSB6B also attenuated hyperactivity caused by a single injection of METH in mice. These results demonstrated that CSB6B, a VGLUTs inhibitor, attenuated acute METH-induced hyperactivity and chronic METH-induced behavioral sensitization, which indicated that VGLUTs were involved in the effect of chronic METH-induced behavioral sensitization and may be a new target against the addiction of METH.

  2. Enhanced Dopamine Release by Dopamine Transport Inhibitors Described by a Restricted Diffusion Model and Fast-Scan Cyclic Voltammetry.

    PubMed

    Hoffman, Alexander F; Spivak, Charles E; Lupica, Carl R

    2016-06-15

    Fast-scan cyclic voltammetry (FSCV) using carbon fiber electrodes is widely used to rapidly monitor changes in dopamine (DA) levels in vitro and in vivo. Current analytical approaches utilize parameters such as peak oxidation current amplitude and decay times to estimate release and uptake processes, respectively. However, peak amplitude changes are often observed with uptake inhibitors, thereby confounding the interpretation of these parameters. To overcome this limitation, we demonstrate that a simple five-parameter, two-compartment model mathematically describes DA signals as a balance of release (r/ke) and uptake (ku), summed with adsorption (kads and kdes) of DA to the carbon electrode surface. Using nonlinear regression, we demonstrate that our model precisely describes measured DA signals obtained in brain slice recordings. The parameters extracted from these curves were then validated using pharmacological manipulations that selectively alter vesicular release or DA transporter (DAT)-mediated uptake. Manipulation of DA release through altering the Ca(2+)/Mg(2+) ratio or adding tetrodotoxin reduced the release parameter with no effect on the uptake parameter. DAT inhibitors methylenedioxypyrovalerone, cocaine, and nomifensine significantly reduced uptake and increased vesicular DA release. In contrast, a low concentration of amphetamine reduced uptake but had no effect on DA release. Finally, the kappa opioid receptor agonist U50,488 significantly reduced vesicular DA release but had no effect on uptake. Together, these data demonstrate a novel analytical approach to distinguish the effects of manipulations on DA release or uptake that can be used to interpret FSCV data.

  3. Effect of periglandular ionic composition and transport inhibitors on rhesus monkey eccrine sweat gland function in vitro.

    PubMed

    Sato, F; Sato, K

    1987-12-01

    1. The effects of peritubular ions and transport inhibitors were studied on methacholine (MCH)-induced sweat secretion by the isolated, cannulated monkey palm sweat glands in vitro and on the transepithelial and basolateral membrane potential (p.d.). 2. Sweat secretory rate was a curvilinear function of peritubular Na+ and Cl- concentration. Among the anion substitutes only Br- was able to totally substitute for Cl-. Presence of HCO3- or H2PO4- in the bath was not essential. 3. Both bumetanide and furosemide inhibited sweat secretion in a dose-dependent manner with the median effective concentration (EC50) of 3 X 10(-6) and 3 X 10(-5) M, respectively. 4. Bumetanide (10(-4) M) had no significant effect on basolateral membrane p.d. but nearly abolished the transepithelial p.d. 5. Hydrochlorothiazide (HCTZ, 3 X 10(-4) M) inhibited sweat secretion by only 35%. Inhibitors of ion exchangers amiloride (10(-4) M) and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 10(-4) M) lowered sweat secretion by less than 20%. 6. Removal of peritubular K+ as well as addition of 5 mM-Ba2+ also inhibited sweat rate. 5 mM-Ba2+ abolished the transepithelial p.d. and depolarized the basolateral p.d. by 26 mV, although the effects of Ba2+ on sweating and the transepithelial p.d. were only transient. 7. The data raise a possibility that either the NaCl or Na+-K+-2Cl- co-transport system or both may be involved in MCH-induced sweat secretion, whereas the role of parallel ion exchangers, if any, may be rather minor.

  4. Dapoxetine, a novel selective serotonin transport inhibitor for the treatment of premature ejaculation

    PubMed Central

    Kendirci, Muammer; Salem, Emad; Hellstrom, Wayne JG

    2007-01-01

    Premature ejaculation (PE) is the most common male sexual disorder, estimated to affect up to 30% of men. Over the past one or two decades, clinical investigators have participated in an increasing number of studies that are helping in our understanding of PE, which will undoubtedly facilitate future treatments. Apart from a number of behavioral approaches, the treatment of PE consists of primarily off-label use of oral selective serotonin reuptake inhibitors (SSRIs) via either on-demand or daily delivery. However, various undesirable side-effects of these medications have led researchers to search for and develop new therapeutic approaches for PE. Dapoxetine is a short-acting SSRI developed specifically for the treatment of PE. Early trials with dapoxetine have documented successful outcomes without serious short- or long-term side-effects. This review addresses the definition, classification, diagnosis, physiology, and neurobiopathology of PE, and evaluates therapeutic strategies with novel treatments for PE. PMID:18360636

  5. Dapoxetine, a novel selective serotonin transport inhibitor for the treatment of premature ejaculation.

    PubMed

    Kendirci, Muammer; Salem, Emad; Hellstrom, Wayne Jg

    2007-06-01

    Premature ejaculation (PE) is the most common male sexual disorder, estimated to affect up to 30% of men. Over the past one or two decades, clinical investigators have participated in an increasing number of studies that are helping in our understanding of PE, which will undoubtedly facilitate future treatments. Apart from a number of behavioral approaches, the treatment of PE consists of primarily off-label use of oral selective serotonin reuptake inhibitors (SSRIs) via either on-demand or daily delivery. However, various undesirable side-effects of these medications have led researchers to search for and develop new therapeutic approaches for PE. Dapoxetine is a short-acting SSRI developed specifically for the treatment of PE. Early trials with dapoxetine have documented successful outcomes without serious short- or long-term side-effects. This review addresses the definition, classification, diagnosis, physiology, and neurobiopathology of PE, and evaluates therapeutic strategies with novel treatments for PE.

  6. N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) is a selective persistent inhibitor of glycine transport

    PubMed Central

    Aubrey, Karin R; Vandenberg, Robert J

    2001-01-01

    The regulation of glycine concentrations within excitatory synapses is poorly understood and it has been proposed that the GLYT1 subtypes of glycine transporters play a critical role in determining resting concentrations of glycine. Selective GLYT1 inhibitors may provide pharmacological tools to probe the dynamics of synaptic glycine concentrations, which may influence the activation properties of NMDA receptor activity.We have characterized the selectivity and mechanism of action of the glycine transport inhibitor N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS). The glycine transporters, GLYT1a, b and c and GLYT2a were expressed in Xenopus laevis oocytes and two electrode voltage clamp techniques and radiolabelled 3H-glycine flux measurements were used to characterize the effects of NFPS on glycine transport.NFPS inhibits glycine transport by the GLYT1a, b and c subtypes of glycine transporters, but has no effect on the GLYT2a subtype of transporter. We show that NFPS does not attain its specificity via an interaction with the Na+, Cl− or glycine site, nor does it act at an intracellular site. NFPS inhibition of glycine transport is time and concentration dependent and inhibition of transport by NFPS persists after washout of NFPS from the bath solution, which suggests that inhibition by NFPS is long lasting. PMID:11724748

  7. Glycine Transporter Type 2 (GlyT2) Inhibitor Ameliorates Bladder Overactivity and Nociceptive Behavior in Rats

    PubMed Central

    Yoshikawa, Satoru; Oguchi, Tomohiko; Funahashi, Yasuhito; de Groat, William C.; Yoshimura, Naoki

    2012-01-01

    Background Glycine is a major inhibitory neurotransmitter in the spinal cord, the concentration of which is regulated by two types of glycine transporters (GlyTs): GlyT1 and GlyT2. We hypothesized that the inhibition of GlyTs could ameliorate bladder overactivity and/or pain sensation in the lower urinary tract. Objective Investigate the effects of GlyT inhibitors on bladder overactivity and pain behavior in rats. Design, setting, and participants Cystometry was performed under urethane anesthesia in cyclophosphamide (CYP)–treated rats. In behavioral studies using conscious rats, nociceptive responses were induced by intravesical administration of resiniferatoxin (3 µM). Selective GlyT1 or GlyT2 inhibitors were administered intrathecally to evaluate their effects. Measurements Cystometric parameters, nociceptive behaviors (licking and freezing), and messenger RNA (mRNA) levels of GlyTs and glycine receptor (GlyR) subunits in the dorsal spinal cord (L6–S1) were measured. Results and limitations During cystometry in CYP-treated rats, significant increases in intercontraction interval and micturition pressure threshold were elicited by ALX-1393, a selective GlyT2 inhibitor, but not by sarcosine, a GlyT1 inhibitor. These effects were completely reversed by strychnine, a GlyR antagonist. ALX-1393 also significantly suppressed nociceptive behaviors in a dose-dependent manner. In sham rats, GlyT2 mRNA was expressed at a much higher level (23-fold) in the dorsal spinal cord than GlyT1 mRNA. In CYP-treated rats, mRNA levels of GlyT2 and the GlyR α1 and β subunits were significantly reduced. Conclusions These results indicate that GlyT2 plays a major role in the clearance of extracellular glycine in the spinal cord and that GlyT2 inhibition leads to amelioration of CYP-induced bladder overactivity and pain behavior. GlyT2 may be a novel therapeutic target for the treatment of overactive bladder and/or bladder hypersensitive disorders such as bladder pain syndrome

  8. The Effect of Anandamide on Uterine Nitric Oxide Synthase Activity Depends on the Presence of the Blastocyst

    PubMed Central

    Sordelli, Micaela S.; Beltrame, Jimena S.; Burdet, Juliana; Zotta, Elsa; Pardo, Romina; Cella, Maximiliano; Franchi, Ana M.; Ribeiro, Maria Laura

    2011-01-01

    Nitric oxide production, catalyzed by nitric oxide synthase (NOS), should be strictly regulated to allow embryo implantation. Thus, our first aim was to study NOS activity during peri-implantation in the rat uterus. Day 6 inter-implantation sites showed lower NOS activity (0.19±0.01 pmoles L-citrulline mg prot−1 h−1) compared to days 4 (0.34±0.03) and 5 (0.35±0.02) of pregnancy and to day 6 implantation sites (0.33±0.01). This regulation was not observed in pseudopregnancy. Both dormant and active blastocysts maintained NOS activity at similar levels. Anandamide (AEA), an endocannabinoid, binds to cannabinoid receptors type 1 (CB1) and type 2 (CB2), and high concentrations are toxic for implantation and embryo development. Previously, we observed that AEA synthesis presents an inverted pattern compared to NOS activity described here. We adopted a pharmacological approach using AEA, URB-597 (a selective inhibitor of fatty acid amide hydrolase, the enzyme that degrades AEA) and receptor selective antagonists to investigate the effect of AEA on uterine NOS activity in vitro in rat models of implantation. While AEA (0.70±0.02 vs 0.40±0.04) and URB-597 (1.08±0.09 vs 0.83±0.06) inhibited NOS activity in the absence of a blastocyst (pseudopregnancy) through CB2 receptors, AEA did not modulate NOS on day 5 pregnant uterus. Once implantation begins, URB-597 decreased NOS activity on day 6 implantation sites via CB1 receptors (0.25±0.04 vs 0.40±0.05). While a CB1 antagonist augmented NOS activity on day 6 inter-implantation sites (0.17±0.02 vs 0.27±0.02), a CB2 antagonist decreased it (0.17±0.02 vs 0.12±0.01). Finally, we described the expression and localization of cannabinoid receptors during implantation. In conclusion, AEA levels close to and at implantation sites seems to modulate NOS activity and thus nitric oxide production, fundamental for implantation, via cannabinoid receptors. This modulation depends on the presence of the blastocyst. These data

  9. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  10. Microwave accelerated synthesis of isoxazole hydrazide inhibitors of the system xc− transporter: Initial homology model

    PubMed Central

    Matti, Afnan A.; Mirzaei, Joseph; Rudolph, John; Smith, Stephen A.; Newell, Jayme L.; Patel, Sarjubhai A.; Braden, Michael R.; Bridges, Richard J.; Natale, Nicholas R.

    2013-01-01

    Microwave accelerated reaction system (MARS) technology provided a good method to obtain selective and open isoxazole ligands that bind to and inhibit the Sxc− antiporter. The MARS provided numerous advantages, including: shorter time, better yield and higher purity of the product. Of the newly synthesized series of isoxazoles the salicyl hydrazide 6 exhibited the highest level of inhibitory activity in the transport assay. A homology model has been developed to summarize the SAR results to date, and provide a working hypothesis for future studies. PMID:24042010

  11. Microwave accelerated synthesis of isoxazole hydrazide inhibitors of the system xc- transporter: Initial homology model.

    PubMed

    Matti, Afnan A; Mirzaei, Joseph; Rudolph, John; Smith, Stephen A; Newell, Jayme L; Patel, Sarjubhai A; Braden, Michael R; Bridges, Richard J; Natale, Nicholas R

    2013-11-01

    Microwave accelerated reaction system (MARS) technology provided a good method to obtain selective and open isoxazole ligands that bind to and inhibit the Sxc- antiporter. The MARS provided numerous advantages, including: shorter time, better yield and higher purity of the product. Of the newly synthesized series of isoxazoles the salicyl hydrazide 6 exhibited the highest level of inhibitory activity in the transport assay. A homology model has been developed to summarize the SAR results to date, and provide a working hypothesis for future studies.

  12. Anandamide Levels Fluctuate in the Bovine Oviduct during the Oestrous Cycle

    PubMed Central

    Gervasi, Maria Gracia; Marczylo, Timothy H.; Lam, Patricia M.; Rana, Shashi; Franchi, Ana M.; Konje, Justin C.; Perez-Martinez, Silvina

    2013-01-01

    Mammalian oviduct acts as a reservoir for spermatozoa and provides an environment in which they may compete for the opportunity to fertilize the oocyte. Whilst in the oviduct spermatozoa undergo capacitation essential for fertilization. Sperm-oviduct interaction is essential for sperm capacitation and is a tightly regulated process influenced by the local microenvironment. Previously we reported that the endocannabinoid anandamide (AEA) regulates sperm release from epithelial oviductal cells by promoting sperm capacitation. The aims of this work were to measure the AEA content and to characterize the main AEA metabolic pathway in the bovine oviduct and determine how these change through the oestrous cycle. In this study, the levels of AEA and two other N-acylethanolamines, N-oleoylethanolamine and N-palmitoylethanolamine, were measured in bovine oviduct collected during different stages of oestrous cycle by ultra high performance liquid chromatography tandem mass spectrometry. Results indicated that intracellular oviductal epithelial levels of all three N-acylethanolamines fluctuate during oestrous cycle. Anandamide from oviductal fluid also varied during oestrous cycle, with the highest values detected during the periovulatory period. Endocannabinoid levels from ipsilateral oviduct to ovulation were higher than those detected in the contralateral one, suggesting that levels of oviductal AEA may be regulated by ovarian hormones. The expression and localization of N-acylethanolamines metabolizing enzymes in bovine oviduct were also determined by RT-PCR, Western blot, and immunohistochemistry but no change was found during the oestrous cycle. Furthermore, nanomolar levels of AEA were detected in follicular fluids, suggesting that during ovulation the mature follicle may contribute to oviductal AEA levels to create an endocannabinoid gradient conducive to the regulation of sperm function for successful fertilization. PMID:23977311

  13. Chronic treatment with krill powder reduces plasma triglyceride and anandamide levels in mildly obese men.

    PubMed

    Berge, Kjetil; Piscitelli, Fabiana; Hoem, Nils; Silvestri, Cristoforo; Meyer, Ingo; Banni, Sebastiano; Di Marzo, Vincenzo

    2013-05-27

    We have previously shown that treatment of Zucker rats and mice with diet-induced obesity with dietary docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in the form of krill oil reduces peripheral levels of endocannabinoids, ectopic fat formation and hyperglycemia. We reported that such treatment reduces plasma endocannabinoid levels also in overweight and obese human individuals, in whom high triglycerides may correlate with high circulating endocannabinoid levels. In this study, we report the effects of krill powder, which contains proteins (34%) in addition to krill oil (61.8%), on these two parameters. We submitted 11 obese men (average BMI of 32.3 kg/m², age of 42.6 years and plasma triglycerides of 192.5 ± 96.3 mg/dl) to a 24 week dietary supplementation with krill powder (4 g/day per os) and measured anthropometric and metabolic parameters, as well as blood endocannabinoid (anandamide and 2-arachidonoylglycerol) and esterified DHA and EPA levels. Six subjects were included as control subjects and not given any supplements. The treatment produced, after 12 and 24 weeks, a significant increase in DHA and EPA in total plasma, a 59 and 84% decrease in anandamide plasma levels, and a 22.5 and 20.6% decrease in triglyceride levels, respectively. There was also a significant decrease in waist/hip ratio and visceral fat/skeletal muscle mass ratio at 24 weeks, but no change in body weight. These data confirm that dietary krill powder reduces peripheral endocannabinoid overactivity in obese subjects, and might ameliorate some parameters of the metabolic syndrome.

  14. The discovery of novel auxin transport inhibitors by molecular modeling and three-dimensional pattern analysis

    NASA Astrophysics Data System (ADS)

    Bures, Mark G.; Black-Schaefer, Candace; Gardner, Gary

    1991-08-01

    Molecular modeling techniques and three-dimensional (3D) pattern analysis have been used to investigate the chemical and steric properties of compounds that inhibit transport of the plant hormone auxin. These compounds bind to a specific site on the plant plasma membrane characterized by its affinity for the herbicide N-1-naphthylphthalamic acid (NPA). A 3D model was derived from critical features of a set of ligands for the NPA receptor, a suggested binding conformation is proposed, and implications for the topographical features of the NPA receptor are discussed. This model, along with 3D structural analysis techniques, was then used to search the Abbott corporate database of chemical structures. Of the 467 compounds that satisfied the criteria of the model, 77 representative molecules were evaluated for their ability to compete for the binding of [3H]NPA to corn microsomal membranes. Nineteen showed activity that ranged from 16 to 85% of the maximum NPA binding. Four of the most active of these, representing chemical classes not included in the original compound set, were also found to inhibit polar auxin transport through corn coleoptile sections. Thus, this study demonstrates that 3D analysis techniques can identify active, novel ligands for biochemical target sites with concomitant physiological activity.

  15. Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor

    NASA Astrophysics Data System (ADS)

    Kong, Lingbing; Harrington, Leon; Li, Qiuhong; Cheley, Stephen; Davis, Benjamin G.; Bayley, Hagan

    2013-08-01

    Capsular polysaccharides form the outermost protective layer around many Gram-negative bacteria. Antibiotics aimed directly at weakening this layer are not yet available. In pathogenic Escherichia coli E69, a protein, Wza, forms a pore in the outer membrane that transports K30 capsular polysaccharide from its site of synthesis to the outside of the cell. This therefore represents a prospective antibiotic target. Here we test a variety of grommet-like mimics of K30 capsular polysaccharide on wild-type Wza and on mutant open forms of the pore by electrical recording in planar lipid bilayers. The most effective glycomimetic was the unnatural cyclic octasaccharide octakis(6-deoxy-6-amino)cyclomaltooctaose (am8γCD), which blocks the α-helix barrel of Wza, a site that is directly accessible from the external medium. This glycomimetic inhibited K30 polysaccharide transport in live E. coli E69. With the protective outer membrane disrupted, the bacteria can be recognized and killed by the human immune system.

  16. Breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport.

    PubMed

    Horie, Kenta; Oba, Takahiro; Motomura, Saori; Isogai, Atsuko; Yoshimura, Takashi; Tsuge, Keisuke; Koganemaru, Kazuyoshi; Kobayashi, Genta; Kitagaki, Hiroshi

    2010-01-01

    Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl alpha-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.

  17. Bicarbonate transport inhibitor SITS modulates pH homeostasis triggering apoptosis of Dalton's lymphoma: implication of novel molecular mechanisms.

    PubMed

    Kant, Shiva; Kumar, Ajay; Singh, Sukh Mahendra

    2014-12-01

    Bicarbonate transporter (BCT) plays a crucial role in maintaining pH homeostasis of tumor cells by import of HCO3(-). This helps the tumor cells in manifesting extracellular tumor acidosis, accompanied by a relative intracellular alkalinization, which in turn promotes tumor progression. Therefore, blocking BCT-mediated HCO3(-) transport is envisaged as a promising anticancer therapeutic approach. Thus, using a murine model of a T cell lymphoma, designated as Dalton's lymphoma (DL), in the present in vitro investigation the antitumor consequences of blocking BCT function by its inhibitor 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) were explored. Treatment of DL cells with SITS resulted in an increase in the extracellular pH, associated with a decline in DL cell survival and augmented induction of apoptosis. BCT inhibition also elevated the expression of cytochrome c, caspase-9, caspase-3, Bax, reactive oxygen species, and nitric oxide along with inhibition of HSP-70 and Bcl2, which regulate tumor cell survival and apoptosis. SITS-treated DL cells displayed upregulated production of IFN-γ and IL-6 along with a decline of IL-10. Treatment of DL cells with SITS also inhibited the expression of fatty acid synthase, which is crucial for membrane biogenesis in neoplastic cells. The expression of lactate transporter MCT-1 and multidrug resistance regulating protein MRP-1 got inhibited along with hampered uptake of glucose and lactate production in SITS-treated DL cells. Thus, the declined tumor cell survival following inhibition of BCT could be the consequence of interplay of several inter-connected regulatory molecular events. The outcome of this study indicates the potential of BCT inhibition as a novel therapeutic approach for treatment of hematological malignancies.

  18. Na(+)/H(+) exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous β-globin knockout thalassemic mice.

    PubMed

    Charoenphandhu, Narattaphol; Kraidith, Kamonshanok; Lertsuwan, Kornkamon; Sripong, Chanakarn; Suntornsaratoon, Panan; Svasti, Saovaros; Krishnamra, Nateetip; Wongdee, Kannikar

    2017-03-01

    Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous β-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe(2+) and H(+) cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na(+)/H(+) exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na(+)/Ca(2+) exchanger (NCX1) and plasma membrane Ca(2+)-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in β-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

  19. The dynein inhibitor Ciliobrevin D inhibits the bidirectional transport of organelles along sensory axons and impairs NGF-mediated regulation of growth cones and axon branches.

    PubMed

    Sainath, Rajiv; Gallo, Gianluca

    2015-07-01

    The axonal transport of organelles is critical for the development, maintenance, and survival of neurons, and its dysfunction has been implicated in several neurodegenerative diseases. Retrograde axon transport is mediated by the motor protein dynein. In this study, using embryonic chicken dorsal root ganglion neurons, we investigate the effects of Ciliobrevin D, a pharmacological dynein inhibitor, on the transport of axonal organelles, axon extension, nerve growth factor (NGF)-induced branching and growth cone expansion, and axon thinning in response to actin filament depolymerization. Live imaging of mitochondria, lysosomes, and Golgi-derived vesicles in axons revealed that both the retrograde and anterograde transport of these organelles was inhibited by treatment with Ciliobrevin D. Treatment with Ciliobrevin D reversibly inhibits axon extension and transport, with effects detectable within the first 20 min of treatment. NGF induces growth cone expansion, axonal filopodia formation and branching. Ciliobrevin D prevented NGF-induced formation of axonal filopodia and branching but not growth cone expansion. Finally, we report that the retrograde reorganization of the axonal cytoplasm which occurs on actin filament depolymerization is inhibited by treatment with Ciliobrevin D, indicating a role for microtubule based transport in this process, as well as Ciliobrevin D accelerating Wallerian degeneration. This study identifies Ciliobrevin D as an inhibitor of the bidirectional transport of multiple axonal organelles, indicating this drug may be a valuable tool for both the study of dynein function and a first pass analysis of the role of axonal transport.

  20. Anandamide inhibits Theiler's virus induced VCAM-1 in brain endothelial cells and reduces leukocyte transmigration in a model of blood brain barrier by activation of CB1 receptors

    PubMed Central

    2011-01-01

    Background VCAM-1 represents one of the most important adhesion molecule involved in the transmigration of blood leukocytes across the blood-brain barrier (BBB) that is an essential step in the pathogenesis of MS. Several evidences have suggested the potential therapeutic value of cannabinoids (CBs) in the treatment of MS and their experimental models. However, the effects of endocannabinoids on VCAM-1 regulation are poorly understood. In the present study we investigated the effects of anandamide (AEA) in the regulation of VCAM-1 expression induced by Theiler's virus (TMEV) infection of brain endothelial cells using in vitro and in vivo approaches. Methods i) in vitro: VCAM-1 was measured by ELISA in supernatants of brain endothelial cells infected with TMEV and subjected to AEA and/or cannabinoid receptors antagonist treatment. To evaluate the functional effect of VCAM-1 modulation we developed a blood brain barrier model based on a system of astrocytes and brain endothelial cells co-culture. ii) in vivo: CB1 receptor deficient mice (Cnr1-/-) infected with TMEV were treated with the AEA uptake inhibitor UCM-707 for three days. VCAM-1 expression and microglial reactivity were evaluated by immunohistochemistry. Results Anandamide-induced inhibition of VCAM-1 expression in brain endothelial cell cultures was mediated by activation of CB1 receptors. The study of leukocyte transmigration confirmed the functional relevance of VCAM-1 inhibition by AEA. In vivo approaches also showed that the inhibition of AEA uptake reduced the expression of brain VCAM-1 in response to TMEV infection. Although a decreased expression of VCAM-1 by UCM-707 was observed in both, wild type and CB1 receptor deficient mice (Cnr1-/-), the magnitude of VCAM-1 inhibition was significantly higher in the wild type mice. Interestingly, Cnr1-/- mice showed enhanced microglial reactivity and VCAM-1 expression following TMEV infection, indicating that the lack of CB1 receptor exacerbated

  1. Rapid chemoenzymatic route to glutamate transporter inhibitor l-TFB-TBOA and related amino acids.

    PubMed

    Fu, Haigen; Younes, Sabry H H; Saifuddin, Mohammad; Tepper, Pieter G; Zhang, Jielin; Keller, Erik; Heeres, André; Szymanski, Wiktor; Poelarends, Gerrit J

    2017-03-21

    The complex amino acid (l-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (l-TFB-TBOA) and its derivatives are privileged compounds for studying the roles of excitatory amino acid transporters (EAATs) in regulation of glutamatergic neurotransmission, animal behavior, and in the pathogenesis of neurological diseases. The wide-spread use of l-TFB-TBOA stems from its high potency of EAAT inhibition and the lack of off-target binding to glutamate receptors. However, one of the main challenges in the evaluation of l-TFB-TBOA and its derivatives is the laborious synthesis of these compounds in stereoisomerically pure form. Here, we report an efficient and step-economic chemoenzymatic route that gives access to enantio- and diastereopure l-TFB-TBOA and its derivatives at multigram scale.

  2. Incretins and selective renal sodium-glucose co-transporter 2 inhibitors in hypertension and coronary heart disease

    PubMed Central

    Sanchez, Ramiro A; Sanabria, Hugo; de los Santos, Cecilia; Ramirez, Agustin J

    2015-01-01

    Hyperglycemia is associated with an increased risk of cardiovascular disease, and the consequences of intensive therapy may depend on the mechanism of the anti-diabetic agent(s) used to achieve a tight control. In animal models, stable analogues of glucagon-like peptide-1 (GLP-1) were able to reduce body weight and blood pressure and also had favorable effects on ischemia following coronary reperfusion. In a similar way, dipeptidyl peptidase IV (DPP-IV) showed to have favorable effects in animal models of ischemia/reperfusion. This could be due to the fact that DPP-IV inhibitors were able to prevent the breakdown of GLP-1 and glucose-dependent insulinotropic polypeptide, but they also decreased the degradation of several vasoactive peptides. Preclinical data for GLP-1, its derivatives and inhibitors of the DPP-IV enzyme degradation suggests that these agents may be able to, besides controlling glycaemia, induce cardio-protective and vasodilator effects. Notwithstanding the many favorable cardiovascular effects of GLP-1/incretins reported in different studies, many questions remain unanswered due the limited number of studies in human beings that aim to examine the effects of GLP-1 on cardiovascular endpoints. For this reason, long-term trials searching for positive cardiovascular effects are now in process, such as the CAROLINA and CARMELINA trials, which are supported by small pilot studies performed in humans (and many more animal studies) with incretin-based therapies. On the other hand, selective renal sodium-glucose co-transporter 2 inhibitors were also evaluated in the prevention of cardiovascular outcomes in type 2 diabetes. However, it is quite early to draw conclusions, since data on cardiovascular outcomes and cardiovascular death are limited and long-term studies are still ongoing. In this review, we will analyze the mechanisms underlying the cardiovascular effects of incretins and, at the same time, we will present a critical position about the real

  3. Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

    PubMed Central

    Srinivas, Ranga V.; Middlemas, David; Flynn, Pat; Fridland, Arnold

    1998-01-01

    The human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs)—saquinavir, ritonavir, nelfinavir, and indinavir—interact with the ABC-type multidrug transporter proteins MDR1 and MRP1 in CEM T-lymphocytic cell lines. Calcein fluorescence was significantly enhanced in MDR1+ CEM/VBL100 and MRP1+ CEM/VM-1-5 cells incubated in the presence of various HIV PIs and calcein acetoxymethyl ester. HIV PIs also enhanced the cytotoxic activity of doxorubicin, a known substrate for MDR1 and MRP1, in both VBL100 and VM-1-5 CEM lines. Saquinavir, ritonavir, and nelfinavir enhanced doxorubicin toxicity in CEM/VBL100 cells by approximately three- to sevenfold. Saquinavir and ritonavir also enhanced doxorubicin toxicity in CEM/VM-1-5 cells. HIV-1 replication was effectively inhibited by the various PIs in all of the cell lines, and the 90% inhibitory concentration for a given compound was comparable between the different cell types. Therefore, overexpression of MDR1 or MRP1 by T lymphocytes is not likely to limit the antiviral efficacy of HIV PI therapy. PMID:9835508

  4. Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytes.

    PubMed

    Hartman, J Craig; Brouwer, Kenneth; Mandagere, Arun; Melvin, Lawrence; Gorczynski, Richard

    2010-06-01

    To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [(3)H]estradiol-17beta-D-glucuronide (for OATP), and [2-D-penicillamine, 5-D-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 micromol*L-1 for inhibition and at 2 micromol*L-1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%-100% of control), whereas bosentan was most affected (32%-58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

  5. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters

    PubMed Central

    Nancolas, Bethany; Guo, Lili; Zhou, Rong; Nath, Kavindra; Nelson, David S.; Leeper, Dennis B.; Blair, Ian A.; Glickson, Jerry D.; Halestrap, Andrew P.

    2016-01-01

    Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitising tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Here we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill Coefficient values of 36–40 μM and 1.65–1.85. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50 ~7 μM) than other substrates including glutamate (IC50 ~20 μM). In isolated DB-1 melanoma cells 1–10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output while increasing intracellular [L-lactate] > five-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND on L-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate. PMID:26831515

  6. Simulation of Swanson's literature-based discovery: anandamide treatment inhibits growth of gastric cancer cells in vitro and in silico.

    PubMed

    Dong, Weiwei; Liu, Yixuan; Zhu, Weijie; Mou, Quan; Wang, Jinliang; Hu, Yi

    2014-01-01

    Swanson's literature-based discovery focus on resurrecting previously published but neglected knowledge. In this study, we propose a two-step model of the discovery process and generate a hypothesis between anandamide and gastric cancer. Further, the potential relationship was confirmed by follow-up experimentation. The anandamide treatment resulted in cell cycle redistribution of gastric cancer cells. Most importantly, the variation of cell cycle was mediated by some genes from the B-terms of the closed discovery, indicating the potential role of the B-terms. Swanson's literature-based discovery not only collates data for possible interactions, but also provides the potential to observe the larger background behind these direct links and is an invaluable discovery tool for investigators.

  7. Synthesis and preliminary pharmacological evaluation of novel derivatives of L-beta-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT3.

    PubMed

    Mavencamp, Terri L; Rhoderick, Joseph F; Bridges, Richard J; Esslinger, C Sean

    2008-08-15

    A series of beta-benzylaspartate derivatives were prepared from N-trityl-L-aspartate dimethyl ester and evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure-activity studies suggests that the position occupied by the aromatic ring of beta-benzylaspartate within the binding site of EAAT3 may be different from that occupied by comparable groups in previously identified inhibitors, such as L-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-position of the aromatic ring of beta-benzylaspartate can increase the potency with which the analogues inhibit EAAT3.

  8. Full Inhibition of Spinal FAAH Leads to TRPV1-Mediated Analgesic Effects in Neuropathic Rats and Possible Lipoxygenase-Mediated Remodeling of Anandamide Metabolism

    PubMed Central

    Starowicz, Katarzyna; Makuch, Wioletta; Korostynski, Michal; Malek, Natalia; Slezak, Michal; Zychowska, Magdalena; Petrosino, Stefania; De Petrocellis, Luciano; Cristino, Luigia; Przewlocka, Barbara; Di Marzo, Vincenzo

    2013-01-01

    Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats. PMID:23573230

  9. Full inhibition of spinal FAAH leads to TRPV1-mediated analgesic effects in neuropathic rats and possible lipoxygenase-mediated remodeling of anandamide metabolism.

    PubMed

    Starowicz, Katarzyna; Makuch, Wioletta; Korostynski, Michal; Malek, Natalia; Slezak, Michal; Zychowska, Magdalena; Petrosino, Stefania; De Petrocellis, Luciano; Cristino, Luigia; Przewlocka, Barbara; Di Marzo, Vincenzo

    2013-01-01

    Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.

  10. Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid

    PubMed Central

    Servadio, M; Melancia, F; Manduca, A; di Masi, A; Schiavi, S; Cartocci, V; Pallottini, V; Campolongo, P; Ascenzi, P; Trezza, V

    2016-01-01

    Autism spectrum disorders (ASD) are characterized by altered sociability, compromised communication and stereotyped/repetitive behaviors, for which no specific treatments are currently available. Prenatal exposure to valproic acid (VPA) is a known, although still underestimated, environmental risk factor for ASD. Altered endocannabinoid activity has been observed in autistic patients, and endocannabinoids are known to modulate behavioral traits that are typically affected in ASD. On this basis, we tested the hypothesis that changes in the endocannabinoid tone contribute to the altered phenotype induced by prenatal VPA exposure in rats, with focus on behavioral features that resemble the core and associated symptoms of ASD. In the course of development, VPA-exposed rats showed early deficits in social communication and discrimination, compromised sociability and social play behavior, stereotypies and increased anxiety, thus providing preclinical proof of the long-lasting deleterious effects induced by prenatal VPA exposure. At the neurochemical level, VPA-exposed rats displayed altered phosphorylation of CB1 cannabinoid receptors in different brain areas, associated with changes in anandamide metabolism from infancy to adulthood. Interestingly, enhancing anandamide signaling through inhibition of its degradation rescued the behavioral deficits displayed by VPA-exposed rats at infancy, adolescence and adulthood. This study therefore shows that abnormalities in anandamide activity may underlie the deleterious impact of environmental risk factors on ASD-relevant behaviors and that the endocannabinoid system may represent a therapeutic target for the core and associated symptoms displayed by autistic patients. PMID:27676443

  11. Small compound inhibitors of basal glucose transport inhibit cell proliferation and induce apoptosis in cancer cells via glucose-deprivation-like mechanisms.

    PubMed

    Liu, Yi; Zhang, Weihe; Cao, Yanyan; Liu, Yan; Bergmeier, Stephen; Chen, Xiaozhuo

    2010-12-08

    Cancer cells depend heavily on glucose as both energy and biosynthesis sources and are found to upregulate glucose transport and switch their main energy supply pathway from oxidative phosphorylation to glycolysis. These molecular and metabolic changes also provide targets for cancer treatment. Here we report that novel small molecules inhibited basal glucose transport and cell proliferation, and induced apoptosis in lung and breast cancer cells without affecting much their normal cell counterparts. Cancer cells survived the compound treatment lost their capability to proliferate. Mechanistic study indicates that the cancer cell inhibition by the test compounds has a component of apoptosis and the induced apoptosis was p53-independent and caspase 3-dependent, similar to those resulted from glucose deprivation. Compound treatment also led to cell cycle arrest in G1/S phase. The inhibition of cancer cell growth was partially relieved when additional glucose was supplied to cells, suggesting that the inhibition was due to, at least in part, the inhibition of basal glucose transport. When used in combination, the test compounds demonstrated synergistic effects with anticancer drugs cisplatin or paclitaxel in inhibition of cancer cell growth. All these results suggest that these glucose transport inhibitors mimic glucose deprivation and work through inhibiting basal glucose transport. These inhibitors have the potential to complement and replace traditional glucose deprivation, which cannot be used in animals, as new tools to study the effects of glucose transport and metabolism on cancer and normal cells.

  12. Pharmacodynamics, efficacy and safety of sodium-glucose co-transporter type 2 (SGLT2) inhibitors for the treatment of type 2 diabetes mellitus.

    PubMed

    Scheen, André J

    2015-01-01

    Inhibitors of sodium-glucose co-transporter type 2 (SGLT2) are proposed as a novel approach for the management of type 2 diabetes mellitus (T2DM). Several compounds are already available in many countries (dapagliflozin, canagliflozin, empagliflozin and ipragliflozin) and some others are in a late phase of development. The available SGLT2 inhibitors share similar pharmacokinetic characteristics, with a rapid oral absorption, a long elimination half-life allowing once-daily administration, an extensive hepatic metabolism mainly via glucuronidation to inactive metabolites, the absence of clinically relevant drug-drug interactions and a low renal elimination as parent drug. SGLT2 co-transporters are responsible for reabsorption of most (90 %) of the glucose filtered by the kidneys. The pharmacological inhibition of SGLT2 co-transporters reduces hyperglycaemia by decreasing renal glucose threshold and thereby increasing urinary glucose excretion. The amount of glucose excreted in the urine depends on both the level of hyperglycaemia and the glomerular filtration rate. Results of numerous placebo-controlled randomised clinical trials of 12-104 weeks duration have shown significant reductions in glycated haemoglobin (HbA1c), resulting in a significant increase in the proportion of patients reaching HbA1c targets, and a significant lowering of fasting plasma glucose when SGLT2 inhibitors were administered as monotherapy or in addition to other glucose-lowering therapies including insulin in patients with T2DM. In head-to-head trials of up to 2 years, SGLT2 inhibitors exerted similar glucose-lowering activity to metformin, sulphonylureas or sitagliptin. The durability of the glucose-lowering effect of SGLT2 inhibitors appears to be better; however, this remains to be more extensively investigated. The risk of hypoglycaemia was much lower with SGLT2 inhibitors than with sulphonylureas and was similarly low as that reported with metformin, pioglitazone or sitagliptin

  13. Behavioral Characterization of the Effects of Cannabis Smoke and Anandamide in Rats

    PubMed Central

    Bruijnzeel, Adriaan W.; Qi, Xiaoli; Guzhva, Lidia V.; Wall, Shannon; Deng, Jie V.; Gold, Mark S.; Febo, Marcelo; Setlow, Barry

    2016-01-01

    Cannabis is the most widely used illicit drug in the world. Delta-9-tetrahydrocannabinol (Δ9-THC) is the main psychoactive component of cannabis and its effects have been well-studied. However, cannabis contains many other cannabinoids that affect brain function. Therefore, these studies investigated the effect of cannabis smoke exposure on locomotor activity, rearing, anxiety-like behavior, and the development of dependence in rats. It was also investigated if cannabis smoke exposure leads to tolerance to the locomotor-suppressant effects of the endogenous cannabinoid anandamide. Cannabis smoke was generated by burning 5.7% Δ9-THC cannabis cigarettes in a smoking machine. The effect of cannabis smoke on the behavior of rats in a small and large open field and an elevated plus maze was evaluated. Cannabis smoke exposure induced a brief increase in locomotor activity followed by a prolonged decrease in locomotor activity and rearing in the 30-min small open field test. The cannabinoid receptor type 1 (CB1) receptor antagonist rimonabant increased locomotor activity and prevented the smoke-induced decrease in rearing. Smoke exposure also increased locomotor activity in the 5-min large open field test and the elevated plus maze test. The smoke exposed rats spent more time in the center zone of the large open field, which is indicative of a decrease in anxiety-like behavior. A high dose of anandamide decreased locomotor activity and rearing in the small open field and this was not prevented by rimonabant or pre-exposure to cannabis smoke. Serum Δ9-THC levels were 225 ng/ml after smoke exposure, which is similar to levels in humans after smoking cannabis. Exposure to cannabis smoke led to dependence as indicated by more rimonabant-precipitated somatic withdrawal signs in the cannabis smoke exposed rats than in the air-control rats. In conclusion, chronic cannabis smoke exposure in rats leads to clinically relevant Δ9-THC levels, dependence, and has a biphasic effect

  14. Behavioral Characterization of the Effects of Cannabis Smoke and Anandamide in Rats.

    PubMed

    Bruijnzeel, Adriaan W; Qi, Xiaoli; Guzhva, Lidia V; Wall, Shannon; Deng, Jie V; Gold, Mark S; Febo, Marcelo; Setlow, Barry

    2016-01-01

    Cannabis is the most widely used illicit drug in the world. Delta-9-tetrahydrocannabinol (Δ9-THC) is the main psychoactive component of cannabis and its effects have been well-studied. However, cannabis contains many other cannabinoids that affect brain function. Therefore, these studies investigated the effect of cannabis smoke exposure on locomotor activity, rearing, anxiety-like behavior, and the development of dependence in rats. It was also investigated if cannabis smoke exposure leads to tolerance to the locomotor-suppressant effects of the endogenous cannabinoid anandamide. Cannabis smoke was generated by burning 5.7% Δ9-THC cannabis cigarettes in a smoking machine. The effect of cannabis smoke on the behavior of rats in a small and large open field and an elevated plus maze was evaluated. Cannabis smoke exposure induced a brief increase in locomotor activity followed by a prolonged decrease in locomotor activity and rearing in the 30-min small open field test. The cannabinoid receptor type 1 (CB1) receptor antagonist rimonabant increased locomotor activity and prevented the smoke-induced decrease in rearing. Smoke exposure also increased locomotor activity in the 5-min large open field test and the elevated plus maze test. The smoke exposed rats spent more time in the center zone of the large open field, which is indicative of a decrease in anxiety-like behavior. A high dose of anandamide decreased locomotor activity and rearing in the small open field and this was not prevented by rimonabant or pre-exposure to cannabis smoke. Serum Δ9-THC levels were 225 ng/ml after smoke exposure, which is similar to levels in humans after smoking cannabis. Exposure to cannabis smoke led to dependence as indicated by more rimonabant-precipitated somatic withdrawal signs in the cannabis smoke exposed rats than in the air-control rats. In conclusion, chronic cannabis smoke exposure in rats leads to clinically relevant Δ9-THC levels, dependence, and has a biphasic effect

  15. Bone effects of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in patients with type 2 diabetes mellitus.

    PubMed

    Blevins, Thomas C; Farooki, Azeez

    2017-01-01

    Canagliflozin, a sodium glucose co-transporter 2 (SGLT2) inhibitor approved for the treatment of type 2 diabetes mellitus (T2DM), lowers blood glucose by inhibiting renal glucose reabsorption and increasing urinary glucose excretion. It has been reported that SGLT2 inhibitors may have potential adverse effects on bone, including increased fracture risk and decreased bone mineral density (BMD). Across clinical studies, canagliflozin was not associated with meaningful changes in serum or urine calcium, vitamin D, or parathyroid hormone. Minimal increases in serum phosphate and magnesium that were within normal limits were seen with canagliflozin versus placebo. Canagliflozin was associated with increases in serum collagen type 1 beta-carboxy telopeptide (beta-CTX), a bone resorption marker, and osteocalcin, a bone formation marker. Decreases in total hip BMD were seen with canagliflozin 100 and 300 mg versus placebo after 2 years (-1.7%, -2.1%, -0.8%; differences of -0.9% and -1.2%), but not at other skeletal sites (normal age-related bone loss, ~0.5-1.0%/year). Changes in beta-CTX and total hip BMD were significantly associated with weight loss, which is known to increase bone resorption markers and decrease BMD. Canagliflozin was associated with a higher fracture incidence in an interim analysis of the CANagliflozin cardioVascular Assessment Study (CANVAS) in patients with a history or high risk of cardiovascular disease (incidence per 100 patient-years of 1.6, 1.6, and 1.1 with canagliflozin 100 and 300 mg and placebo), but not in other clinical studies of patients with T2DM. Fractures tended to occur as early as 12 weeks after initiating treatment and were primarily located in the distal parts of the upper and lower extremities. The reason for increased fracture risk with canagliflozin treatment is unknown, but is likely not related to a direct effect of canagliflozin on bone-related biomarkers. Data from ongoing canagliflozin studies, including CANVAS, will

  16. Loratadine and analogues: discovery and preliminary structure-activity relationship of inhibitors of the amino acid transporter B(0)AT2.

    PubMed

    Cuboni, Serena; Devigny, Christian; Hoogeland, Bastiaan; Strasser, Andrea; Pomplun, Sebastian; Hauger, Barbara; Höfner, Georg; Wanner, Klaus T; Eder, Matthias; Buschauer, Armin; Holsboer, Florian; Hausch, Felix

    2014-11-26

    B(0)AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B(0)AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B(0)AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure-activity relationships. Our studies provide the first chemical tool for B(0)AT2.

  17. Targeting Type 2 Diabetes with C-Glucosyl Dihydrochalcones as Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: Synthesis and Biological Evaluation.

    PubMed

    Jesus, Ana R; Vila-Viçosa, Diogo; Machuqueiro, Miguel; Marques, Ana P; Dore, Timothy M; Rauter, Amélia P

    2017-01-26

    Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of C-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC50 = 9-23 nM) were considerably weaker inhibitors of SGLT1 (IC50 = 10-19 μM). They showed no effect on the sodium independent GLUT family of glucose transporters, and the most potent ones were not acutely toxic to cultured cells. The interaction of a C-glucosyl dihydrochalcone with a POPC membrane was modeled computationally, providing evidence that it is not a pan-assay interference compound. These results point toward the discovery of structures that are potent and highly selective inhibitors of SGLT2.

  18. Anandamide and decidual remodelling: COX-2 oxidative metabolism as a key regulator.

    PubMed

    Almada, M; Piscitelli, F; Fonseca, B M; Di Marzo, V; Correia-da-Silva, G; Teixeira, N

    2015-11-01

    Recently, endocannabinoids have emerged as signalling mediators in reproduction. It is widely accepted that anandamide (AEA) levels must be tightly regulated, and that a disturbance in AEA levels may impact decidual stability and regression. We have previously characterized the endocannabinoid machinery in rat decidual tissue and reported the pro-apoptotic action of AEA on rat decidual cells. Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a crucial role in early pregnancy, and is also a key modulator in the crosstalk between endocannabinoids and prostaglandins. On the other hand, AEA-oxidative metabolism by COX-2 is not merely a mean to inactivate its action, but it yields the formation of a new class of mediators, named prostaglandin-ethanolamides, or prostamides. In this study we found that AEA-induced apoptosis in decidual cells involves COX-2 metabolic pathway. AEA induced COX-2 expression through p38 MAPK, resulting in the formation of prostamide E2 (PME2). Our findings also suggest that AEA-induced effect is associated with NF-kB activation. Finally, we describe the involvement of PME2 in the induction of the intrinsic apoptotic pathway in rat decidual cells. Altogether, our findings highlight the role of COX-2 as a gatekeeper in the uterine environment and clarify the impact of the deregulation of AEA levels on the decidual remodelling process.

  19. Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

    PubMed Central

    McDougle, Daniel R.; Kambalyal, Amogh; Meling, Daryl D.

    2014-01-01

    The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEA-epoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stopped-flow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase. PMID:25277139

  20. Physiological and behavioural effects of the endogenous cannabinoid, arachidonylethanolamide (anandamide), in the rat.

    PubMed Central

    Stein, E. A.; Fuller, S. A.; Edgemond, W. S.; Campbell, W. B.

    1996-01-01

    1. Arachidonylethanolamide (AEA; anandamide) has been isolated from mammalian brain and found to bind to, and is thought to be, an endogenous ligand for the cannabinoid receptor. In order to understand better its behavioural and physiological properties, we have examined its acute effects in unanaesthetized freely behaving rats. 2. Intravenous AEA caused dose-related decreases in locomotor behaviour, a pronounced hyperreflexia, and a moderate antinociceptive state. At doses between 3 and 30 mg kg-1, a dose-dependent hypothermia and profound, time-dependent cardiovascular changes were also observed. 3. An immediate bradycardia exceeding 50% was seen within 10-15 s of administration and lasted up to 11 min following the highest dose of the drug. In contrast, the change in mean arterial pressure was biphasic: an immediate 20% decrease in mean arterial pressure followed by a significant increase in blood pressure that lasted about 13 min after the highest dose. 4. These data demonstrate that AEA in the unanaesthetized rat exerts behavioural and physiological effects generally similar to those seen following natural cannabinoids and synthetic cannabimimetic agents and suggests a role for AEA in regulation of various physiological processes. Images Figure 2 Figure 4 Figure 5 Figure 7 PMID:8872363

  1. Alterations in the Anandamide Metabolism in the Development of Neuropathic Pain

    PubMed Central

    Malek, Natalia; Kucharczyk, Mateusz; Starowicz, Katarzyna

    2014-01-01

    Endocannabinoids (EC), particularly anandamide (AEA), released constitutively in pain pathways might be accountable for the inhibitory effect on nociceptors. Pathogenesis of neuropathic pain may reflect complex remodeling of the dorsal root ganglia (DRGs) and spinal cord EC system. Multiple pathways involved both in the biosynthesis and degradation of AEA have been suggested. We investigated the local synthesis and degradation features of AEA in DRGs and spinal cord during the development and maintenance of pain in a model of chronic constriction injury (CCI). All AEA synthesis and degradation enzymes are present on the mRNA level in DRGs and lumbar spinal cord of intact as well as CCI-treated animals. Deregulation of EC system components was consistent with development of pain phenotype at days 3, 7, and 14 after CCI. The expression levels of enzymes involved in AEA degradation was significantly upregulated ipsilateral in DRGs and spinal cord at different time points. Expression of enzymes of the alternative, sPLA2-dependent and PLC-dependent, AEA synthesis pathways was elevated in both of the analyzed structures at all time points. Our data have shown an alteration of alternative AEA synthesis and degradation pathways, which might contribute to the variation of AEA levels and neuropathic pain development. PMID:25276812

  2. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    PubMed

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway.

  3. A Role for Accumbal Glycine Receptors in Modulation of Dopamine Release by the Glycine Transporter-1 Inhibitor Org25935

    PubMed Central

    Lidö, Helga Höifödt; Ericson, Mia; Marston, Hugh; Söderpalm, Bo

    2010-01-01

    Accumbal glycine modulates basal and ethanol-induced dopamine levels in the nucleus accumbens (nAc) as well as voluntary ethanol consumption. Also, systemic administration of the glycine transporter-1 inhibitor Org25935 elevates dopamine levels in nAc, prevents a further ethanol-induced dopamine elevation and robustly and dose-dependently decreases ethanol consumption in rats. Here we investigated whether Org25935 applied locally in nAc modulates dopamine release, and whether accumbal glycine receptors or NMDA receptors are involved in this tentative effect. We also addressed whether Org25935 and ethanol applied locally in nAc interact with dopamine levels, as seen after systemic administration. We used in vivo microdialysis coupled to HPLC-ED in freely moving male Wistar rats to monitor dopamine output in nAc after local perfusion of Org25935 alone, with ethanol, or Org25935-perfusion after pre-treatment with the glycine receptor antagonist strychnine or the NMDA receptor glycine site antagonist L-701.324. Local Org25935 increased extracellular dopamine levels in a subpopulation of rats. Local strychnine, but not systemic L-701.324, antagonized the dopamine-activating effect of Org25935. Ethanol failed to induce a dopamine overflow in the subpopulation responding to Org25935 with a dopamine elevation. The study supports a role for accumbal glycine receptors rather than NMDA receptor signaling in the dopamine-activating effect of Org25935. The results further indicate that the previously reported systemic Org25935–ethanol interaction with regard to accumbal dopamine is localized to the nAc. This adds to the growing evidence for the glycine receptor as an important player in the dopamine reward circuitry and in ethanol's effects within this system. PMID:21556278

  4. Increasing influence of the glutamate transporter inhibitor on glutamate release in low [Na +] media under extremal conditions.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Krisanova, N.; Himmelreich, N.

    The effect of the competitive nontransportable inhibitor DL-threo-beta-benzyloxyaspartate DL-TBOA on the release of glutamate in Ca 2 -free Na - and NMDG-supplemented media was evaluated after exposure of rats to extremal conditions 6 min incubation of synaptosomes with 10 mu M DL-TBOA in low Na media resulted in the increase in extracellular L- 14 C glutamate level for control animals by 2 0 pm 0 5 of total accumulated label and 100 mu M DL-TBOA - 3 5 pm 0 5 respectively The experimental data for animals subjected to centrifuge-induced hypergravity showed 4 0 pm 1 0 and 9 0 pm 2 0 increase in L- 14 C glutamate level for 10 mu M and 100 mu M DL-TBOA respectively D le 0 05 The enhancement of the extracellular level of L- 14 C glutamate after application of DL-TBOA would be expected to connect with the inhibition of L- 14 C glutamate uptake process It appears that DL-TBOA inhibited uptake more potently after hypergravity The effect of DL-TBOA on depolarization-induced carrier-mediated L- 14 C glutamate release increased after hypergravity loading in Na - and low Na NMDG- supplemented media 10 mu M DL-TBOA-induced decrease in L- 14 C glutamate release in Na - supplemented medium was 15 2 pm 2 2 in the control experiments and 26 2 pm 3 9 after loading D le 0 05 and in low Na medium was 37 0 pm 2 5 and 45 0 pm 3 4 respectively DL-TBOA was demonstrated to better inhibit the transporter-mediated

  5. Sodium glucose co-transport 2 inhibitors in the treatment of type 2 diabetes mellitus: a meta-analysis of randomized double-blind controlled trials

    PubMed Central

    2013-01-01

    Background The discovery of sodium-glucose co-transporter 2 (SGLT2) inhibitors, with a novel mechanism independent of insulin secretion or sensitization, bring about a new therapeutic approach to the management of type 2 diabetes mellitus. The aim of this meta-analysis was to evaluate the safety and efficacy of SGLT2 inhibitors at different doses in randomized double blind clinical trials. Methods This meta-analysis was conducted by including randomized double-blind controlled trials of SGLT2 inhibitors in patients with type 2 diabetes irrespective of their antidiabetic drug exposure history but with an inadequate glycemic control. All the effect sizes were computed using the random effects model. Standardized mean differences (SMDs) and odds ratios (OR) were computed for continuous and dichotomous variables, respectively. Additional analyses like sensitivity analysis, subgroup analysis and meta-regression were also performed. Results The pooled analyses demonstrated a significant reduction in mean changes in Hemoglobin A1c (HbA1c) (SMD = −0.78%, 95% CI, -0.87 to −0.69), fasting plasma glucose (FPG) (SMD = −0.70 mg/dl, 95% CI, -0.79 to −0.61), body weight (overall SMD = −0.59 kg, 95% CI, -0.65 to −0.52) and blood pressure from baseline with SGLT2 inhibitors based therapy. Consistently a significant number of patients treated with SGLT2 inhibitors achieved HbA1c < 7% (OR = 2.09, 95% CI, 1.77 to 2.46). SGLT2 inhibitors based therapy was associated with adverse events like genital and urinary tract infections. Conclusion All studied doses of SGLT2 inhibitors, either as monotherapy or in combination with other antidiabetic agents, consistently improved glycemic control in patients with type 2 diabetes. However, a small percentage of patients suffer from genital and urinary tract infections. PMID:24341330

  6. Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors.

    PubMed

    Takeda, Michio; Noshiro, Rie; Onozato, Maristela Lika; Tojo, Akihiro; Hasannejad, Habib; Huang, Xiu-Lin; Narikawa, Shinichi; Endou, Hitoshi

    2004-01-12

    The purpose of this study was to elucidate the role of human organic anion transporters (human OATs) in the induction of drug-induced skeletal muscle abnormalities. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have been clinically used for lowering plasma cholesterol levels, and are known to induce various forms of skeletal muscle abnormalities including myopathy and rhabdomyolysis. Immunohistochemical analysis revealed that human OAT1 and human OAT3 are localized in the cytoplasmic membrane of the human skeletal muscles. The activities of human OATs were measured using mouse cell lines from renal proximal tubules stably expressing human OATs. Human OAT3, but not human OAT1, mediates the transport of pravastatin. Fluvastatin inhibited organic anion uptake mediated by human OAT1 in a mixture of competitive and noncompetitive manner, whereas simvastatin and fluvastatin noncompetitively inhibited the organic anion uptake mediated by human OAT3. In conclusion, the organic anion transporters OAT1 and OAT3 are localized in the cytoplasmic membrane of human skeletal muscles. Pravastatin, simvasatin, and fluvasatin inhibit human OATs activity. These results suggest that muscle organic anion transporters play a role in the muscular side effects of HMG-CoA reductase inhibitors.

  7. A novel class of apical sodium--dependent bile salt transporter inhibitors: 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides.

    PubMed

    Liu, Hongtao; Pang, Guoxun; Ren, Jinfeng; Zhao, Yue; Wang, Juxian

    2017-03-01

    The apical sodium--dependent bile acid transporter (ASBT) is the main transporter to promote re-absorption of bile acids from the intestinal tract into the enterohepatic circulation. Inhibition of ASBT could increase the excretion of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. Therefore, ASBT is an attractive target for developing new cholesterol-lowering drugs. In this report, a series of 1-(2,4-bifluorophenyl)-7-dialkylamino-1,8-naphthyridine-3-carboxamides were designed as inhibitors of ASBT. Most of them demonstrated potency against ASBT transport of bile acids. In particular, compound 4a1 was found to have the best activity, resulting in 80.1% inhibition of ASBT at 10 μmol/L.

  8. Delta9-tetrahydrocannabinol and endogenous cannabinoid anandamide directly potentiate the function of glycine receptors.

    PubMed

    Hejazi, Nadia; Zhou, Chunyi; Oz, Murat; Sun, Hui; Ye, Jiang Hong; Zhang, Li

    2006-03-01

    Anandamide (AEA) and delta9-tetrahydrocannabinol (THC) are endogenous and exogenous ligands, respectively, for cannabinoid receptors. Whereas most of the pharmacological actions of cannabinoids are mediated by CB1 receptors, there is also evidence that these compounds can produce effects that are not mediated by the activation of identified cannabinoid receptors. Here, we report that THC and AEA, in a CB1 receptor-independent manner, cause a significant potentiation of the amplitudes of glycine-activated currents (I(Gly)) in acutely isolated neurons from rat ventral tegmental area (VTA) and in Xenopus laevis oocytes expressing human homomeric (alpha1) and heteromeric (alpha1beta1) subunits of glycine receptors (GlyRs). The potentiation of I(Gly) by THC and AEA is concentration-dependent, with respective EC50 values of 86 +/- 9 and 319 +/- 31 nM for alpha1 homomeric receptors, 73 +/- 8 and 318 +/- 24 nM for alpha1beta1 heteromeric receptors, and 115 +/- 13 and 230 +/- 29 nM for native GlyRs in VTA neurons. The effects of THC and AEA are selective for I(Gly), because GABA-activated current in VTA neurons or in X. laevis oocytes expressing alpha2beta3gamma2 GABA(A) receptor subunits were unaffected by these compounds. The maximal potentiation by THC and AEA was observed at the lowest concentration of glycine; with increasing concentrations of glycine, the potentiation significantly decreased. The site for THC and AEA seems to be distinct from that of the alcohol and volatile anesthetics. The results indicate that THC and AEA, in pharmacologically relevant concentrations, directly potentiate the function of GlyRs through an allosteric mechanism.

  9. Dietary linoleic acid elevates endogenous 2-AG and anandamide and induces obesity.

    PubMed

    Alvheim, Anita R; Malde, Marian K; Osei-Hyiaman, Douglas; Lin, Yu Hong; Pawlosky, Robert J; Madsen, Lise; Kristiansen, Karsten; Frøyland, Livar; Hibbeln, Joseph R

    2012-10-01

    Suppressing hyperactive endocannabinoid tone is a critical target for reducing obesity. The backbone of both endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA) is the ω-6 fatty acid arachidonic acid (AA). Here we posited that excessive dietary intake of linoleic acid (LA), the precursor of AA, would induce endocannabinoid hyperactivity and promote obesity. LA was isolated as an independent variable to reflect the dietary increase in LA from 1 percent of energy (en%) to 8 en% occurring in the United States during the 20th century. Mice were fed diets containing 1 en% LA, 8 en% LA, and 8 en% LA + 1 en% eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) in medium-fat diets (35 en% fat) and high-fat diets (60 en%) for 14 weeks from weaning. Increasing LA from 1 en% to 8 en% elevated AA-phospholipids (PL) in liver and erythrocytes, tripled 2-AG + 1-AG and AEA associated with increased food intake, feed efficiency, and adiposity in mice. Reducing AA-PL by adding 1 en% long-chain ω-3 fats to 8 en% LA diets resulted in metabolic patterns resembling 1 en% LA diets. Selectively reducing LA to 1 en% reversed the obesogenic properties of a 60 en% fat diet. These animal diets modeled 20th century increases of human LA consumption, changes that closely correlate with increasing prevalence rates of obesity. In summary, dietary LA increased tissue AA, and subsequently elevated 2-AG + 1-AG and AEA resulting in the development of diet-induced obesity. The adipogenic effect of LA can be prevented by consuming sufficient EPA and DHA to reduce the AA-PL pool and normalize endocannabinoid tone.

  10. The Volitional Nature of Nicotine Exposure Alters Anandamide and Oleoylethanolamide Levels in the Ventral Tegmental Area

    PubMed Central

    Buczynski, Matthew W; Polis, Ilham Y; Parsons, Loren H

    2013-01-01

    Cannabinoid-1 receptors (CB1) have an important role in nicotine reward and their function is disrupted by chronic nicotine exposure, suggesting nicotine-induced alterations in endocannabinoid (eCB) signaling. However, the effects of nicotine on brain eCB levels have not been rigorously evaluated. Volitional intake of nicotine produces physiological and behavioral effects distinct from forced drug administration, although the mechanisms underlying these effects are not known. This study compared the effects of volitional nicotine self-administration (SA) and forced nicotine exposure (yoked administration (YA)) on levels of eCBs and related neuroactive lipids in the ventral tegmental area (VTA) and other brain regions. Brain lipid levels were indexed both by in vivo microdialysis in the VTA and lipid extractions from brain tissues. Nicotine SA, but not YA, reduced baseline VTA dialysate oleoylethanolamide (OEA) levels relative to nicotine-naïve controls, and increased anandamide (AEA) release during nicotine intake. In contrast, all nicotine exposure paradigms increased VTA dialysate 2-arachidonoyl glycerol (2-AG) levels. Thus, nicotine differentially modulates brain lipid (2-AG, AEA, and OEA) signaling, and these modulations are influenced by the volitional nature of the drug exposure. Corresponding bulk tissue analysis failed to identify these lipid changes. Nicotine exposure had no effect on fatty acid amide hydrolase activity in the VTA, suggesting that changes in AEA and OEA signaling result from alterations in their nicotine-induced biosynthesis. Both CB1 (by AEA and 2-AG) and non-CB1 (by OEA) targets can alter the excitability and activity of the dopaminergic neurons in the VTA. Collectively, these findings implicate disrupted lipid signaling in the motivational effects of nicotine. PMID:23169348

  11. The volitional nature of nicotine exposure alters anandamide and oleoylethanolamide levels in the ventral tegmental area.

    PubMed

    Buczynski, Matthew W; Polis, Ilham Y; Parsons, Loren H

    2013-03-01

    Cannabinoid-1 receptors (CB(1)) have an important role in nicotine reward and their function is disrupted by chronic nicotine exposure, suggesting nicotine-induced alterations in endocannabinoid (eCB) signaling. However, the effects of nicotine on brain eCB levels have not been rigorously evaluated. Volitional intake of nicotine produces physiological and behavioral effects distinct from forced drug administration, although the mechanisms underlying these effects are not known. This study compared the effects of volitional nicotine self-administration (SA) and forced nicotine exposure (yoked administration (YA)) on levels of eCBs and related neuroactive lipids in the ventral tegmental area (VTA) and other brain regions. Brain lipid levels were indexed both by in vivo microdialysis in the VTA and lipid extractions from brain tissues. Nicotine SA, but not YA, reduced baseline VTA dialysate oleoylethanolamide (OEA) levels relative to nicotine-naïve controls, and increased anandamide (AEA) release during nicotine intake. In contrast, all nicotine exposure paradigms increased VTA dialysate 2-arachidonoyl glycerol (2-AG) levels. Thus, nicotine differentially modulates brain lipid (2-AG, AEA, and OEA) signaling, and these modulations are influenced by the volitional nature of the drug exposure. Corresponding bulk tissue analysis failed to identify these lipid changes. Nicotine exposure had no effect on fatty acid amide hydrolase activity in the VTA, suggesting that changes in AEA and OEA signaling result from alterations in their nicotine-induced biosynthesis. Both CB(1) (by AEA and 2-AG) and non-CB(1) (by OEA) targets can alter the excitability and activity of the dopaminergic neurons in the VTA. Collectively, these findings implicate disrupted lipid signaling in the motivational effects of nicotine.

  12. A Randomized Placebo-Controlled Phase IIb Trial of A3309, A Bile Acid Transporter Inhibitor, for Chronic Idiopathic Constipation

    PubMed Central

    Chey, William D; Camilleri, Michael; Chang, Lin; Rikner, Leif; Graffner, Hans

    2011-01-01

    OBJECTIVES: A3309 is a minimally absorbed ileal bile acid (BA) transporter (IBAT) inhibitor. We conducted an 8-week, multicenter, randomized, double-blind, placebo-controlled, parallel group, phase IIb study, which evaluated A3309 in patients with chronic idiopathic constipation (CIC). METHODS: Patients with CIC (modified Rome III criteria and <3 complete (CSBM) spontaneous bowel movements (SBMs)/week during the 2-week baseline) were randomized to 5, 10, or 15 mg A3309 or placebo once daily. The primary end point was change in SBM number during week 1 compared with baseline. Other bowel and abdominal symptoms were assessed as secondary end points. Serum 7αC4 and lipids were evaluated as biomarkers of BA synthesis/loss. RESULTS: In all, 190 patients (mean 48 years, 90% female) were randomized. Mean increase (95% confidence interval) in SBM for week 1 were 1.7 (0.7–2.8) for placebo vs. 2.5 (1.5–3.5), 4.0 (2.9–5.0), and 5.4 (4.4–6.4) for 5 mg, 10 mg (P<0.002), and 15 mg (P<0.001) A3309, respectively. Increased stool frequency was maintained over 8 weeks. Time to first SBM and CSBM were significantly reduced in the 10- and 15-mg A3309 groups compared with placebo. Straining and bloating decreased with A3309 compared with placebo (P<0.05). Increased 7αC4 and reduced low-density lipoprotein cholesterol with A3309 suggested increased BA synthesis and BA loss. The most common adverse events (AEs) were abdominal pain and diarrhea, which occurred most commonly in the 15-mg A3309 group. No drug-related serious AEs were observed. CONCLUSIONS: A3309 increased stool frequency and improved constipation-related symptoms in CIC; effects were maintained over 8 weeks of treatment. PMID:21606974

  13. Interactions of human P-glycoprotein transport substrates and inhibitors at the drug binding domain: Functional and molecular docking analyses.

    PubMed

    Kadioglu, Onat; Saeed, Mohamed E M; Valoti, Massimo; Frosini, Maria; Sgaragli, Giampietro; Efferth, Thomas

    2016-03-15

    Rhodamine 123 (R123) transport substrate sensitizes P-glycoprotein (P-gp) to inhibition by compound 2c (cis-cis) N,N-bis(cyclohexanolamine)aryl ester isomer in a concentration-dependent manner in human MDR1-gene transfected mouse T-lymphoma L5178 cells as shown previously. By contrast, epirubicin (EPI) concentration changes left unaltered 2c IC50 values of EPI efflux. To clarify this discrepancy, defined molecular docking (DMD) analyses of 12 N,N-bis(cyclohexanolamine)aryl esters, the highly flexible aryl ester analog 4, and several P-gp substrate/non-substrate inhibitors were performed on human P-gp drug- or nucleotide-binding domains (DBD or NBD). DMD measurements yielded lowest binding energy (LBE, kcal/mol) values (mean ± SD) ranging from -11.8 ± 0.54 (valspodar) to -3.98 ± 0.01 (4). Lys234, Ser952 and Tyr953 residues formed H-bonds with most of the compounds. Only 2c docked also at ATP binding site (LBE value of -6.9 ± 0.30 kcal/mol). Inhibition of P-gp-mediated R123 efflux by 12 N,N-bis(cyclohexanolamine)aryl esters and 4 significantly correlated with LBE values. DMD analysis of EPI, (3)H-1EPI, (3)H-2EPI, (14)C-1EPI, (14)C-2EPI, R123 and 2c before and after previous docking of each of them indicated that pre-docking of either 2c or EPI significantly reduced LBE of both EPI and R123, and that of both (3)H-2EPI and (14)C-2EPI, respectively. Since the clusters of DBD amino acid residues interacting with EPI were different, if EPI docked alone or after pre-docking of EPI or 2c, the existence of alternative secondary binding site for EPI on P-gp is credible. In conclusion, 2c may allocate the drug-binding pocket and reduce strong binding of EPI and R123 in agreement with P-gp inhibition experiments, where 2c reduced efflux of EPI and R123.

  14. The role of hepatic transport and metabolism in the interactions between pravastatin or repaglinide and two rOatp inhibitors in rats.

    PubMed

    Badolo, Lassina; Bundgaard, Christoffer; Garmer, Mats; Jensen, Bente

    2013-07-16

    A change in the function or expression of hepatic drug transporters may have significant effect on the efficacy or safety of orally administered drugs. Although a number of clinical drug-drug interactions associated with hepatic transport proteins have been reported, in practice it is not always straightforward to discriminate other pathways (e.g. drug metabolism) from being involved in these interactions. The present study was designed to assess the interactions between organic anion transporting polypeptide (Oatp) substrates (pravastatin or repaglinide) and inhibitors (spironolactone or diphenhydramine) in vivo in rats. The mechanisms behind the interactions were then investigated using in vitro tools (isolated hepatocytes and rat liver microsomes). The results showed a significant increase in the systemic exposures of pravastatin (2.5-fold increase in AUC) and repaglinide (1.8-fold increase in AUC) after co-administration of spironolactone to rats. Diphenhydramine increased the AUC of repaglinide by 1.4-fold. The in vivo interactions observed in rats between Oatp substrates and inhibitors may a priori be classified as transport-mediated drug-drug interactions. However, mechanistic studies performed in vitro using both isolated rat hepatocytes and rat liver microsomes showed that the interaction between pravastatin and spironolactone may be solely linked to the inhibition of pravastatin uptake in liver. On the contrary, the inhibition of cytochrome P450 seemed to be the reason for the interactions observed between repaglinide and spironolactone. Although the function and structure of transport proteins may vary between rats and humans, the approach used in the present study can be applied to humans and help to understand the role of drug transport and drug metabolism in a given drug-drug interaction. This is important to predict and mitigate the risk of drug-drug interactions for a candidate drug in pre-clinical development, it is also important for the optimal

  15. Sperm Release From the Oviductal Epithelium Depends on Ca(2+) Influx Upon Activation of CB1 and TRPV1 by Anandamide.

    PubMed

    Gervasi, M G; Osycka-Salut, C; Sanchez, T; Alonso, C A I; Llados, C; Castellano, L; Franchi, A M; Villalón, M; Perez-Martinez, S

    2016-02-01

    The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway.

  16. Phase I/II clinical trial of 2-difluoromethyl-ornithine (DFMO) and a novel polyamine transport inhibitor (MQT 1426) for feline oral squamous cell carcinoma.

    PubMed

    Skorupski, K A; O'Brien, T G; Guerrero, T; Rodriguez, C O; Burns, M R

    2011-12-01

    Polyamines are essential for cell proliferation. Their production is dysregulated in many cancers and polyamine depletion leads to tumour regression in mouse models of squamous cell carcinoma (SCC). The purpose of this study was to determine the maximally tolerated dose of the polyamine transport inhibitor, MQT 1426, when combined with the ornithine decarboxylase (ODC) inhibitor, DFMO, and to determine whether this therapy results in reduction in tumour polyamine levels. Thirteen cats with oral SCC received both drugs orally and serial tumour biopsies were obtained for polyamine measurement. Cats were monitored for response to therapy and toxicity. A maximum tolerated dose (MTD) of MQT 1426 when combined with DFMO was determined. Dose-limiting toxicity was vestibular in nature, but was fully reversible. Spermidine and total polyamine levels decreased significantly in tissues, two cats experienced objective tumour regression and six cats had stable disease. These results suggest that further study of polyamine depletion therapies is warranted.

  17. Exoplasmic cysteine Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-molecule inhibitor and normal lipid transport activity

    PubMed Central

    Yu, Miao; Romer, Katherine A.; Nieland, Thomas J. F.; Xu, Shangzhe; Saenz-Vash, Veronica; Penman, Marsha; Yesilaltay, Ayce; Carr, Steven A.; Krieger, Monty

    2011-01-01

    The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC50 ∼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1’s thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor’s intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses. PMID:21746906

  18. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  19. TRANSPORT

    EPA Science Inventory

    Presentation outline: transport principles, effective solubility; gasoline composition; and field examples (plume diving).
    Presentation conclusions: MTBE transport follows from - phyiscal and chemical properties and hydrology. Field examples show: MTBE plumes > benzene plu...

  20. Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack

    PubMed Central

    1994-01-01

    The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway. PMID:8089173

  1. Identification of novel natural inhibitor for NorM - a multidrug and toxic compound extrusion transporter - an insilico molecular modeling and simulation studies.

    PubMed

    Kesherwani, Manish; Michael Gromiha, M; Fukui, Kazuhiko; Velmurugan, Devadasan

    2017-01-01

    The emergence of bacterial multidrug resistance is an increasing problem in treatment of infectious diseases. An important cause for the multidrug resistance of bacteria is the expression of multidrug efflux transporters. The multidrug and toxic compound extrusion (MATE) transporters are most recently recognized as unique efflux system for extrusion of antimicrobials and therapeutic drugs due to energy stored in either Na(+) or H(+) electrochemical gradient. In the present study, high throughput virtual screening of natural compound collections against NorM - a MATE transporter from Neisseria gonorrhea (NorM-NG) has been carried out followed by flexible docking. The molecular simulation in membrane environment has been performed for understanding the stability and binding energetic of top lead compounds. Results identified a compound from the Indian medicinal plant "Terminalia chebula" which has good binding free energy compared to substrates (rhodamine 6 g, ethidium) and more favorable interactions with the central cavity forming active site residues. The compound has restricted movement in TM7, TM8, and TM1, thus blocking the disruption of Na+ - coordination along with equilibrium state bias towards occlude state of NorM transporter. Thus, this compound blocks the effluxing pathway of antimicrobial drugs and provides as a natural bioactive lead inhibitor against NorM transporter in drug-resistant gonorrhea.

  2. Discovery of novel conformationally constrained tropane-based biaryl and arylacetylene ligands as potent and selective norepinephrine transporter inhibitors and potential antidepressants.

    PubMed

    Zhou, Jia; Kläss, Thomas; Johnson, Kenneth M; Giberson, Kelly M; Kozikowski, Alan P

    2005-05-16

    To further explore the structure-activity relationships of conformationally constrained tropanes, a number of new biaryl and arylacetylene analogs were designed and synthesized. Some of these compounds such as 3a-b, 3d, 3f-h, 5b, and 7g were found to be highly potent and selective or mixed norepinephrine transporter (NET) inhibitors with Ki values of 0.8-9.4 nM. Moreover, all of these compounds display weak to extremely weak muscarinic receptor binding affinity, indicating that as potential antidepressants, they may overcome certain side effects that are of concern with other antidepressants, which are thought to be mediated by their anticholinergic properties.

  3. [Sodium-glucose co-transporter-2 inhibitors: from the bark of apple trees and familial renal glycosuria to the treatment of type 2 diabetes mellitus].

    PubMed

    Mauricio, Dídac

    2013-09-01

    The therapeutic armamentarium for the treatment of hyperglycemia in type 2 diabetes mellitus is still inadequate. We are currently witnessing the introduction of a new mode of hypoglycemic treatment through induction of glycosuria to decrease the availability of the metabolic substrate, i.e. glucose. Clinical trials have shown that sodium-glucose co-transporter-2 (SGLT2) inhibitors are as efficacious as other oral hypoglycemic drugs. This article discusses the basic features of this new treatment concept and the efficacy and safety of this new drug group.

  4. Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

    PubMed Central

    Al Kury, Lina T; Voitychuk, Oleg I; Yang, Keun-Hang Susan; Thayyullathil, Faisal T; Doroshenko, Petro; Ramez, Ali M; Shuba, Yaroslav M; Galadari, Sehamuddin; Howarth, Frank Christopher; Oz, Murat

    2014-01-01

    BACKGROUND AND PURPOSE The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS In the presence of 1–10 μM AEA, suppression of both Na+ and L-type Ca2+ channels was observed. Inhibition of Na+ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [3H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd. Further studies on L-type Ca2+ channels indicated that AEA potently inhibited these channels (IC50 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba2+ currents. MetAEA also inhibited Na+ and L-type Ca2+ currents. Radioligand studies indicated that specific binding of [3H]isradipine, was inhibited significantly by metAEA. (10 μM), changing Bmax but not Kd. CONCLUSION AND IMPLICATIONS Results indicate that AEA inhibited the function of voltage-dependent Na+ and L-type Ca2+ channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation. PMID:24758718

  5. Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells.

    PubMed Central

    Di Marzo, V; De Petrocellis, L; Sepe, N; Buono, A

    1996-01-01

    Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N-aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N-aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role

  6. A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter, LeuT.

    PubMed

    Simmons, Katie J; Gotfryd, Kamil; Billesbølle, Christian B; Loland, Claus J; Gether, Ulrik; Fishwick, Colin W G; Johnson, A Peter

    2013-03-01

    Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.

  7. The potential of inhibitors of endocannabinoid metabolism as anxiolytic and antidepressive drugs--A practical view.

    PubMed

    Fowler, Christopher J

    2015-06-01

    The endocannabinoid system, comprising cannabinoid CB1 and CB2 receptors, their endogenous ligands anandamide and 2-arachidonoylglyerol, and their synthetic and metabolic enzymes, are involved in many biological processes in the body, ranging from appetite to bone turnover. Compounds inhibiting the breakdown of anandamide and 2-arachidonoylglycerol increase brain levels of these lipids and thus modulate endocannabinoid signalling. In the present review, the preclinical evidence that these enzymes are good targets for development of novel therapies for anxiety and depression are discussed from a practical, rather than mechanistic, point of view. It is concluded that the preclinical data are promising, albeit tempered by problems of tolerance as well as effects upon learning and memory for irreversible monoacylglycerol lipase inhibitors, and limited by a focus upon male rodents alone. Clinical data so far has been restricted to safety studies with inhibitors of anandamide hydrolysis and a hitherto unpublished study on such a compound in elderly patients with major depressive disorders, but under the dose regimes used, they are well tolerated and show no signs of "cannabis-like" behaviours.

  8. Regulation of divalent metal transporter-1 by serine phosphorylation

    PubMed Central

    Seo, Young Ah; Kumara, Ruvin; Wetli, Herbert; Wessling-Resnick, Marianne

    2016-01-01

    Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake. PMID:27681840

  9. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    PubMed Central

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  10. Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport.

    PubMed

    Mironov, Alexander A; Colanzi, Antonino; Polishchuk, Roman S; Beznoussenko, Galina V; Mironov, Alexander A; Fusella, Aurora; Di Tullio, Giuseppe; Silletta, Maria Giuseppina; Corda, Daniela; De Matteis, Maria Antonietta; Luini, Alberto

    2004-07-01

    Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.

  11. Pharmacological characterization of RS-1259, an orally active dual inhibitor of acetylcholinesterase and serotonin transporter, in rodents: possible treatment of Alzheimer's disease.

    PubMed

    Abe, Yasuyuki; Aoyagi, Atsushi; Hara, Takao; Abe, Kazumi; Yamazaki, Reina; Kumagae, Yoshihiro; Naruto, Shunji; Koyama, Kazuo; Marumoto, Shinji; Tago, Keiko; Toda, Narihiro; Takami, Kazuko; Yamada, Naho; Ori, Mayuko; Kogen, Hiroshi; Kaneko, Tsugio

    2003-09-01

    A dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT), RS-1259 (4-[1S)-methylamino-3-(4-nitrophenoxy)]propylphenyl N,N-dimethylcarbamate (fumaric acid)(1/2)salt), was newly synthesized. RS-1259 simultaneously inhibited AChE and SERT in the brain following an oral administration in mice and rats. Actual simultaneous elevation of extracellular levels of 5-HT and ACh in the rat hippocampus was confirmed by microdialysis. The compound was as effective as SERT inhibitors such as fluoxetine and fluvoxamine in a 5-hydroxytryptophan-enhancing test in mice. Spatial memory deficits in the two-platform task of a water maze in aged rats were ameliorated by RS-1259 as well as donepezil. Both RS-1259 and donepezil increased the awake episodes in the daytime electroencephalogram of rats. Although RS-1259 was weaker than donepezil in enhancing central cholinergic transmission, as observed by ACh elevation in the hippocampus and memory enhancement in aged rats, the efficacy of RS-1259 on the consciousness level, which reflects the whole activity in the brain, was almost the same as that of donepezil. These results suggest that both cholinergic and serotonergic systems are involved in maintaining brain arousal and that a dual inhibitor of AChE and SERT may be useful for the treatment of cognitive disorders associated with reduced brain activity such as in Alzheimer's disease.

  12. Avertin®, but Not Volatile Anesthetics Addressing the Two-Pore Domain K+ Channel, TASK-1, Slows Down Cilia-Driven Particle Transport in the Mouse Trachea

    PubMed Central

    Murtaza, Ghulam; Mermer, Petra; Pfeil, Uwe; Kummer, Wolfgang

    2016-01-01

    Rationale Volatile anesthetics inhibit mucociliary clearance in the airways. The two-pore domain K+ channel, TASK-1, represents one of their molecular targets in that they increase its open probability. Here, we determine whether particle transport speed (PTS) at the mucosal surface of the mouse trachea, an important factor of the cilia-driven mechanism in mucociliary clearance, is regulated by TASK-1. Methodology/Results RT-PCR analysis revealed expression of TASK-1 mRNA in the manually dissected and laser-assisted microdissected tracheal epithelium of the mouse. Effects of anesthetics (isoflurane and Avertin®) and TASK-1 inhibitors (anandamide and A293) on ciliary activity were investigated by assessment of PTS at the mucosal surface of the explanted and opened murine trachea. Neither TASK-1 inhibitors nor isoflurane had any impact on basal and ATP-stimulated PTS. Avertin® reduced basal PTS, and ATP-stimulated PTS decreased in its presence in wild-type (WT) mice. Avertin®-induced decrease in basal PTS persisted in WT mice in the presence of TASK-1 inhibitors, and in two different strains of TASK-1 knockout mice. Conclusions/Significance Our findings indicate that TASK-1 is expressed by the tracheal epithelium but is not critically involved in the regulation of tracheal PTS in mice. Avertin® reduces PTS independent of TASK-1. PMID:27930725

  13. β-Sulfonamido Functionalized Aspartate Analogues as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype Selectivity Profiles Arising from Subtle Structural Differences.

    PubMed

    Hansen, Jacob C; Bjørn-Yoshimoto, Walden E; Bisballe, Niels; Nielsen, Birgitte; Jensen, Anders A; Bunch, Lennart

    2016-10-13

    In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC50 values ∼1 μM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC50 of 0.8 μM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF3-phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC50 = 2.8 μM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these β-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.

  14. Characterization of the K+ (Na+)/H+ monovalent cation exchanger in the human red blood cell membrane: effects of transport inhibitors.

    PubMed

    Bernhardt, I; Bogdanova, A Y; Kummerow, D; Kiessling, K; Hamann, J; Ellory, J C

    1999-06-01

    The (ouabain + bumetanide + EGTA)-insensitive K+ influx (defined as residual K+ influx) in the human erythrocyte was investigated with respect to the characterization of the recently identified K+(Na+)/H+ exchanger (Richter et al. 1997). In particular, the effects of selected ion transport inhibitors on this flux in physiological ionic strength (high ionic strength, HIS) as well as low ionic strength (LIS) solutions were qstudied. The stimulation of the K+ influx observed in LIS medium was further enhanced when DIDS, phloretin, eosin-5-maleimide, furosemide, DIOA, NPPB, or DCDPC was present at a concentration of 0.1 mmol/l. This paradoxical, inhibitor-induced increase of the K+ influx was more pronounced in LIS media where chloride (7.5 mmol/l) was replaced by nitrate. For DNDS, niflumic acid, and MK-196 (0.1 mmol/l) an enhanced K+ transport could only be observed in nitrate-containing LIS solution. Bumetanide and purine riboside, at a concentration of 0.1 mmol/l, did not cause significant changes of the K+ influx in either chloride- or nitrate-containing LIS media. Dipyridamole and ruthenium red (0.1 mmol/l), which are positively charged, significantly reduced the K+ influx in both chloride- and nitrate-containing LIS media. In nitrate-containing HIS solution only dipyridamole inhibited the K+ influx. The residual K+ influx in LIS solution was significantly increased by removing internal [Mg2+], and decreased by quinacrine (1 mmol/l). In HIS solution, no effect of altering intracellular Mg2+ occurred but a stimulation of the flux by quinacrine was observed. The results are discussed in terms of a more general surface charge effect of the used inhibitors on the K+(Na+)/H+ exchanger.

  15. Transcriptional Responses of Escherichia coli to a Small-Molecule Inhibitor of LolCDE, an Essential Component of the Lipoprotein Transport Pathway

    PubMed Central

    Lorenz, Christian; Dougherty, Thomas J.

    2016-01-01

    ABSTRACT In Gram-negative bacteria, a dedicated machinery consisting of LolABCDE components targets lipoproteins to the outer membrane. We used a previously identified small-molecule inhibitor of the LolCDE complex of Escherichia coli to assess the global transcriptional consequences of interference with lipoprotein transport. Exposure of E. coli to the LolCDE inhibitor at concentrations leading to minimal and significant growth inhibition, followed by transcriptome sequencing, identified a small group of genes whose transcript levels were decreased and a larger group whose mRNA levels increased 10- to 100-fold compared to those of untreated cells. The majority of the genes whose mRNA concentrations were reduced were part of the flagellar assembly pathway, which contains an essential lipoprotein component. Most of the genes whose transcript levels were elevated encode proteins involved in selected cell stress pathways. Many of these genes are involved with envelope stress responses induced by the mislocalization of outer membrane lipoproteins. Although several of the genes whose RNAs were induced have previously been shown to be associated with the general perturbation of the cell envelope by antibiotics, a small subset was affected only by LolCDE inhibition. Findings from this work suggest that the efficiency of the Lol system function may be coupled to a specific monitoring system, which could be exploited in the development of reporter constructs suitable for use for screening for additional inhibitors of lipoprotein trafficking. IMPORTANCE Inhibition of the lipoprotein transport pathway leads to E. coli death and subsequent lysis. Early significant changes in the levels of RNA for a subset of genes identified to be associated with some periplasmic and envelope stress responses were observed. Together these findings suggest that disruption of this key pathway can have a severe impact on balanced outer membrane synthesis sufficient to affect viability. PMID

  16. Sarcosine-Based Glycine Transporter Type-1 (GlyT-1) Inhibitors Containing Pyridazine Moiety: A Further Search for Drugs with Potential to Influence Schizophrenia Negative Symptoms.

    PubMed

    Harsing, Laszlo G; Timar, Julia; Szabo, Geza; Udvari, Szabolcs; Nagy, Katalin M; Marko, Bernadett; Zsilla, Gabriella; Czompa, Andrea; Tapolcsanyi, Pal; Kocsis, Akos; Matyus, Peter

    2015-01-01

    We have synthesized a novel series of N-substituted sarcosines, analogues of NFPS (N-[3-(biphenyl-4- yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine), as type-1 glycine transporter (GlyT-1) inhibitors. Several compounds incorporated a diazine ring inhibited recombinant hGlyT-1b expressed permanently in CHO cells and GlyT-1 in rat brain synaptosomal preparations. A structure-activity relationship for the newly synthesized compounds was obtained and discussed on the ground of their GlyT-1 inhibitory potencies. Replacement of the biphenyl-4-yloxy moiety in NFPS with a 5-pyridazinylphenoxy moiety (compounds 3, 4, 5, and 6) or a 2-phenyl-5- pyridazinyloxy moiety (compounds 10, 11, and 12) afforded compounds exhibiting potent inhibition on GlyT-1 activity. The GlyT-1 inhibitory properties of NFPS analogues, in which sarcosine was closed into a ring forming (methylamino)pyridazine-3-(2H)-one, were markedly reduced (compounds 13 and 14). The pyridazine-containing GlyT-1 inhibitors with in vitro GlyT-1 inhibitory potency also enhanced extracellular glycine concentrations in conscious rat striatum as was measured by microdialysis technique. In contrast to NFPS, sarcosine-based pyridazine containing GlyT-1 inhibitors failed to evoke compulsive running behavior whereas they inhibited phencyclidine- induced hypermotility in mice. It is believed that increase of extracellular concentrations of glycine by inhibition of its reuptake may probably influence positively glutamate N-methyl-D-aspartate (NMDA)-type ionotropic receptors in the central nervous system. This may have importance in the treatment of neuropsychiatric disorders associated with hypofunctional NMDA receptor-mediated glutamatergic neurochemical transmission. Thus, impaired NMDA receptor functions have been shown to be involved in the development of the negative symptoms and the cognitive deficit of schizophrenia and the treatment of these symptoms is the possible clinical indication of GlyT-1 inhibitors including

  17. The effect of electron transport (ET) inhibitors and thiabendazole on the fumarate reductase (FR) and succinate dehydrogenase (SDH) of Strongyloides ratti infective (L3) larvae.

    PubMed

    Armson, A; Grubb, W B; Mendis, A H

    1995-02-01

    The fumarate reductase (FR) and succinate dehydrogenase (SDH) activities of isolated submitochondrial particles (SMPs) prepared from axenised L3 larvae of S. ratti were characterised with respect to their response to a selected range of inhibitors. Rotenone (a specific inhibitor of electron transport Complex I) inhibited the S. ratti FR (EC50 = 3.0 x 10(-7) M) but not SDH. This strongly suggests that the S. ratti FR is functionally linked with the S. ratti ET-Complex I. 2-Thenoyltrifluoroacetone (TTFA, an inhibitor of ET-Complex II) inhibited FR (EC50 = 2.6 x 10(-5) M) and SDH (EC50 = 2.8 x 10(-5) M) with similar effectiveness. Sodium malonate (substrate analogue of succinate) had a greater affinity for SDH (EC50 = 6.8 x 10(-4) M), than FR (EC50 = 1.9 x 10(-2) M). Sodium fumarate was ca. 8-fold more effective in inhibiting the S. ratti FR (EC50 = 6.0 x 10(-4) M) than SDH (EC50 = 4.8 x 10(-3) M). The S. ratti FR was more sensitive to inhibition by thiabendazole (TBZ; EC50 = 4.6 x 10(-4) M) than SDH (EC50 > 1.0 x 10(-3) M), suggesting that one of the sites-of-action of TBZ to be the FR of S. ratti mitochondria. More potent inhibitors of S. ratti FR, if developed, may prove to be effective chemotherapeutic agents in the management of human strongloidiasis.

  18. Stimulation of glucose metabolism in human blood cells by inhibitors of carnitine-dependent fatty acid transport.

    PubMed

    Haeckel, R; Colic, D; Binder, L; Oellerich, M

    1990-05-01

    According to a well accepted hypothesis, increased fatty acid oxidation can lead to hyperglycaemia by stimulating gluconeogenesis and reducing glycolysis. Therefore, inhibitors of fatty acid metabolism should cause hypoglycaemia by inhibiting gluconeogenesis and activating glycolysis. Various substances were tested to validate this hypothesis with regard to glucose oxidation in human mononuclear leukocytes and thrombocytes. 2-(3-Methyl-cinnamyl-hydrazono)-propionate, an inhibitor of the carnitine acyltransfer system was found to cause hypoglycaemia in whole animals and to inhibit gluconeogensis in the perfused guinea pig liver, while the acetyl-CoA/CoASH ratio was decreased. This substance stimulated the metabolism of glucose to CO2 in human mononuclear leukocytes and especially in platelets. This effect could be potentiated if concanavalin A and 2-(3-methyl-cinnamyl-hydrazono)-propionate were applied simultaneously. Under these conditions, however, fatty acid oxidation was no longer inhibited. From these results, it can be concluded that the activation of glucose oxidation by 2-(3-methyl-cinnamyl-hydrazono)-propionate is independent of its effect on fatty acid metabolism. Other inhibitors of fatty acid metabolism which were also investigated behaved similarly.

  19. Analysis of anandamide- and lysophosphatidylinositol-induced inhibition of the vasopressor responses produced by sympathetic stimulation or noradrenaline in pithed rats.

    PubMed

    Marichal-Cancino, Bruno A; Manrique-Maldonado, Guadalupe; Altamirano-Espinoza, Alain H; Ruiz-Salinas, Inna; González-Hernández, Abimael; Maassenvandenbrink, Antoinette; Villalón, Carlos M

    2013-12-05

    The endocannabinoid system exhibits multiple functions in cardiovascular regulation mainly by cannabinoid (CB1 and CB2) receptors, vanilloid TRPV1 receptors and, probably, by the orphan G protein-coupled receptor 55 (GPR55). Hence, the role of these receptors was investigated in Wistar pithed rats on anandamide- and lysophosphatidylinositol (LPI)-induced inhibition of the vasopressor responses induced by preganglionic (T7-T9) stimulation of the vasopressor sympathetic outflow or i.v. bolus injections of noradrenaline. The corresponding frequency- and dose-dependent vasopressor responses were analyzed before and during i.v. continuous infusions of anandamide (CB1, CB2, TRPV1 and GPR55), JWH-015 (CB2) and LPI (GPR55) in animals receiving (i.v.) the antagonists NIDA41020 (CB1), AM630 (CB2), capsazepine (TRPV1) and/or cannabidiol (GPR55). Anandamide (0.1-3.1 μg/kg min) inhibited the vasopressor responses by electrical stimulation, but not those by noradrenaline; while LPI (5.6-10 μg/kg min) inhibited both responses. In contrast, JWH-015 (5.6-10 μg/kg min) failed to induce sympatho-inhibition. Anandamide-induced sympatho-inhibition was: (i) dose-dependently blocked by 31 and 100 μg/kg NIDA41020; (ii) slightly blocked by 310 μg/kg AM630 or 31 μg/kg cannabidiol; and (iii) unaffected by 310 μg/kg capsazepine. Moreover, LPI-induced inhibition of both vasopressor responses was blocked and abolished by 10 and 31 μg/kg cannabidiol, respectively, and weakly blocked by 100 μg/kg NIDA41020. Thus, the sympatho-inhibition by anandamide is primarily mediated by cannabinoid CB1 and, minimally, by cannabidiol-sensitive receptors. In contrast, LPI-induced inhibition of both responses seems to be mainly mediated by postjunctional cannabidiol-sensitive (presumably endothelial GPR55) receptors.

  20. Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study

    PubMed Central

    Björklund, Emmelie; Blomqvist, Anders; Hedlin, Joel; Persson, Emma; Fowler, Christopher J.

    2014-01-01

    Background The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process of cellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces the observed uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentration gradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differences are methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the role of FAAH in gating the uptake of AEA. Methodology/Principal Findings The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1 rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. Semi-quantitative PCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of a transcript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four could accumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in the sensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor, with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. The four cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggesting that the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5. Conclusions/Significance When assayed using the same methodology, different FAAH-expressing cells display different sensitivities of uptake to FAAH inhibition. PMID:25078278

  1. The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding.

    PubMed

    Knauf, P A; Raha, N M; Spinelli, L J

    2000-02-01

    WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.

  2. Assessment of the in vitro binding of JHW 007, a dopamine transport inhibitor that blocks the effects of cocaine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Benztropine (BZT) and its analogues, like cocaine, bind to the dopamine transporter and block dopamine uptake. However, while BZT analogues bind the DAT with high affinity, they generally do not have cocaine-like behavioral effects. JHW 007 is a BZT analogue that displaces [3H]WIN 35,428 from the D...

  3. Ginsenosides, ingredients of the root of Panax ginseng, are not substrates but inhibitors of sodium-glucose transporter 1.

    PubMed

    Gao, Shengli; Kushida, Hirotaka; Makino, Toshiaki

    2017-01-01

    Recent pharmacokinetic studies have revealed that ginsenosides, the major ingredients of ginseng (the roots of Panax ginseng), are present in the plasma collected from subjects receiving ginseng, and speculated that ginsenosides might be actively transported via glucose transporters. We evaluated whether ginsenosides Rb1 and Rg1, and their metabolites from enteric bacteria act as substrates of sodium-glucose cotransporter (SGLT) 1, the major glucose transporter expressed on the apical side of intestinal epithelial cells. First, we evaluated the competing effects of ginseng extract and ginsenosides on the uptake of [(14)C]methyl-glucose, a substrate of SGLT1, by SGLT1-overexpressing HEK293 cells. A boiling water extract of ginseng inhibited SGLT1 in a concentration-dependent manner with an IC50 value of 0.85 mg/ml. By activity-guided fractionation, we determined that the fraction containing ginsenosides displayed an inhibitory effect on SGLT1. Of the ginsenosides evaluated, protopanaxatriol-type ginsenosides were not found to inhibit SGLT1, whereas protopanaxadiol-type ginsenosides, including ginsenosides Rd, Rg3, Rh2, F2 and compound K, exhibited significant inhibitory effects on SGLT1, with ginsenoside F2 having the highest activity with an IC50 value of 23.0 µM. Next, we measured the uptake of ginsenoside F2 and compound K into Caco-2 cells, a cell line frequently used to evaluate the intestinal absorption of drugs. The uptake of ginsenoside F2 and compound K into Caco-2 cells was not competitively inhibited by glucose. Furthermore, the uptake of ginsenoside F2 and compound K into SGLT1-overexpressing HEK293 cells was not significantly higher than into mock cells. Ginsenoside F2 and compound K did not appear to be substrates of SGLT1, although these compounds could inhibit SGLT1. Ginsenosides might be absorbed by passive diffusion through the intestinal membrane or actively transported via unknown transporters other than SGLT1.

  4. Alterations in brain extracellular dopamine and glycine levels following combined administration of the glycine transporter type-1 inhibitor Org-24461 and risperidone.

    PubMed

    Nagy, Katalin; Marko, Bernadett; Zsilla, Gabriella; Matyus, Peter; Pallagi, Katalin; Szabo, Geza; Juranyi, Zsolt; Barkoczy, Jozsef; Levay, Gyorgy; Harsing, Laszlo G

    2010-12-01

    The most dominant hypotheses for the pathogenesis of schizophrenia have focused primarily upon hyperfunctional dopaminergic and hypofunctional glutamatergic neurotransmission in the central nervous system. The therapeutic efficacy of all atypical antipsychotics is explained in part by antagonism of the dopaminergic neurotransmission, mainly by blockade of D(2) dopamine receptors. N-methyl-D-aspartate (NMDA) receptor hypofunction in schizophrenia can be reversed by glycine transporter type-1 (GlyT-1) inhibitors, which regulate glycine concentrations at the vicinity of NMDA receptors. Combined drug administration with D(2) dopamine receptor blockade and activation of hypofunctional NMDA receptors may be needed for a more effective treatment of positive and negative symptoms and the accompanied cognitive deficit in schizophrenia. To investigate this type of combined drug administration, rats were treated with the atypical antipsychotic risperidone together with the GlyT-1 inhibitor Org-24461. Brain microdialysis was applied in the striatum of conscious rats and determinations of extracellular dopamine, DOPAC, HVA, glycine, glutamate, and serine concentrations were carried out using HPLC/electrochemistry. Risperidone increased extracellular concentrations of dopamine but failed to influence those of glycine or glutamate measured in microdialysis samples. Org-24461 injection reduced extracellular dopamine concentrations and elevated extracellular glycine levels but the concentrations of serine and glutamate were not changed. When risperidone and Org-24461 were added in combination, a decrease in extracellular dopamine concentrations was accompanied with sustained elevation of extracellular glycine levels. Interestingly, the extracellular concentrations of glutamate were also enhanced. Our data indicate that coadministration of an antipsychotic with a GlyT-1 inhibitor may normalize hypofunctional NMDA receptor-mediated glutamatergic neurotransmission with reduced

  5. Fetzima (levomilnacipran), a drug for major depressive disorder as a dual inhibitor for human serotonin transporters and beta-site amyloid precursor protein cleaving enzyme-1.

    PubMed

    Rizvi, Syed Mohd Danish; Shaikh, Sibhghatulla; Khan, Mahiuddin; Biswas, Deboshree; Hameed, Nida; Shakil, Shazi

    2014-01-01

    Pharmacological management of Major Depressive Disorder includes the use of serotonin reuptake inhibitors which targets serotonin transporters (SERT) to increase the synaptic concentrations of serotonin. Beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) is responsible for amyloid β plaque formation. Hence it is an interesting target for Alzheimer's disease (AD) therapy. This study describes molecular interactions of a new Food and Drug Administration approved antidepressant drug named 'Fetzima' with BACE-1 and SERT. Fetzima is chemically known as levomilnacipran. The study has explored a possible link between the treatment of Depression and AD. 'Autodock 4.2' was used for docking study. The free energy of binding (ΔG) values for 'levomilnacipran-SERT' interaction and 'levomilnacipran-BACE1' interaction were found to be -7.47 and -8.25 kcal/mol, respectively. Levomilnacipran was found to interact with S438, known to be the most important amino acid residue of serotonin binding site of SERT during 'levomilnacipran-SERT' interaction. In the case of 'levomilnacipran-BACE1' interaction, levomilnacipran interacted with two very crucial aspartic acid residues of BACE-1, namely, D32 and D228. These residues are accountable for the cleavage of amyloid precursor protein and the subsequent formation of amyloid β plaques in AD brain. Hence, Fetzima (levomilnacipran) might act as a potent dual inhibitor of SERT and BACE-1 and expected to form the basis of a future dual therapy against depression and AD. It is an established fact that development of AD is associated with Major Depressive Disorder. Therefore, the design of new BACE-1 inhibitors based on antidepressant drug scaffolds would be particularly beneficial.

  6. Design, synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease.

    PubMed

    Toda, Narihiro; Tago, Keiko; Marumoto, Shinji; Takami, Kazuko; Ori, Mayuko; Yamada, Naho; Koyama, Kazuo; Naruto, Shunji; Abe, Kazumi; Yamazaki, Reina; Hara, Takao; Aoyagi, Atsushi; Abe, Yasuyuki; Kaneko, Tsugio; Kogen, Hiroshi

    2003-05-01

    We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.

  7. Benefits/risks of sodium-glucose co-transporter 2 inhibitor canagliflozin in women for the treatment of Type 2 diabetes.

    PubMed

    Kushner, Pamela

    2016-06-01

    Sodium-glucose co-transporter 2 (SGLT2) inhibitors, such as canagliflozin, are used in patients with Type 2 diabetes mellitus (T2DM). In clinical studies, canagliflozin significantly reduced A1C, bodyweight and blood pressure, and was generally well tolerated with no increased risk of hypoglycemia. Most common adverse effects observed were genital mycotic infections and urinary tract infections, and increased urination. Approximately 10% of women treated with canagliflozin experienced a genital mycotic infection compared with 3% treated with placebo; those with a prior history were at greater risk. Approximately 9% of women treated with canagliflozin reported a urinary tract infection compared with 7% treated with placebo. Most adverse events were considered mild to moderate in intensity and responded to standard therapy. Treatment with canagliflozin was effective and generally well tolerated in both women (and men) with T2DM.

  8. Phencyclidine-induced cognitive deficits in mice are improved by subsequent subchronic administration of the glycine transporter-1 inhibitor NFPS and D-serine.

    PubMed

    Hashimoto, Kenji; Fujita, Yuko; Ishima, Tamaki; Chaki, Shigeyuki; Iyo, Masaomi

    2008-06-01

    Accumulating evidence suggests that the glycine modulatory site on the NMDA receptor could be potential therapeutic target for cognitive deficits in schizophrenia. The present study was undertaken to examine the effects of the glycine transporter-1 (GlyT-1) inhibitor, (R)-(N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine (NFPS), on cognitive deficits in mice after repeated administration of the NMDA receptor antagonist phencyclidine (PCP). PCP (10 mg/kg/day for 10 days)-induced cognitive deficits were significantly improved by subsequent subchronic (2-week) administration of NFPS (1.0 and 3.0 mg/kg/day) or D-serine (600 mg/kg/day). However, PCP-induced cognitive deficits were not improved by a single administration of NFPS (3.0 mg/kg). Furthermore, Western blot analysis revealed that levels of GlyT-1 in the hippocampus, but not frontal cortex, of the PCP (10 mg/kg/day for 10 days)-treated mice were significantly higher than those of saline-treated mice. An in vivo microdialysis study revealed that repeated PCP administration significantly decreased the extracellular levels of glycine in the hippocampus, but not frontal cortex, of mice. These findings suggest that repeated PCP administration increased the density of GlyT-1 in the hippocampus of mouse brain, and that the GlyT-1 inhibitor NFPS could ameliorate cognitive deficits in mice after repeated administration of PCP.

  9. The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB▿ †

    PubMed Central

    Garvey, Mark I.; Piddock, Laura J. V.

    2008-01-01

    One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer. PMID:18362193

  10. L-Cysteine/D,L-homocysteine-regulated ileum motility via system L and B°(,+) transporter: Modification by inhibitors of hydrogen sulfide synthesis and dietary treatments.

    PubMed

    Yamane, Satoshi; Nomura, Ryouya; Yanagihara, Madoka; Nakamura, Hiroyuki; Fujino, Hiromichi; Matsumoto, Kenjiro; Horie, Syunji; Murayama, Toshihiko

    2015-10-05

    Previous studies including ours demonstrated that L-cysteine treatments decreased motility in gastrointestinal tissues including the ileum via hydrogen sulfide (H2S), which is formed from sulfur-containing amino acids such as L-cysteine and L-homocysteine. However, the amino acid transport systems involved in L-cysteine/L-homocysteine-induced responses have not yet been elucidated in detail; therefore, we investigated these systems pharmacologically by measuring electrical stimulation (ES)-induced contractions with amino acids in mouse ileum preparations. The treatments with L-cysteine and D,L-homocysteine inhibited ES-induced contractions in ileum preparations from fasted mice, and these responses were decreased by the treatment with 2-aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), an inhibitor of systems L and B°(,+). The results obtained using ileum preparations and a model cell line (PC12 cells) with various amino acids and BCH showed that not only L-cysteine, but also aminooxyacetic acid and D,L-propargylglycine, which act as H2S synthesis inhibitors, appeared to be taken up by these preparations/cells in L and B°(,+) system-dependent manners. The L-cysteine and D,L-homocysteine responses were delayed and abolished, respectively, in ileum preparations from fed mice. Our results suggested that the regulation of ileum motility by L-cysteine and D,L-homocysteine was dependent on BCH-sensitive systems, and varied depending on feeding in mice. Therefore, the effects of aminooxyacetic acid and D,L-propargylglycine on transport systems need to be considered in pharmacological analyses.

  11. TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.

    PubMed

    Kalverda, Arnout P; Gowdy, James; Thompson, Gary S; Homans, Steve W; Henderson, Peter J F; Patching, Simon G

    2014-06-01

    Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

  12. The small-molecule iron transport inhibitor ferristatin/NSC306711 promotes degradation of the transferrin receptor.

    PubMed

    Horonchik, Lior; Wessling-Resnick, Marianne

    2008-07-21

    Iron delivery by transferrin (Tf) is accomplished through clathrin-mediated endocytosis of Tf receptors. The small molecule NSC306711 inhibits iron uptake from the Tf-TfR pathway. Here we show that the drug's mechanism of action is to induce internalization and degradation of unoccupied Tf receptors through an unexpected endocytic pathway. Unlike classical clathrin-mediated Tf receptor endocytosis, internalization promoted by NSC306711 is independent of clathrin and dynamin, and is sensitive to the cholesterol-depleting agents filipin and nystatin. The finding of this cholesterol-dependent Tf receptor internalization pathway through use of the small-molecule inhibitor sheds light on the pleiotropic nature of membrane trafficking dynamics and adds a complex dimension to our understanding of receptor regulation. Because of its unusual properties to inhibit iron uptake, we refer to NSC306711 as "ferristatin."

  13. Effect of electron-transport inhibitors on the generation of reactive oxygen species by pea mitochondria during succinate oxidation.

    PubMed

    Popov, V N; Ruuge, E K; Starkov, A A

    2003-07-01

    The effect of inhibitors of the cytochrome pathway and alternative oxidase on the rate of respiration and generation of reactive oxygen species by pea mitochondria was studied. Respiration of mitochondria from pea cotyledons was inhibited by 70-80% by salicylhydroxamate (SHAM). The rate of hydrogen peroxide production by pea cotyledon mitochondria during succinate oxidation was 0.15 nmol/min per mg protein. SHAM considerably accelerated the hydrogen peroxide production. The SHAM-dependent H2O2 production was stimulated by 2 micro M antimycin A and inhibited by 5 mM KCN and 1 micro M myxothiazol. The study of the rate of O2*- generation by pea mitochondria using EPR spin traps and epinephrine oxidation showed that H2O2 accumulation can be accounted for by a significant increase in the rate of O2*- production.

  14. Coupling between H+ transport and anaerobic glycolysis in turtle urinary bladder: effect of inhibitors of H+ ATPase

    SciTech Connect

    Steinmetz, P.R.; Husted, R.F.; Mueller, A.; Beauwens, R.

    1981-03-15

    The coupling between H+ transport (JH) and anaerobic glycolysis was examined in vitro in an anaerobic preparation of turtle urinary bladder. JH was measured as the short-circuit current after Na+ transport was abolished with ouabain and by pH stat titration. The media were gassed with N2 and 1% CO2 (PO2 less than 0.5 mm Hg) and contained 10 mM glucose. Under these conditions, JH was not inhibited by 3 mM serosal (S) cyanide or by 0.1 mM mucosal (M) dinitrophenol. Control anaerobic lactate production (Jlac) of 47 bladders was plotted as a function of simultaneously measured JH. The slope of Jlac on JH was 0.58

  15. Novel compound, organic cation transporter 3 detection agent and organic cation transporter 3 activity inhibitor, WO2015002150 A1: a patent evaluation.

    PubMed

    Hu, Tao; Wang, Li; Pan, Xiaolei; Qi, Hualin

    2016-08-01

    Increasing pharmacological studies have demonstrated that organic cation transporter 3 (OCT3) plays an important role in controlling the extracellular concentrations of released monoamine neurotransmitter, suggesting that OCT3 might be a promising target in the treatment of depression. As a consequence, compounds showing inhibitory effects on the function of OCT3 have the potential for depression treatment. The current patent WO2015002150 A1 described the synthesis of 59 novel guanidine derivatives. All investigated compounds exhibited significant inhibitory effects (41.9-88.2%) on human OCT3 activity at 30 µM, using human OCT3-transfected human embryonic kidney 293 cell. Concentration-response curves (IC50 values) were determined for seven compounds with higher inhibition potency from the initial screening. IC50 values ranged from 1.9 to 24 µM. In addition, the concentration of these compound in aqueous solution with artificial membranes containing human OCT3 protein was measured. The concentration of compound 6 (SR-2045) was significantly reduced in the presence of human OCT3. Therefore, these compounds have the potential to be further developed as novel antidepressant and human OCT3 detection agent. Future investigations are needed to study the pharmacokinetic and pharmacological properties of these compounds and potential interaction with other transporters.

  16. Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain

    PubMed Central

    Parvathy, Subramanian S.

    2016-01-01

    Background There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1) whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP) might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. Methods The reaction latency to thermal stimuli (hot plate test; at 55 °C) and cold stimuli (cold plate test; at 4 °C) of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. Results The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. Conclusions These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711’s antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1 inhibitors

  17. Effect of Sodium-Glucose Co-Transporter 2 Inhibitor, Dapagliflozin, on Renal Renin-Angiotensin System in an Animal Model of Type 2 Diabetes

    PubMed Central

    Shin, Seok Joon; Chung, Sungjin; Kim, Soo Jung; Lee, Eun-Mi; Yoo, Young-Hye; Kim, Ji-Won; Ahn, Yu-Bae; Kim, Eun-Sook; Moon, Sung-Dae; Kim, Myung-Jun

    2016-01-01

    Background Renal renin-angiotensin system (RAS) activation is one of the important pathogenic mechanisms in the development of diabetic nephropathy in type 2 diabetes. The aim of this study was to investigate the effects of a sodium-glucose co-transporter 2 (SGLT-2) inhibitor, dapagliflozin, on renal RAS in an animal model with type 2 diabetes. Methods Dapagliflozin (1.0 mg/kg, OL-DA) or voglibose (0.6 mg/kg, OL-VO, diabetic control) (n = 10 each) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats for 12 weeks. We used voglibose, an alpha-glucosidase inhibitor, as a comparable counterpart to SGLT2 inhibitor because of its postprandial glucose-lowering effect without proven renoprotective effects. Control Long-Evans Tokushima Otsuka (LT) and OLETF (OL-C) rats received saline (n = 10, each). Changes in blood glucose, urine albumin, creatinine clearance, and oxidative stress were measured. Inflammatory cell infiltration, mesangial widening, and interstitial fibrosis in the kidney were evaluated by histological analysis. The effects of dapagliflozin on renal expression of the RAS components were evaluated by quantitative RT-PCR in renal tissue. Results After treatment, hyperglycemia and urine microalbumin levels were attenuated in both OL-DA and OL-VO rather than in the OL-C group (P < 0.05). The urine angiotensin II (Ang II) and angiotensinogen levels were significantly decreased following treatment with dapagliflozin or voglibose, but suppression of urine Ang II level was more prominent in the OL-DA than the OL-VO group (P < 0.05). The expressions of angiotensin type 1 receptor and tissue oxidative stress markers were markedly increased in OL-C rats, which were reversed by dapagliflozin or voglibose (P < 0.05, both). Inflammatory cell infiltration, mesangial widening, interstitial fibrosis, and total collagen content were significantly increased in OL-C rats, which were attenuated in OL-DA group (P < 0.05). Conclusion Dapagliflozin treatment showed

  18. Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram

    PubMed Central

    2015-01-01

    Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (Ki = 24–227 nM) for hSERT, as assessed by [3H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [125I]15 compared to that by [125I]22 and [125I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug–protein binding interactions for (S)-citalopram at hSERT. PMID:26153715

  19. Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram.

    PubMed

    Kumar, Vivek; Yarravarapu, Nageswari; Lapinsky, David J; Perley, Danielle; Felts, Bruce; Tomlinson, Michael J; Vaughan, Roxanne A; Henry, L Keith; Lever, John R; Newman, Amy Hauck

    2015-07-23

    Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

  20. Interaction of chloroplasts with inhibitors: effects of two diphenylether herbicides, fomesafen and nitrofluorfen, on electron transport, and some comparisons with dibromothymoquinone, diuron, and paraquat.

    PubMed

    Ridley, S M

    1983-06-01

    Several effects on pea (Pisum sativum L. var Onwards) chloroplasts of a new diphenylether herbicide, fomesafen (5-[2-chloro-4-trifluoromethyl-phenoxy]-N-methanesulfonyl-2 -nitrobenzamide) have been compared with those of a herbicide of related structure, nitrofluorfen (2-chloro-1-[4-nitrophenoxy]-4-[trifluoromethyl]benzene). Although both compounds produce the same light-dependent symptoms of desiccation and chlorosis indicative of a common primary mechanism of action, this study is concerned with a more broadly based investigation of different effects on the electron transport system. Comparisons have also been made with other compounds interacting with the chloroplast. Unlike nitrofluorfen, fomesafen has little effect as an inhibitor of electron flow or energy transfer. Both compounds have the ability to stimulate superoxide production through a functional electron transport system, and this involves specifically the p-nitro substituent. The stimulation, which is not likely to be an essential part of the primary herbicidal effect, is diminished under conditions that remove the coupling factor. Evidence suggests that both diphenylethers may be able to bind to the coupling factor, and kinetic studies reveal this for dibromothymoquinone as well. Such a binding site might be an important feature in allowing the primary effect of the diphenylether herbicides to be expressed.

  1. Human organic cation transporter 2 (hOCT2): Inhibitor studies using S2-hOCT2 cells.

    PubMed

    Chiba, Shoetsu; Ikawa, Toru; Takeshita, Hiroshi; Kanno, Sanae; Nagai, Tomonori; Takada, Meri; Mukai, Toshiji; Wempe, Michael F

    2013-08-09

    Highly expressed in kidney and located on the basolateral membrane, human organic cation transporter 2 (hOCT2) can transport various compounds (i.e. drugs and toxins) into the proximal tubular cell. Using cultured proximal tubule cells stably expressing hOCT2 (i.e. S2-hOCT2 cells), we sought to probe different compound classes (e.g. analgesics, anti-depressants, anti-psychotics, disinfectant, herbicides, insecticides, local anesthetic, muscarinic acetylcholine receptor antagonist, sedatives, steroid hormone, stimulants and toxins) for their ability to inhibit (14)C-TEA uptake, a prototypical OCT2 substrate. Aconitine, amitriptyline, atropine, chlorpyrifos, diazepam, fenitrothion, haloperidol, lidocaine, malathion, mianserin, nicotine and triazolam significantly inhibited (14)C-TEA uptake; IC50 values were 59.2, 2.4, 2.0, 20.7, 32.3, 13.2, 32.5, 104.6, 71.1, 17.7, 52.8 and 65.5μM, respectively. In addition, aconitine, amitriptyline, atropine, chlorpyrifos, fenitrothion, haloperidol, lidocaine, and nicotine displayed competitive inhibition with Ki values of 145.6, 2.5, 2.4, 24.8, 16.9, 51.6, 86.8 and 57.7μM, respectively. These in vitro data support the notion that compounds pertaining to a wide variety of different drug classes have the potential to decrease renal clearance of drugs transported via hOCT2. Consequently, these data warrant additional studies to probe hOCT2 and its role to influence drug pharmacokinetics.

  2. Substrate-analogous inhibitors exert antimalarial action by targeting the Plasmodium lactate transporter PfFNT at nanomolar scale

    PubMed Central

    Golldack, André; Henke, Björn; Wiechert, Marie; Blancke Soares, Alexandra

    2017-01-01

    Resistance against all available antimalarial drugs calls for novel compounds that hit unexploited targets in the parasite. Here, we show that the recently discovered Plasmodium falciparum lactate/proton symporter, PfFNT, is a valid druggable target, and describe a new class of fluoroalkyl vinylogous acids that potently block PfFNT and kill cultured parasites. The original compound, MMV007839, is derived from the malaria box collection of potent antimalarials with unknown targets and contains a unique internal prodrug principle that reversibly switches between a lipophilic transport form and a polar, substrate-analogous active form. Resistance selection of cultured P. falciparum parasites with sub-lethal concentrations of MMV007839 produced a single nucleotide exchange in the PfFNT gene; this, and functional characterization of the resulting PfFNT G107S validated PfFNT as a novel antimalarial target. From quantitative structure function relations we established the compound binding mode and the pharmacophore. The pharmacophore largely circumvents the resistance mutation and provides the basis for a medicinal chemistry program that targets lactate and proton transport as a new mode of antimalarial action. PMID:28178358

  3. Metabolic and hemodynamic effects of sodium-dependent glucose co-transporter 2 inhibitors on cardio-renal protection in the treatment of patients with type 2 diabetes mellitus.

    PubMed

    Kashiwagi, Atsunori; Maegawa, Hiroshi

    2017-02-08

    The specific Na+/glucose co-transporter 2 inhibitors (SGLT2 inhibitors) inhibit glucose reabsorption in proximal renal tubular cells, and both fasting and postprandial glucose significantly decrease due to urinary glucose loss. As a result, pancreatic β-cell function and peripheral insulin action significantly improve with relief from glucose toxicity. Furthermore, whole body energy metabolism changes to relative glucose deficiency and triggers increased lipolysis in fat cells, and fatty acid oxidation and then ketone body production in the liver during treatment with SGLT2 inhibitors. In addition, SGLT2 inhibitors have profound hemodynamic effects including diuresis, dehydration, weight loss, and lowering blood pressure. The most recent findings on SGLT2 inhibitors come from results of the Empagliflozin, cardiovascular outcomes and mortality in type 2 diabetes trial. SGLT2 inhibitors exert extremely unique and cardio-renal protection through metabolic and hemodynamic effects with long-term durability on the reduction of blood glucose, body weight, and blood pressure. Although a site of action of SGLT2 inhibitors is highly specific to inhibit renal glucose reabsorption, whole body energy metabolism and hemodynamic and renal functions are profoundly modulated during the treatment of SGLT2 inhibitors. Previous studies suggest multifactorial clinical benefits and safety concerns of SGLT2 inhibitors. Although ambivalent clinical results of this drug are still under active discussion, the present review summarizes promising recent evidences on the cardio-renal and metabolic benefits of SGLT2 inhibitors in the treatment of type 2 diabetes. This article is protected by copyright. All rights reserved.

  4. Volume-activated amino acid efflux from term human placental tissue: stimulation of efflux via a pathway sensitive to anion transport inhibitors.

    PubMed

    Shennan, D B; McNeillie, S A

    1995-04-01

    The effect of a hyposmotic challenge and hence cell-swelling upon the efflux of a variety of solutes from isolated human placental tissue has been examined. A hyposmotic shock increased the fractional release of taurine, the most abundant free amino acid in placental tissue, via a pathway sensitive to niflumic acid, DIDS (4,4'-Diisothiocyanatostilbene-2',2'-disulphonic acid,) NPPB (5-Nitro-2(3-phenylpropylamino)benzoic acid) and DIOA (R(+)[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden -5-y) oxy] acetic acid). In contrast, tamoxifen was without effect. The cell-swelling induced efflux of taurine was attenuated (40 per cent) by replacing external Cl- with NO3-. The efflux of glutamic acid was also markedly increased by a hyposmotic challenge. Niflumic acid inhibited both basal and volume-activated glutamic acid efflux. A hyposmotic shock also increased alpha-aminoisobutyric acid efflux but not that of 3-O-methylglucose and SO4(2)-. The results suggest that the human placenta can respond to cell-swelling by releasing organic osmolytes such as amino acids via a pathway which is sensitive to anion transport inhibitors. However, it appears that the volume-activated amino acid transport system is independent from the placental anion-exchange pathways. The efflux of these compounds may act with K+ and Cl- efflux to effect a regulatory volume decrease in placental tissue. In addition, volume-activated transport may play a role in transplacental amino acid transfer.

  5. Tesofensine, a novel triple monoamine re-uptake inhibitor with anti-obesity effects: dopamine transporter occupancy as measured by PET.

    PubMed

    Appel, Lieuwe; Bergström, Mats; Buus Lassen, Jørgen; Långström, Bengt

    2014-02-01

    Tesofensine (TE) is a novel triple monoamine re-uptake inhibitor inducing a potent inhibition of the re-uptake process in the synaptic cleft of the neurotransmitters dopamine, norepinephrine, and serotonin. In recent preclinical and clinical evaluations TE showed a robust anti-obesity effect, but the specific mechanism of this triple monoamine re-uptake inhibitor still needs to be further elucidated. This positron emission tomography (PET) study, using [¹¹C]βCIT-FE, aimed to assess the degree of the dopamine transporter (DAT) occupancy, at constant TE plasma levels, following different oral, multiple doses of TE during totally 8-12 days. In addition, the relationships between DAT occupancy and TE plasma concentrations, or doses, were investigated to enable assessment of DAT occupancies in subsequent clinical trials. The results demonstrated that TE induced a dose-dependent blockade of DAT following multiple doses of 0.125-1 mg TE at anticipated steady-state conditions. The mean striatal DAT occupancy varied dose-dependently between 18% and 77%. A sigmoid E(max) model well described the relationship between striatal DAT occupancy and TE plasma concentrations or doses. It was estimated that the maximum achievable DAT occupancy was about 80% and that half of this effect was accomplished by approximately 0.25 mg TE and a plasma drug concentration of 4 ng/ml. The results indicated an important mechanism of action of TE on DAT. Further, these results suggest that the previously reported dose-dependent weight loss, in TE treated subjects, was in part mediated by an up-regulation of dopaminergic pathways due to enhanced amounts of synaptic dopamine after blockade of DAT.

  6. Dynamic Nuclear Polarization Study of Inhibitor Binding to the M218–60 Proton Transporter from Influenza A

    PubMed Central

    Andreas, Loren B.; Barnes, Alexander B.; Corzilius, Björn; Chou, James J.; Miller, Eric A.; Caporini, Marc; Rosay, Melanie; Griffin, Robert G

    2013-01-01

    We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M218–60 from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the 15N labeled ammonium of rimantadine was observed near A30 13Cβ and G34 13Cα suggesting a possible hydrogen bond to A30 Carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4–6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å. PMID:23480101

  7. FLUID AND ION SECRETION BY MALPIGHIAN TUBULES OF LARVAL CHIRONOMIDS, Chironomus riparius: EFFECTS OF REARING SALINITY, TRANSPORT INHIBITORS, AND SEROTONIN.

    PubMed

    Zadeh-Tahmasebi, Melika; Bui, Phuong; Donini, Andrew

    2016-10-01

    Larvae of Chironomus riparius respond to ion-poor and brackish water (IPW, BW) conditions by activating ion uptake mechanisms in the anal papillae and reducing ion absorption at the rectum, respectively. The role that the Malpighian tubules play in ion and osmoregulation under these conditions is not known in this species. This study examines rates of fluid secretion and major cation composition of secreted fluid from tubules of C. riparius reared in IPW, freshwater (FW) and BW. Fluid secretion of tubules from FW and BW larvae was similar but tubules from IPW larvae secrete fluid at higher rates, are more sensitive to serotonin stimulation, and the secreted fluid contains less Na(+) . Therefore in IPW, tubules work in concert with anal papillae to eliminate excess water while conserving Na(+) in the hemolymph. Tubules do not appear to play a significant role in ion/osmoregulation under BW. Serotonin immunoreactivity in the nervous system and gastrointestinal tract of larval C. riparius was similar to that seen in mosquito larvae with the exception that the hindgut was devoid of staining. Hemolymph serotonin titer was similar in FW and IPW; hence, serotonin is not responsible for the observed high rates of fluid secretion in IPW. Instead, it is suggested that serotonin may work in a synergistic manner with an unidentified hormonal factor in IPW. Ion transport mechanisms in the tubules of C. riparius are pharmacologically similar to those of other insects.

  8. Dynamic nuclear polarization study of inhibitor binding to the M2(18-60) proton transporter from influenza A.

    PubMed

    Andreas, Loren B; Barnes, Alexander B; Corzilius, Björn; Chou, James J; Miller, Eric A; Caporini, Marc; Rosay, Melanie; Griffin, Robert G

    2013-04-23

    We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the (15)N-labeled ammonium of rimantadine was observed near A30 (13)Cβ and G34 (13)Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ~10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å.

  9. A re-evaluation of 9-HODE activity at TRPV1 channels in comparison with anandamide: enantioselectivity and effects at other TRP channels and in sensory neurons

    PubMed Central

    De Petrocellis, Luciano; Schiano Moriello, Aniello; Imperatore, Roberta; Cristino, Luigia; Starowicz, Katarzyna; Di Marzo, Vincenzo

    2012-01-01

    Background and Purpose Two oxidation products of linoleic acid, 9- and 13-hydroxy-octadecadienoic acids (HODEs), have recently been suggested to act as endovanilloids, that is, endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) channels, thereby contributing to inflammatory hyperalgesia in rats. However, HODE activity at rat TRPV1 in comparison with the best established endovanilloid, anandamide, and its enantioselectivity and selectivity towards other TRP channels that are also abundant in sensory neurons have never been investigated. Experimental Approach We studied the effect of 9(R)-HODE, 9(S)-HODE, (+/–)13-HODE, 15(S)-hydroxyanandamide and anandamide on [Ca2+]i in HEK-293 cells stably expressing the rat or human recombinant TRPV1, or rat recombinant TRPV2, TRPA1 or TRPM8, and also the effect of 9(S)-HODE in rat dorsal root ganglion (DRG) neurons by calcium imaging. Key Results Anandamide and 15(S)-hydroxyanandamide were the most potent endovanilloids at human TRPV1, whereas 9(S)-HODE was approximately threefold less efficacious and 75- and 3-fold less potent, respectively, and did not perform much better at rat TRPV1. The 9(R)-HODE and (+/–)13-HODE were almost inactive at TRPV1. Unlike anandamide and 15(S)-hydroxyanandamide, all HODEs were very weak at desensitizing TRPV1 to the action of capsaicin, but activated rat TRPV2 [only (+/–)13-HODE] and rat TRPA1, and antagonized rat TRPM8, at concentrations higher than those required to activate TRPV1. Finally, 9(S)-HODE elevated [Ca2+]i in DRG neurons almost exclusively in capsaicin-sensitive cells but only at concentrations between 25 and 100 μM. Conclusions and Implications The present data suggest that HODEs are less important endovanilloids than anandamide. Linked Articles This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8 PMID:22861649

  10. Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

    PubMed Central

    Martell, R. L.; Slapak, C. A.; Levy, S. B.

    1997-01-01

    The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

  11. Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells.

    PubMed

    Chen, Nianhang; Reith, Maarten E A

    2007-04-01

    The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.

  12. The inhibitory effect of anandamide on oxytocin and vasopressin secretion from neurohypophysis is mediated by nitric oxide.

    PubMed

    Luce, Valeria; Fernandez Solari, Javier; Rettori, Valeria; De Laurentiis, Andrea

    2014-01-10

    The neurohypophyseal hormones oxytocin (OT) and vasopressin (VP) are involved in behavioral, autonomic and neuroendocrine functions. Both peptides are synthesized in magnocellular neurons of paraventricular and supraoptic nuclei at hypothalamic level whose axons terminate in the neurohypophysis (NH), from where OT and VP are released into the systemic circulation. NH contains abundant nitric oxide (NO) synthase suggesting that NO plays a role in the release of these neuropeptides. The endocannabinoid system is present in magnocellular neurons of the hypothalamic neurohypophyseal system, and we have previously demonstrated that endocannabinoids modulate OT secretion at hypothalamic level. In the present work, we investigated the in vitro effect of the endocannabinoid anandamide (AEA) on OT and VP release from NH of untreated adult male rats and the involvement of NO in this action. Our results showed that AEA decreased OT and VP secretion from NH. AEA action was mediated by NO, since the inhibition of NO synthesis completely blocked this inhibitory effect. We found that cannabinoid receptor type 2 (CB2) and transient receptor potential cation channel subfamily V member 1 (TRPV1) are involved in the inhibitory effect of AEA because AM630 and capsazepine, CB2 and TRPV1 antagonists respectively, but not AM251, a CB1 antagonist, blocked AEA effect at neurohypophyseal level. These findings revealed an interaction between endocannabinoid, nitric oxide and oxytocin/vasopressin systems that could be involved in the modulation of homeostatic, behavioral and reproductive processes.

  13. Membrane-mediated action of the endocannabinoid anandamide on membrane proteins: implications for understanding the receptor-independent mechanism

    PubMed Central

    Medeiros, Djalma; Silva-Gonçalves, Laíz da Costa; da Silva, Annielle Mendes Brito; dos Santos Cabrera, Marcia Perez; Arcisio-Miranda, Manoel

    2017-01-01

    Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function. PMID:28128290

  14. Vaccenic acid suppresses intestinal inflammation by increasing anandamide and related N-acylethanolamines in the JCR:LA-cp rat.

    PubMed

    Jacome-Sosa, Miriam; Vacca, Claudia; Mangat, Rabban; Diane, Abdoulaye; Nelson, Randy C; Reaney, Martin J; Shen, Jianheng; Curtis, Jonathan M; Vine, Donna F; Field, Catherine J; Igarashi, Miki; Piomelli, Daniele; Banni, Sebastiano; Proctor, Spencer D

    2016-04-01

    Vaccenic acid (VA), the predominant ruminant-derivedtransfat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cprats were assigned to a control diet with or without VA (1% w/w),cis-9,trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P< 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P> 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P< 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1β (P< 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine.

  15. Membrane-mediated action of the endocannabinoid anandamide on membrane proteins: implications for understanding the receptor-independent mechanism

    NASA Astrophysics Data System (ADS)

    Medeiros, Djalma; Silva-Gonçalves, Laíz Da Costa; da Silva, Annielle Mendes Brito; Dos Santos Cabrera, Marcia Perez; Arcisio-Miranda, Manoel

    2017-01-01

    Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.

  16. Membrane-mediated action of the endocannabinoid anandamide on membrane proteins: implications for understanding the receptor-independent mechanism.

    PubMed

    Medeiros, Djalma; Silva-Gonçalves, Laíz da Costa; da Silva, Annielle Mendes Brito; Dos Santos Cabrera, Marcia Perez; Arcisio-Miranda, Manoel

    2017-01-27

    Endocannabinoids are amphiphilic molecules that play crucial neurophysiological functions acting as lipid messengers. Antagonists and knockdown of the classical CB1 and CB2 cannabinoid receptors do not completely abolish many endocannabinoid activities, supporting the idea of a mechanism independent of receptors whose mode of action remains unclear. Here we combine gramicidin A (gA) single channel recordings and membrane capacitance measurements to investigate the lipid bilayer-modifying activity of endocannabinoids. Single channel recordings show that the incorporation of endocannabinoids into lipid bilayers reduces the free energy necessary for gramicidin channels to transit from the monomeric to the dimeric conformation. Membrane capacitance demonstrates that the endocannabinoid anandamide has limited effects on the overall structure of the lipid bilayers. Our results associated with the theory of membrane elastic deformation reveal that the action of endocannabinoids on membrane proteins can involve local adjustments of the lipid/protein hydrophobic interface. The current findings shed new light on the receptor-independent mode of action of endocannabinoids on membrane proteins, with important implications towards their neurobiological function.

  17. Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats

    PubMed Central

    Cil, Onur; Esteva-Font, Cristina; Tas, Sadik Taskin; Su, Tao; Lee, Sujin; Anderson, Marc O.; Ertunc, Mert; Verkman, A. S.

    2015-01-01

    Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics (‘urearetics’) with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10–20 μM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2–3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 hours, and a urine concentration of 20–40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of Syndrome of Inappropriate Antidiuretic Hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition. PMID:25993324

  18. Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters

    PubMed Central

    Kwatra, Deep; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Khurana, Varun; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Background The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). Methods LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. Results Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. Conclusions In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition. PMID:24399676

  19. HIV-Protease Inhibitors Suppress Skeletal Muscle Fatty Acid Oxidation by Reducing CD36 and CPT-I Fatty Acid Transporters

    PubMed Central

    Richmond, Scott R.; Carper, Michael J.; Lei, Xiaoyong; Zhang, Sheng; Yarasheski, Kevin E.; Ramanadham, Sasanka

    2010-01-01

    Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5 μmol/l) plus 20 μmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase I (CPTI) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV. PMID:20117238

  20. Pharmacophore modeling of nilotinib as an inhibitor of ATP-binding cassette drug transporters and BCR-ABL kinase using a three-dimensional quantitative structure-activity relationship approach.

    PubMed

    Shukla, Suneet; Kouanda, Abdul; Silverton, Latoya; Talele, Tanaji T; Ambudkar, Suresh V

    2014-07-07

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure-activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [(125)I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

  1. Pharmacophore Modeling of Nilotinib as an Inhibitor of ATP-Binding Cassette Drug Transporters and BCR-ABL Kinase Using a Three-Dimensional Quantitative Structure–Activity Relationship Approach

    PubMed Central

    2015-01-01

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure–activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [125I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

  2. A sodium-glucose co-transporter 2 inhibitor empagliflozin prevents abnormality of circadian rhythm of blood pressure in salt-treated obese rats.

    PubMed

    Takeshige, Yui; Fujisawa, Yoshihide; Rahman, Asadur; Kittikulsuth, Wararat; Nakano, Daisuke; Mori, Hirohito; Masaki, Tsutomu; Ohmori, Koji; Kohno, Masakazu; Ogata, Hiroaki; Nishiyama, Akira

    2016-06-01

    Studies were performed to examine the effects of the selective sodium-glucose co-transporter 2 (SGLT2) inhibitor empagliflozin on urinary sodium excretion and circadian blood pressure in salt-treated obese Otsuka Long Evans Tokushima Fatty (OLETF) rats. Fifteen-week-old obese OLETF rats were treated with 1% NaCl (in drinking water), and vehicle (0.5% carboxymethylcellulose, n=10) or empagliflozin (10 mg kg(-1)per day, p.o., n=11) for 5 weeks. Blood pressure was continuously measured by telemetry system. Glucose metabolism and urinary sodium excretion were evaluated by oral glucose tolerance test and high salt challenge test, respectively. Vehicle-treated OLETF rats developed non-dipper type blood pressure elevation with glucose intolerance and insulin resistance. Compared with vehicle-treated animals, empagliflozin-treated OLETF rats showed an approximately 1000-fold increase in urinary glucose excretion and improved glucose metabolism and insulin resistance. Furthermore, empagliflozin prevented the development of blood pressure elevation with normalization of its circadian rhythm to a dipper profile, which was associated with increased urinary sodium excretion. These data suggest that empagliflozin elicits beneficial effects on both glucose homeostasis and hypertension in salt-replete obese states.

  3. Human therapeutic plasma levels of the selective serotonin reuptake inhibitor (SSRI) sertraline decrease serotonin reuptake transporter binding and shelter-seeking behavior in adult male fathead minnows.

    PubMed

    Valenti, Theodore W; Gould, Georgianna G; Berninger, Jason P; Connors, Kristin A; Keele, N Bradley; Prosser, Krista N; Brooks, Bryan W

    2012-02-21

    Selective serotonin reuptake inhibitors (SSRIs) represent a class of pharmaceuticals previously reported in aquatic ecosystems. SSRIs are designed to treat depression and other disorders in humans, but are recognized to elicit a variety of effects on aquatic organisms, ranging from neuroendocrine disruption to behavioral perturbations. However, an understanding of the relationships among mechanistic responses associated with SSRI targets and ecologically important behavioral responses of fish remains elusive. Herein, linking Adverse Outcomes Pathways (AOP) models with internal dosimetry represent potential approaches for developing an understanding of pharmaceutical risks to aquatic life. We selected sertraline as a model SSRI for a 28-d study with adult male fathead minnows. Binding activity of the serotonin reuptake transporter (SERT), previously demonstrated in mammals and fish models to respond to sertraline exposure, was selected as an endpoint associated with therapeutic activity. Shelter-seeking behavior was monitored using digital tracking software to diagnose behavioral abnormalities. Fish plasma levels of sertraline exceeding human therapeutic doses were accurately modeled from external exposure concentrations when pH influences on ionization and log D were considered. We observed statistically significant decreases in binding at the therapeutic target (SERT) and shelter-seeking behavior when fish plasma levels exceeded human therapeutic thresholds. Such observations highlights the strengths of coupling physiologically based pharmacokinetic modeling and AOP approaches and suggest that internal dosimetry should be monitored to advance an understanding of the ecological consequences of SSRI exposure to aquatic vertebrates.

  4. Evaluation of drug-drug interaction between henagliflozin, a novel sodium-glucose co-transporter 2 inhibitor, and metformin in healthy Chinese males.

    PubMed

    Wang, Liupeng; Wu, Chunyong; Shen, Lu; Liu, Haiyan; Chen, Ying; Liu, Fang; Wang, Youqun; Yang, Jin

    2016-08-01

    1. Henagliflozin is a novel sodium-glucose transporter 2 inhibitor and presents a complementary therapy to metformin for patients with T2DM due to its insulin-independent mechanism of action. This study evaluated the potential pharmacokinetic drug-drug interaction between henagliflozin and metformin in healthy Chinese male subjects. 2. In open-label, single-center, single-arm, two-period, three-treatment self-control study, 12 subjects received 25 mg henagliflozin, 1000 mg metformin or the combination. Lack of PK interaction was defined as the ratio of geometric means and 90% confidence interval (CI) for combination: monotherapy being within the range of 0.80-1.25. 3. Co-administration of henagliflozin with metformin had no effect on henagliflozin area under the plasma concentration-time curve (AUC0-24) (GRM: 1.08; CI: 1.05, 1.10) and peak plasma concentration (Cmax) (GRM: 0.99; CI: 0.92, 1.07). Reciprocally, co-administration of metformin with henagliflozin had no clinically significant on metformin AUC0-24 (GRM: 1.09, CI: 1.02, 1.16) although there was an 11% increase in metformin Cmax (GRM 1.12; CI 1.02, 1.23). All monotherapies and combination therapy were well tolerated. 4. Henagliflozin can be co-administered with metformin without dose adjustment of either drug.

  5. Altered response to the selective serotonin reuptake inhibitor escitalopram in mice heterozygous for the serotonin transporter: an electrophysiological and neurochemical study.

    PubMed

    Guiard, Bruno P; Mansari, Mostafa El; Murphy, Dennis L; Blier, Pierre

    2012-04-01

    A serotonin (5-HT) transporter (5-HTT; SERT) polymorphism has been associated with depressive states and poor responses to selective serotonin reuptake inhibitors (SSRIs). Given the similar attenuation of SERT activity in SERT+/- mice and in humans with short allele(s) of SERT in its promoter region, it is conceivable that SERT+/- mice offer an adequate model to mimic the human subpopulation with respect to their altered response to SSRIs. This study investigated the effects of the most selective SSRI escitalopram, in heterozygous SERT+/- mice using a combined electrophysiological and neurochemical approach. Results indicated that administration of escitalopram for 2 d resulted in a 72% and 63% decrease in dorsal raphe 5-HT neuronal firing rate in SERT+/+ and SERT+/- mice, respectively. In contrast, administration of escitalopram for 21 d produced a gradual recovery of 5-HT neuronal firing rate to basal level in SERT+/+, but not in SERT+/- mice. In the hippocampus, microdialysis revealed that sustained administration of escitalopram produced a greater increase in extracellular 5-HT ([5-HT]ext) outflow in SERT+/- than in the wild-types with or without a washout of the SSRI. Nevertheless, the ability of microiontophoretically applied 5-HT to inhibit the firing rate of CA3 pyramidal neurons was not different between SERT+/+ and SERT+/- mice given escitalopram for 21 d. The data indicate that the poor response to SSRIs of depressive patients with short allele(s) of SERT is not attributable to a lesser increase in 5-HT transmission in the hippocampus.

  6. Safe and pragmatic use of sodium–glucose co-transporter 2 inhibitors in type 2 diabetes mellitus: South Asian Federation of Endocrine Societies consensus statement

    PubMed Central

    Kalra, Sanjay; Ghosh, Sujoy; Aamir, A. H.; Ahmed, Md. Tofail; Amin, Mohammod Feroz; Bajaj, Sarita; Baruah, Manash P.; Bulugahapitiya, Uditha; Das, A. K.; Giri, Mimi; Gunatilake, Sonali; Mahar, Saeed A.; Pathan, Md. Faruque; Qureshi, Nazmul Kabir; Raza, S. Abbas; Sahay, Rakesh; Shakya, Santosh; Shreshta, Dina; Somasundaram, Noel; Sumanatilleke, Manilka; Unnikrishnan, A. G.; Wijesinghe, Achini Madushani

    2017-01-01

    Diabetes prevalence shows a continuous increasing trend in South Asia. Although well-established treatment modalities exist for type 2 diabetes mellitus (T2DM) management, they are limited by their side effect profile. Sodium–glucose co-transporter 2 inhibitors (SGLT2i) with their novel insulin-independent renal action provide improved glycemic control, supplemented by reduction in weight and blood pressure, and cardiovascular safety. Based on the clinical outcomes with SGLT2i in patients with T2DM, treatment strategies that make a “good clinical sense” are desirable. Considering the peculiar lifestyle, body types, dietary patterns (long duration religious fasts), and the hot climate of the South Asian population, a unanimous decision was taken to design specific, customized guidelines for T2DM treatment strategies in these regions. The panel met for a discussion three times so as to get a consensus for the guidelines, and only unanimous consensus was included. After careful consideration of the quality and strength of the available evidence, the executive summary of this consensus statement was developed based on the American Association of Clinical Endocrinologists/American College of Endocrinology protocol. PMID:28217523

  7. Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate.

    PubMed

    Keith, John M; Apodaca, Rich; Tichenor, Mark; Xiao, Wei; Jones, William; Pierce, Joan; Seierstad, Mark; Palmer, James; Webb, Michael; Karbarz, Mark; Scott, Brian; Wilson, Sandy; Luo, Lin; Wennerholm, Michelle; Chang, Leon; Brown, Sean; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra; Breitenbucher, J Guy

    2012-10-11

    A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a significant role in regulating plasma levels of fatty acid ethanolamides in primates.

  8. The Potential of Inhibitors of Endocannabinoid Metabolism for Drug Development: A Critical Review.

    PubMed

    Fowler, Christopher J

    2015-01-01

    The endocannabinoids anandamide and 2-arachidonoylglycerol are metabolised by both hydrolytic enzymes (primarily fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL)) and oxygenating enzymes (e.g. cyclooxygenase-2, COX-2). In the present article, the in vivo data for compounds inhibiting endocannabinoid metabolism have been reviewed, focussing on inflammation and pain. Potential reasons for the failure of an FAAH inhibitor in a clinical trial in patients with osteoarthritic pain are discussed. It is concluded that there is a continued potential for compounds inhibiting endocannabinoid metabolism in terms of drug development, but that it is wise not to be unrealistic in terms of expectations of success.

  9. Lack of Contribution of Multidrug Resistance-associated Protein and Organic Anion-transporting Polypeptide to Pharmacokinetics of Regorafenib, a Novel Multi-Kinase Inhibitor, in Rats.

    PubMed

    Hotta, Kazuo; Ueyama, Jun; Tatsumi, Yasuaki; Tsukiyama, Ikuto; Sugiura, Yuka; Saito, Hiroko; Matsuura, Katsuhiko; Hasegawa, Takaaki

    2015-09-01

    We investigated whether hepatic multidrug resistance-associated protein 2 (ABCC2) is involved in the hepatobiliary excretion of regorafenib, a novel multi-kinase inhibitor, using Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR) lacking the efflux transporter ABCC2. The involvement of organic anion-transporting polypeptide 1 (OATP1; OATP in humans) and OATP2 in the hepatic uptake of regorafenib and their protein levels in the liver were also investigated in the two rat groups. When regorafenib (5 mg/kg) was administered intravenously, the plasma concentrations of regorafenib were higher in EHBR than those in SD rats. However, the slope of the plasma concentration-time curves was the same for the two groups. Although the apparent biliary clearance of regorafenib in EHBR was lower than that of SD rats, no significant difference in the biliary excretion rate was observed between them, suggesting that regorafenib is not a substrate for ABCC2 and is not excreted into bile by ABCC2. It was also found that the contribution of biliary excretion to the systemic elimination of regorafenib is small. The protein-binding profiles of regorafenib were found to be linear in both rat groups. The binding potency, which was very high in both rat groups (>99.5%), was significantly higher in EHBR than that in SD rats. No significant differences in the plasma concentrations of unbound regorafenib were observed between the two rat groups, suggesting that the differences observed in the pharmacokinetic behaviors of regorafenib between the two rat groups were due to differences in protein-binding. When the protein levels of hepatic OATP1 and OATP2 were measured by immunoblot analysis, the expression of both transporters in EHBR was less than 40% of that in SD rats. The present results suggest that regorafenib is not a substrate for OATP1 and OATP2. These findings suggest the possibility that ABCC2-mediated hepatobiliary excretion and OATP1/OATP2-mediated hepatic uptake do

  10. Benefits and Harms of Sodium-Glucose Co-Transporter 2 Inhibitors in Patients with Type 2 Diabetes: A Systematic Review and Meta-Analysis

    PubMed Central

    Gluud, Lise L.; Bennett, Cathy; Grøndahl, Magnus F.; Christensen, Mikkel B.; Knop, Filip K.; Vilsbøll, Tina

    2016-01-01

    Objective Sodium-glucose co-transporter 2 inhibitors (SGLT2-i) are a novel drug class for the treatment of diabetes. We aimed at describing the maximal benefits and risks associated with SGLT2-i for patients with type 2 diabetes. Design Systematic review and meta-analysis. Data Sources and Study Selection We included double-blinded, randomised controlled trials (RCTs) evaluating SGLT2-i administered in the highest approved therapeutic doses (canagliflozin 300 mg/day, dapagliflozin 10 mg/day, and empagliflozin 25 mg/day) for ≥12 weeks. Comparison groups could receive placebo or oral antidiabetic drugs (OAD) including metformin, sulphonylureas (SU), or dipeptidyl peptidase 4 inhibitors (DPP-4-i). Trials were identified through electronic databases and extensive manual searches. Primary outcomes were glycated haemoglobin A1c (HbA1c) levels, serious adverse events, death, severe hypoglycaemia, ketoacidosis and CVD. Secondary outcomes were fasting plasma glucose, body weight, blood pressure, heart rate, lipids, liver function tests, creatinine and adverse events including infections. The quality of the evidence was assessed using GRADE. Results Meta-analysis of 34 RCTs with 9,154 patients showed that SGLT2-i reduced HbA1c compared with placebo (mean difference -0.69%, 95% confidence interval -0.75 to -0.62%). We downgraded the evidence to ‘low quality’ due to variability and evidence of publication bias (P = 0.015). Canagliflozin was associated with the largest reduction in HbA1c (-0.85%, -0.99% to -0.71%). There were no differences between SGLT2-i and placebo for serious adverse events. SGLT2-i increased the risk of urinary and genital tract infections and increased serum creatinine, and exerted beneficial effects on bodyweight, blood pressure, lipids and alanine aminotransferase (moderate to low quality evidence). Analysis of 12 RCTs found a beneficial effect of SGLT2-i on HbA1c compared with OAD (-0.20%, -0.28 to -0.13%; moderate quality evidence). Conclusion

  11. Effects of antidiabetic drugs on the incidence of macrovascular complications and mortality in type 2 diabetes mellitus: a new perspective on sodium-glucose co-transporter 2 inhibitors.

    PubMed

    Rahelić, Dario; Javor, Eugen; Lucijanić, Tomo; Skelin, Marko

    2017-02-01

    Elevated hemoglobin A1c (HbA1c) values correlate with microvascular and macrovascular complications. Thus, patients with type 2 diabetes mellitus (T2DM) are at an increased risk of developing macrovascular events. Treatment of T2DM should be based on a multifactorial approach because of its evidence regarding reduction of macrovascular complications and mortality in T2DM. It is well known that intensive glucose control reduces the risk of microvascular complications in T2DM, but the effects of antidiabetic drugs on macrovascular complications and mortality in T2DM are less clear. The results of recent trials have demonstrated clear evidence that empagliflozin and liraglutide reduce cardiovascular (CV) and all-cause mortality in T2DM, an effect that is absent in other members of antidiabetic drugs. Empagliflozin is a member of a novel class of antidiabetic drugs, the sodium-glucose co-transporter 2 (SGLT2) inhibitors. Two ongoing randomized clinical trials involving other SGLT2 inhibitors, canagliflozin and dapagliflozin, will provide additional evidence of the beneficial effects of SGLT2 inhibitors in T2DM population. The aim of this paper is to systematically present the latest evidence regarding the usage of antidiabetic drugs, and the reduction of macrovascular complications and mortality. A special emphasis is put on the novel class of antidiabetic drugs, of SGLT2 inhibitors. Key messages Macrovascular complications and mortality are best clinical trial endpoints for evaluating the efficacy of antidiabetic drugs. The first antidiabetic drug that demonstrated a reduction in mortality in the treatment of type 2 diabetes mellitus (T2DM) was empagliflozin, a sodium-glucose co-transporter 2 (SGLT2) inhibitor. SGLT2 inhibitors are novel class of antidiabetic drugs that play a promising role in the treatment of T2DM.

  12. Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

    PubMed

    Di Scala, Coralie; Mazzarino, Morgane; Yahi, Nouara; Varini, Karine; Garmy, Nicolas; Fantini, Jacques; Chahinian, Henri

    2017-04-04

    Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

  13. FAAH-/- mice display differential tolerance, dependence, and cannabinoid receptor adaptation after delta 9-tetrahydrocannabinol and anandamide administration.

    PubMed

    Falenski, Katherine W; Thorpe, Andrew J; Schlosburg, Joel E; Cravatt, Benjamin F; Abdullah, Rehab A; Smith, Tricia H; Selley, Dana E; Lichtman, Aron H; Sim-Selley, Laura J

    2010-07-01

    Repeated administration of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and downregulation of CB(1) cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH(-/-) mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose-effect curves for antinociception, catalepsy, and hypothermia than in AEA dose-effect curves. Subchronic THC significantly attenuated agonist-stimulated [(35)S]GTP gamma S binding in brain and spinal cord, and reduced [(3)H]WIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH(-/-) mice showed less CB(1) receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB(1) receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB(1) receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH(-/-) mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB(1) receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB(1) receptor agonists.

  14. Development and Validation of a High-Content Screening Assay to Identify Inhibitors of Cytoplasmic Dynein-Mediated Transport of Glucocorticoid Receptor to the Nucleus

    PubMed Central

    Shinde, Sunita N.; Hua, Yun; Shun, Tong Ying; Lazo, John S.; Day, Billy W.

    2012-01-01

    Abstract Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to

  15. Inhibition of leukocyte function and interleukin-2 gene expression by 2-methylarachidonyl-(2'-fluoroethyl)amide, a stable congener of the endogenous cannabinoid receptor ligand anandamide

    SciTech Connect

    Kaplan, Barbara L.F.; Ouyang Yanli; Herring, Amy; Yea, Sung Su; Razdan, Raj; Kaminski, Norbert E. . E-mail: kamins11@msu.edu

    2005-06-01

    Arachidonylethanolamide (anandamide, AEA) has been identified as an endogenous ligand for cannabinoid receptors CB1 and CB2. Characterization of the direct cannabimimetic actions of anandamide has been hampered by its short duration of action and rapid degradation in in vivo and in vitro systems to arachidonic acid, a precursor in the biosynthesis of a broad range of biologically active molecules. In the present studies, we utilized 2-methylarachidonyl-(2'-fluoroethyl)amide (F-Me-AEA), an analog of anandamide resistant to enzymatic degradation, to determine whether F-Me-AEA modulated T cell function similar to that of plant-derived cannabinoids. Indeed, F-Me-AEA at low micromolar concentrations exhibited a marked inhibition of phorbol ester plus calcium ionophore (PMA/Io)-induced IL-2 protein secretion and steady state mRNA expression. Likewise, a modest suppression of the mixed lymphocyte response was observed in the presence of F-Me-AEA indicating an alteration in T cell responsiveness to allogeneic MHC class II antigens. F-Me-AEA was also found to modestly inhibit forskolin-stimulated adenylate cyclase activity in thymocytes and splenocytes, a hallmark of cannabinoid receptor agonists. Further characterization of the influence of F-Me-AEA on the cAMP signaling cascade revealed an inhibition of CREB-1/ATF-1 phosphorylation and subsequently, an inhibition of CRE DNA binding activity. Characterization of nuclear binding proteins further revealed that NF-AT and, to a lesser extent, NF-{kappa}B DNA binding activities were also suppressed. These studies demonstrate that F-Me-AEA modulates T cell function in a similar manner to plant-derived and endogenous cannabinoids and therefore can be utilized as an amidase- and hydrolysis-resistant endogenous cannabinoid.

  16. Quinine dimers are potent inhibitors of the Plasmodium falciparum chloroquine resistance transporter and are active against quinoline-resistant P. falciparum.

    PubMed

    Hrycyna, Christine A; Summers, Robert L; Lehane, Adele M; Pires, Marcos M; Namanja, Hilda; Bohn, Kelsey; Kuriakose, Jerrin; Ferdig, Michael; Henrich, Philipp P; Fidock, David A; Kirk, Kiaran; Chmielewski, Jean; Martin, Rowena E

    2014-03-21

    Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

  17. Three-dimensional quantitative structure-activity relationship (3D QSAR) and pharmacophore elucidation of tetrahydropyran derivatives as serotonin and norepinephrine transporter inhibitors

    NASA Astrophysics Data System (ADS)

    Kharkar, Prashant S.; Reith, Maarten E. A.; Dutta, Aloke K.

    2008-01-01

    Three-dimensional quantitative structure-activity relationship (3D QSAR) using comparative molecular field analysis (CoMFA) was performed on a series of substituted tetrahydropyran (THP) derivatives possessing serotonin (SERT) and norepinephrine (NET) transporter inhibitory activities. The study aimed to rationalize the potency of these inhibitors for SERT and NET as well as the observed selectivity differences for NET over SERT. The dataset consisted of 29 molecules, of which 23 molecules were used as the training set for deriving CoMFA models for SERT and NET uptake inhibitory activities. Superimpositions were performed using atom-based fitting and 3-point pharmacophore-based alignment. Two charge calculation methods, Gasteiger-Hückel and semiempirical PM3, were tried. Both alignment methods were analyzed in terms of their predictive abilities and produced comparable results with high internal and external predictivities. The models obtained using the 3-point pharmacophore-based alignment outperformed the models with atom-based fitting in terms of relevant statistics and interpretability of the generated contour maps. Steric fields dominated electrostatic fields in terms of contribution. The selectivity analysis (NET over SERT), though yielded models with good internal predictivity, showed very poor external test set predictions. The analysis was repeated with 24 molecules after systematically excluding so-called outliers (5 out of 29) from the model derivation process. The resulting CoMFA model using the atom-based fitting exhibited good statistics and was able to explain most of the selectivity (NET over SERT)-discriminating factors. The presence of -OH substituent on the THP ring was found to be one of the most important factors governing the NET selectivity over SERT. Thus, a 4-point NET-selective pharmacophore, after introducing this newly found H-bond donor/acceptor feature in addition to the initial 3-point pharmacophore, was proposed.

  18. Effects of rifampin, cyclosporine A, and probenecid on the pharmacokinetic profile of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in healthy participants

    PubMed Central

    Devineni, Damayanthi; Vaccaro, Nicole; Murphy, Joe; Curtin, Christopher; Mamidi, Rao N.V.S.; Weiner, Sveta; Wang, Shean-Sheng; Ariyawansa, Jay; Stieltjes, Hans; Wajs, Ewa; Di Prospero, Nicholas A.; Rothenberg, Paul

    2015-01-01

    Objective: Canagliflozin, a sodium-glucose co-transporter 2 inhibitor, approved for the treatment of type-2 diabetes mellitus (T2DM), is metabolized by uridine diphosphate-glucuronosyltransferases (UGT) 1A9 and UGT2B4, and is a substrate of P-glycoprotein (P-gp). Canagliflozin exposures may be affected by coadministration of drugs that induce (e.g., rifampin for UGT) or inhibit (e.g. probenecid for UGT; cyclosporine A for P-gp) these pathways. The primary objective of these three independent studies (single-center, open-label, fixed-sequence) was to evaluate the effects of rifampin (study 1), probenecid (study 2), and cyclosporine A (study 3) on the pharmacokinetics of canagliflozin in healthy participants. Methods: Participants received; in study 1: canagliflozin 300 mg (days 1 and 10), rifampin 600 mg (days 4 – 12); study 2: canagliflozin 300 mg (days 1 – 17), probenecid 500 mg twice daily (days 15 – 17); and study 3: canagliflozin 300 mg (days 1 – 8), cyclosporine A 400 mg (day 8). Pharmacokinetics were assessed at pre-specified intervals on days 1 and 10 (study 1); on days 14 and 17 (study 2), and on days 2 – 8 (study 3). Results: Rifampin decreased the maximum plasma canagliflozin concentration (Cmax) by 28% and its area under the curve (AUC) by 51%. Probenecid increased the Cmax by 13% and the AUC by 21%. Cyclosporine A increased the AUC by 23% but did not affect the Cmax. Conclusion: Coadministration of canagliflozin with rifampin, probenecid, and cyclosporine A was well-tolerated. No clinically meaningful interactions were observed for probenecid or cyclosporine A, while rifampin coadministration modestly reduced canagliflozin plasma concentrations and could necessitate an appropriate monitoring of glycemic control. PMID:25407255

  19. NBI‐98854, a selective monoamine transport inhibitor for the treatment of tardive dyskinesia: A randomized, double‐blind, placebo‐controlled study

    PubMed Central

    Jimenez, Roland; Hauser, Robert A.; Factor, Stewart A.; Burke, Joshua; Mandri, Daniel; Castro‐Gayol, Julio C.

    2015-01-01

    ABSTRACT Background Tardive dyskinesia is a persistent movement disorder induced by chronic neuroleptic exposure. NBI‐98854 is a novel, highly selective, vesicular monoamine transporter 2 inhibitor. We present results of a randomized, 6‐week, double‐blind, placebo‐controlled, dose‐titration study evaluating the safety, tolerability, and efficacy of NBI‐98854 for the treatment of tardive dyskinesia. Methods Male and female adult subjects with moderate or severe tardive dyskinesia were included. NBI‐98854 or placebo was given once per day starting at 25 mg and then escalated by 25 mg to a maximum of 75 mg based on dyskinesia and tolerability assessment. The primary efficacy endpoint was the change in Abnormal Involuntary Movement Scale from baseline at week 6 scored by blinded, central video raters. The secondary endpoint was the Clinical Global Impression of Change—Tardive Dyskinesia score assessed by the blinded investigator. Results Two hundred five potential subjects were screened, and 102 were randomized; 76% of NBI‐98854 subjects and 80% of placebo subjects reached the maximum allowed dose. Abnormal Involuntary Movement Scale scores for NBI‐98854 compared with placebo were significantly reduced (p = 0.0005). Active drug was also superior on the Clinical Global Impression of Change—Tardive Dyskinesia (p < 0.0001). Treatment‐emergent adverse event rates were 49% in the NBI‐98854 and 33% in the placebo subjects. The most common adverse events (active vs. placebo) were fatigue and headache (9.8% vs. 4.1%) and constipation and urinary tract infection (3.9% vs. 6.1%). No clinically relevant changes in safety assessments were noted. Conclusion NBI‐98854 significantly improved tardive dyskinesia and was well tolerated in patients. These results support the phase 3 clinical trials of NBI‐98854 now underway. © 2015 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder

  20. Effects of the Indole-3-Acetic Acid (IAA) Transport Inhibitors N-1-Naphthylphthalamic Acid and Morphactin on Endogenous IAA Dynamics in Relation to Compression Wood Formation in 1-Year-Old Pinus sylvestris (L.) Shoots.

    PubMed Central

    Sundberg, B.; Tuominen, H.; Little, CHA.

    1994-01-01

    Both N-1-naphthylphthalamic acid (NPA) and methyl-2-chloro-9-hydroxyfluorene-9-carboxylic acid (CF) inhibit the polar transport of indole-3-acetic acid (IAA) and, therefore, are attractive tools for investigating IAA's role in the regulation of plant growth. Ringing an intact conifer shoot with lanolin containing NPA or CF induces the formation of compression wood above the ring. This induction has been attributed to a postulated accumulation of IAA above the application site of the IAA transport inhibitor, but the validity of this postulation has never been confirmed. Using gas chromatography-selected ion monitoring-mass spectroscopy with [13C6]IAA as an internal standard, we measured the levels of endogenous free and conjugated IAA in 1-year-old Pinus sylvestris (L.) shoots ringed with NPA or CF. The level of free IAA was dramatically decreased below the ring, indicating that the polar transport of endogenous IAA was inhibited by the treatment. However, the free IAA level above the ring, where compression wood was formed, was also slightly lower than in control shoots. The lack of IAA accumulation above the site of the IAA transport inhibitor could not be explained by an increase in IAA conjugation. Furthermore, the turnover of [2-14C]IAA, measured using high-performance liquid chromatography with on-line radioactivity monitoring, was the same in NPA-treated and control shoots. The decrease in IAA level above a NPA or CF ring is attributed to these substances being transported acropetally and interfering with polar IAA transport along the shoot. It is concluded that compression wood formation above a NPA or CF ring is not associated with an overall increase in cambial region IAA level or increased IAA turnover. Instead, we suggest that acropetally transported NPA and CF induce compression wood formation by interacting with the NPA receptor in differentiating tracheids, thereby locally increasing IAA in these cells. PMID:12232343

  1. Short-term exposure to alcohol in rats affects brain levels of anandamide, other N-acylethanolamines and 2-arachidonoyl-glycerol.

    PubMed

    Rubio, Marina; McHugh, Douglas; Fernández-Ruiz, Javier; Bradshaw, Heather; Walker, J Michael

    2007-06-29

    Chronic alcohol exposure leads to significant changes in the levels of endocannabinoids and their receptors in the brains of humans and laboratory animals, as well as in cultured neuronal cells. However, little is known about the effects of short-term periods of alcohol exposure. In the present study, we examined the changes in endocannabinoid levels (anandamide and 2-arachidonoylglycerol), as well as four additional N-acylethanolamines, in four brain regions of rats exposed to alcohol through the liquid diet for a period of 24h. The levels of N-acylethanolamines were diminished 24h after the onset of alcohol exposure. This was particularly evident for anandamide in the hypothalamus, amygdala and caudate-putamen, for N-palmitoylethanolamine in the caudate-putamen, for N-oleoylethanolamine in the hypothalamus, caudate-putamen and prefrontal cortex, and for N-stearoylethanolamine in the amygdala. The only exception was N-linoleoylethanolamine for which the levels increased in the amygdala after the exposure to alcohol. The levels of the other major endocannabinoid, 2-arachidonoylglycerol, were also reduced with marked effects in the prefrontal cortex. These results support the notion that short-term alcohol exposure reduces endocannabinoid levels in the brain accompanied by a reduction in several related N-acylethanolamines.

  2. Low Level Chlorpyrifos Exposure Increases Anandamide Accumulation in Juvenile Rat Brain in the Absence of Brain Cholinesterase Inhibition

    PubMed Central

    Carr, Russell L.; Graves, Casey A.; Mangum, Lee C.; Nail, Carole A.; Ross, Matthew K.

    2014-01-01

    The prevailing dogma is that chlorpyrifos (CPF) mediates its toxicity through inhibition of cholinesterase (ChE). However, in recent years, the toxicological effects of developmental CPF exposure have been attributed to an unknown non-cholinergic mechanism of action. We hypothesize that the endocannabinoid system may be an important target because of its vital role in nervous system development. We have previously reported that repeated exposure to CPF results in greater inhibition of fatty acid amide hydrolase (FAAH), the enzyme that metabolizes the endocannabinoid anandamide (AEA), than inhibition of either forebrain ChE or monoacylglycerol lipase (MAGL), the enzyme that metabolizes the endocannabinoid 2-arachidonylglycerol (2-AG). This exposure resulted in the accumulation of 2-AG and AEA in the forebrain of juvenile rats; however, even at the lowest dosage level used (1.0 mg/kg), forebrain ChE inhibition was still present. Thus, it is not clear if FAAH activity would be inhibited at dosage levels that do not inhibit ChE. To determine this, 10 day old rat pups were exposed daily for 7 days to either corn oil or 0.5 mg/kg CPF by oral gavage. At 4 and 12 h post-exposure on the last day of administration, the activities of serum ChE and carboxylesterase (CES) and forebrain ChE, MAGL, and FAAH were determined as well as the forebrain AEA and 2-AG levels. Significant inhibition of serum ChE and CES was present at both 4 and 12 h. There was no significant inhibition of the activities of forebrain ChE or MAGL and no significant change in the amount of 2-AG at either time point. On the other hand, while no statistically significant effects were observed at 4 h, FAAH activity was significantly inhibited at 12 h resulting in a significant accumulation of AEA. Although it is not clear if this level of accumulation impacts brain maturation, this study demonstrates that developmental CPF exposure at a level that does not inhibit brain ChE can alter components of

  3. Prenatal serotonin reuptake inhibitor (SRI) antidepressant exposure and serotonin transporter promoter genotype (SLC6A4) influence executive functions at 6 years of age

    PubMed Central

    Weikum, Whitney M.; Brain, Ursula; Chau, Cecil M. Y.; Grunau, Ruth E.; Boyce, W. Thomas; Diamond, Adele; Oberlander, Tim F.

    2013-01-01

    Prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressants and maternal depression may affect prefrontal cognitive skills (executive functions; EFs) including self-control, working memory and cognitive flexibility. We examined long-term effects of prenatal SRI exposure on EFs to determine whether effects are moderated by maternal mood and/or genetic variations in SLC6A4 (a gene that codes for the serotonin transporter [5-HTT] central to the regulation of synaptic serotonin levels and behavior). Children who were exposed to SRIs prenatally (SRI-exposed N = 26) and non-exposed (N = 38) were studied at age 6 years (M = 6.3; SD = 0.5) using the Hearts & Flowers task (H&F) to assess EFs. Maternal mood was measured during pregnancy (3rd trimester) and when the child was age 6 years (Hamilton Depression Scale). Parent reports of child behavior were also obtained (MacArthur Health & Behavior Questionnaire). Parents of prenatally SRI-exposed children reported fewer child externalizing and inattentive (ADHD) behaviors. Generalized estimate equation modeling showed a significant 3-way interaction between prenatal SRI exposure, SLC6A4 variant, and maternal mood at the 6-year time-point on H&F accuracy. For prenatally SRI-exposed children, regardless of maternal mood, the H&F accuracy of children with reduced 5HTT expression (a short [S] allele) remained stable. Even with increasing maternal depressive symptoms (though all below clinical threshold), EFs of children with at least one short allele were comparable to children with the same genotype whose mothers reported few if any depressive symptoms—in this sense they showed resilience. Children with two long (L) alleles were more sensitive to context. When their mothers had few depressive symptoms, LL children showed extremely good EF performance—better than any other group. When their mothers reported more depressive symptoms, LL children's EF performance was worse than that of any other group. In the face of

  4. Pharmacodynamic Effects of Canagliflozin, a Sodium Glucose Co-Transporter 2 Inhibitor, from a Randomized Study in Patients with Type 2 Diabetes

    PubMed Central

    Sha, Sue; Devineni, Damayanthi; Ghosh, Atalanta; Polidori, David; Hompesch, Marcus; Arnolds, Sabine; Morrow, Linda; Spitzer, Heike; Demarest, Keith; Rothenberg, Paul

    2014-01-01

    Introduction This randomized, double-blind, placebo-controlled, single and multiple ascending-dose study evaluated the pharmacodynamic effects and safety/tolerability of canagliflozin, a sodium glucose co-transporter 2 inhibitor, in patients with type 2 diabetes. Methods Patients (N = 116) discontinued their antihyperglycemic medications 2 weeks before randomization. Patients received canagliflozin 30, 100, 200, or 400 mg once daily or 300 mg twice daily, or placebo at 2 study centers in the United States and Germany, or canagliflozin 30 mg once daily or placebo at 1 study center in Korea, while maintaining an isocaloric diet for 2 weeks. On Days –1, 1, and 16, urinary glucose excretion (UGE), plasma glucose (PG), fasting PG (FPG), and insulin were measured. The renal threshold for glucose (RTG) was calculated from UGE, PG, and estimated glomerular filtration rate. Safety was evaluated based on adverse event (AE) reports, vital signs, electrocardiograms, clinical laboratory tests, and physical examinations. Results Canagliflozin increased UGE dose-dependently (∼80–120 g/day with canagliflozin ≥100 mg), with increases maintained over the 14-day dosing period with each dose. Canagliflozin dose-dependently decreased RTG, with maximal reductions to ∼4–5 mM (72–90 mg/dL). Canagliflozin also reduced FPG and 24-hour mean PG; glucose reductions were seen on Day 1 and maintained over 2 weeks. Plasma insulin reductions with canagliflozin were consistent with observed PG reductions. Canagliflozin also reduced body weight. AEs were transient, mild to moderate in intensity, and balanced across groups; 1 canagliflozin-treated female reported an episode of vaginal candidiasis. Canagliflozin did not cause hypoglycemia, consistent with the RTG values remaining above the hypoglycemia threshold. At Day 16, there were no clinically meaningful changes in urine volume, urine electrolyte excretion, renal function, or routine laboratory test values. Conclusions

  5. Identification of Novel Inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) Using a Consensus Vote of Six Classification Models

    PubMed Central

    2015-01-01

    Organic anion transporting polypeptides 1B1 and 1B3 are transporters selectively expressed on the basolateral membrane of the hepatocyte. Several studies reveal that they are involved in drug–drug interactions, cancer, and hyperbilirubinemia. In this study, we developed a set of classification models for OATP1B1 and 1B3 inhibition based on more than 1700 carefully curated compounds from literature, which were validated via cross-validation and by use of an external test set. After combining several sets of descriptors and classifiers, the 6 best models were selected according to their statistical performance and were used for virtual screening of DrugBank. Consensus scoring of the screened compounds resulted in the selection and purchase of nine compounds as potential dual inhibitors and of one compound as potential selective OATP1B3 inhibitor. Biological testing of the compounds confirmed the validity of the models, yielding an accuracy of 90% for OATP1B1 and 80% for OATP1B3, respectively. Moreover, at least half of the new identified inhibitors are associated with hyperbilirubinemia or hepatotoxicity, implying a relationship between OATP inhibition and these severe side effects. PMID:26469880

  6. 1-[N, O-bis-(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), an inhibitor of calcium-dependent camodulin protein kinase II, inhibits both insulin- and hypoxia-stimulated glucose transport in skeletal muscle.

    PubMed Central

    Brozinick, J T; Reynolds, T H; Dean, D; Cartee, G; Cushman, S W

    1999-01-01

    Previous studies have indicated a role for calmodulin in hypoxia-and insulin-stimulated glucose transport. However, since calmodulin interacts with multiple protein targets, it is unknown which of these targets is involved in the regulation of glucose transport. In the present study, we have used the calcium-dependent calmodulin protein kinase II (CAMKII) inhibitor 1-[N, O-bis-(5-isoquinolinesulphonyl) -N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) to investigate the possible role of this enzyme in the regulation of glucose transport in isolated rat soleus and epitrochlearis muscles. KN-62 did not affect basal 2-deoxyglucose transport, but it did inhibit both insulin- and hypoxia-stimulated glucose transport activity by 46 and 40% respectively. 1-[N,O-Bis-(1, 5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-04), a structural analogue of KN-62 that does not inhibit CAMKII, had no effect on hypoxia-or insulin-stimulated glucose transport. Accordingly, KN-62 decreased the stimulated cell-surface GLUT4 labelling by a similar extent as the inhibition of glucose transport (insulin, 49% and hypoxia, 54%). Additional experiments showed that KN-62 also inhibited insulin- and hypoxia-stimulated transport by 37 and 40% respectively in isolated rat epitrochlearis (a fast-twitch muscle), indicating that the effect of KN-62 was not limited to the slow-twitch fibres of the soleus. The inhibitory effect of KN-62 on hypoxia-stimulated glucose transport appears to be specific to CAMKII, since KN-62 did not inhibit hypoxia-stimulated 45Ca efflux from muscles pre-loaded with 45Ca, or hypoxia-stimulated glycogen breakdown. Additionally, KN-62 affected neither insulin-stimulated phosphoinositide 3-kinase nor Akt activity, suggesting that the effects of KN-62 are not due to non-specific effects of this inhibitor on these regions of the insulin-signalling cascade. The results of the present study suggest that CAMKII might have a distinct role in insulin- and hypoxia

  7. Review of insulin-dependent and insulin-independent agents for treating patients with type 2 diabetes mellitus and potential role for sodium-glucose co-transporter 2 inhibitors.

    PubMed

    Freeman, Jeffrey S

    2013-05-01

    Diabetes, especially type 2 diabetes mellitus (T2DM), continues to be a global health care problem. Although the beneficial effects of glycemic control are well established, in the United States, > 40% of adults with diabetes fail to achieve target glycated hemoglobin levels. Antidiabetic drug classes vary with respect to their mechanisms of action, glucose-lowering potential, and safety and tolerability profiles. Antidiabetic drug classes include some agents that depend on the presence or action of insulin for their therapeutic effect. As the disease state of T2DM progresses, patient pancreatic β-cell function declines, and therapies that stimulate insulin secretion or improve insulin sensitivity become less effective for this population. Therefore, the development of additional antidiabetic agents with novel mechanisms of action that can be used alone or in combination with currently approved medications may help patients achieve glycemic control. Agents that have comparable glucose-lowering capabilities but different mechanisms of action may fill treatment gaps or meet the needs of patient subpopulations. For example, inhibitors of sodium-glucose co-transporter 2 (SGLT2) represent an emerging class of glucose-lowering agents. The SGLT2 inhibitors reduce glucose reabsorption by the kidney, leading to increased urinary glucose excretion and caloric loss. In clinical trials, these agents have been shown to improve glycemic control and to reduce body weight in patients with T2DM. Additionally, SGLT2 inhibitors pose a low risk for hypoglycemia and are generally well tolerated; however, their use has been associated with an increase in the frequency of genital infections and, in some studies, urinary tract infections. Sodium-glucose co-transporter 2 inhibitors may provide an alternative or an addition to existing therapies for the treatment of patients with T2DM.

  8. Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: a possible target for combination therapy with anti-proliferative aminopeptidase inhibitors.

    PubMed

    Fan, Xuetao; Ross, Douglas D; Arakawa, Hiroshi; Ganapathy, Vadivel; Tamai, Ikumi; Nakanishi, Takeo

    2010-09-15

    Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC(50) value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.

  9. Role of pre-junctional CB1, but not CB2 , TRPV1 or GPR55 receptors in anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow in pithed rats.

    PubMed

    Marichal-Cancino, Bruno A; Altamirano-Espinoza, Alain H; Manrique-Maldonado, Guadalupe; MaassenVanDenBrink, Antoinette; Villalón, Carlos M

    2014-03-01

    Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1 , CB2 , TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non-selective), JWH-015 (CB2 ) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre-treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9 -T12 ) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The infusions of anandamide in a dose-dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH-015 or lysophosphatidylinositol), but not those by α-CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31-100 μg/kg NIDA41020 (CB1 ), (ii) unaffected by 180 μg/kg AM630 (CB2 ), 31 μg/kg cannabidiol (GPR55) or 31-100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre-junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2 , TRPV1 or GPR55 receptors.

  10. Discovery and characterization of novel inhibitors of the sodium-coupled citrate transporter (NaCT or SLC13A5)

    PubMed Central

    Huard, Kim; Brown, Janice; Jones, Jessica C.; Cabral, Shawn; Futatsugi, Kentaro; Gorgoglione, Matthew; Lanba, Adhiraj; Vera, Nicholas B.; Zhu, Yimin; Yan, Qingyun; Zhou, Yingjiang; Vernochet, Cecile; Riccardi, Keith; Wolford, Angela; Pirman, David; Niosi, Mark; Aspnes, Gary; Herr, Michael; Genung, Nathan E.; Magee, Thomas V.; Uccello, Daniel P.; Loria, Paula; Di, Li; Gosset, James R.; Hepworth, David; Rolph, Timothy; Pfefferkorn, Jeffrey A.; Erion, Derek M.

    2015-01-01

    Citrate is a key regulatory metabolic intermediate as it facilitates the integration of the glycolysis and lipid synthesis pathways. Inhibition of hepatic extracellular citrate uptake, by blocking the sodium-coupled citrate transporter (NaCT or SLC13A5), has been suggested as a potential therapeutic approach to treat metabolic disorders. NaCT transports citrate from the blood into the cell coupled to the transport of sodium ions. The studies herein report the identification and characterization of a novel small dicarboxylate molecule (compound 2) capable of selectively and potently inhibiting citrate transport through NaCT, both in vitro and in vivo. Binding and transport experiments indicate that 2 specifically binds NaCT in a competitive and stereosensitive manner, and is recognized as a substrate for transport by NaCT. The favorable pharmacokinetic properties of 2 permitted in vivo experiments to evaluate the effect of inhibiting hepatic citrate uptake on metabolic endpoints. PMID:26620127

  11. CB1 cannabinoid receptor-mediated anandamide signaling mechanisms of the inferior colliculus modulate the haloperidol-induced catalepsy.

    PubMed

    Medeiros, P; de Freitas, R L; Silva, M O; Coimbra, N C; Melo-Thomas, L

    2016-11-19

    The inferior colliculus (IC), a midbrain structure that processes acoustic information of aversive nature, is distinguished from other auditory nuclei in the brainstem by its connections with structures of the motor system. Previous evidence relating the IC to motor behavior shows that glutamatergic and GABAergic mechanisms in the IC exert influence on systemic haloperidol-induced catalepsy. There is substantial evidence supporting a role played by the endocannabinoid system as a modulator of the glutamatergic neurotransmission, as well as the dopaminergic activity in the basal nuclei and therefore it may be considered as a potential pharmacological target for the treatment of movement disorders. The present study evaluated if the endocannabinoid system in the IC plays a role in the elaboration of systemic haloperidol-induced catalepsy. Male Wistar rats received intracollicular microinjection of either the endogenous cannabinoid anandamide (AEA) at different concentrations (5, 50 or 100pmol/0.2μl), the CB1 cannabinoid receptor antagonist AM251 at 50, 100 or 200pmol/0.2μl or vehicle, followed by intraperitoneal (IP) administration of either haloperidol at 0.5 or 1mg/kg or physiological saline. Systemic injection of haloperidol at both doses (0.5 or 1mg/kg, IP) produced a cataleptic state, compared to vehicle/physiological saline-treated group, lasting 30 and 50min after systemic administration of the dopaminergic receptors non-selective antagonist. The midbrain microinjection of AEA at 50pmol/0.2μl increased the latency for stepping down from the horizontal bar after systemic administration of haloperidol. Moreover, the intracollicular administration of AEA at 50pmol/0.2μl was able to increase the duration of catalepsy as compared to AEA at 100pmol/0.2-μl-treated group. Intracollicular pretreatment with AM251 at the intermediate concentration (100pmol/0.2μl) was able to decrease the duration of catalepsy after systemic administration of haloperidol. However

  12. A Systems Pharmacology Perspective on the Clinical Development of Fatty Acid Amide Hydrolase Inhibitors for Pain

    PubMed Central

    Benson, N; Metelkin, E; Demin, O; Li, G L; Nichols, D; van der Graaf, P H

    2014-01-01

    The level of the endocannabinoid anandamide is controlled by fatty acid amide hydrolase (FAAH). In 2011, PF-04457845, an irreversible inhibitor of FAAH, was progressed to phase II clinical trials for osteoarthritic pain. This article discusses a prospective, integrated systems pharmacology model evaluation of FAAH as a target for pain in humans, using physiologically based pharmacokinetic and systems biology approaches. The model integrated physiological compartments; endocannabinoid production, degradation, and disposition data; PF-04457845 pharmacokinetics and pharmacodynamics, and cannabinoid receptor CB1-binding kinetics. The modeling identified clear gaps in our understanding and highlighted key risks going forward, in particular relating to whether methods are in place to demonstrate target engagement and pharmacological effect. The value of this modeling exercise will be discussed in detail and in the context of the clinical phase II data, together with recommendations to enable optimal future evaluation of FAAH inhibitors. PMID:24429592

  13. Piperidinyl thiazole isoxazolines: A new series of highly potent, slowly reversible FAAH inhibitors with analgesic properties.

    PubMed

    Pember, Stephen O; Mejia, Galo L; Price, Theodore J; Pasteris, Robert J

    2016-06-15

    Fatty acid amide hydrolase (FAAH) is a membrane anchored serine hydrolase that has a principle role in the metabolism of the endogenous cannabinoid anandamide. Docking studies using representative FAAH crystal structures revealed that compounds containing a novel piperidinyl thiazole isoxazoline core fit within the ligand binding domains. New potential FAAH inhibitors were designed and synthesized incorporating urea, carbamate, alkyldione and thiourea reactive centers as potential pharmacophores. A small library of candidate compounds (75) was then screened against human FAAH leading to the identification of new carbamate and urea based inhibitors (Ki=pM and nM, respectively). Representative carbamate and urea based chemotypes displayed slow, time dependent inhibition kinetics leading to enzyme inactivation which was slowly reversible. However, evidence indicated that features of the mechanism of inactivation differ between the two pharmacophore types. Selected compounds were also evaluated for analgesic activity in the mouse-tail flick test.

  14. Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

    PubMed

    Merino, Gracia; Real, Rebeca; Baro, Marta F; Gonzalez-Lobato, Lucia; Prieto, Julio G; Alvarez, Ana I; Marques, Margarita M

    2009-01-01

    ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

  15. Efflux Transporters at the Blood-Brain Barrier Limit Delivery and Efficacy of Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib (PD-0332991) in an Orthotopic Brain Tumor Model

    PubMed Central

    Parrish, Karen E.; Pokorny, Jenny; Mittapalli, Rajendar K.; Bakken, Katrina; Sarkaria, Jann N.

    2015-01-01

    6-Acetyl-8-cyclopentyl-5-methyl-2-([5-(piperazin-1-yl)pyridin-2-yl]amino)pyrido(2,3-d)pyrimidin-7(8H)-one [palbociclib (PD-0332991)] is a cyclin-dependent kinase 4/6 inhibitor approved for the treatment of metastatic breast cancer and is currently undergoing clinical trials for many solid tumors. Glioblastoma (GBM) is the most common primary brain tumor in adults and has limited treatment options. The cyclin-dependent kinase 4/6 pathway is commonly dysregulated in GBM and is a promising target in treating this devastating disease. The blood-brain barrier (BBB) limits the delivery of drugs to invasive regions of GBM, where the efflux transporters P-glycoprotein and breast cancer resistance protein can prevent treatments from reaching the tumor. The purpose of this study was to examine the mechanisms limiting the effectiveness of palbociclib therapy in an orthotopic xenograft model. The in vitro intracellular accumulation results demonstrated that palbociclib is a substrate for both P-glycoprotein and breast cancer resistance protein. In vivo studies in transgenic mice confirmed that efflux transport is responsible for the limited brain distribution of palbociclib. There was an ∼115-fold increase in brain exposure at steady state in the transporter deficient mice when compared with wild-type mice, and the efflux inhibitor elacridar significantly increased palbociclib brain distribution. Efficacy studies demonstrated that palbociclib is an effective therapy when GBM22 tumor cells are implanted in the flank, but ineffective in an orthotopic (intracranial) model. Moreover, doses designed to mimic brain exposure were ineffective in treating flank tumors. These results demonstrate that efflux transport in the BBB is involved in limiting the brain distribution of palbociclib and this has critical implications in determining effective dosing regimens of palbociclib therapy in the treatment of brain tumors. PMID:26354993

  16. Cannabinoid-Induced Changes in the Activity of Electron Transport Chain Complexes of Brain Mitochondria.

    PubMed

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2015-08-01

    The aim of this study was to investigate changes in the activity of individual mitochondrial respiratory chain complexes (I, II/III, IV) and citrate synthase induced by pharmacologically different cannabinoids. In vitro effects of selected cannabinoids on mitochondrial enzymes were measured in crude mitochondrial fraction isolated from pig brain. Both cannabinoid receptor agonists, Δ(9)-tetrahydrocannabinol, anandamide, and R-(+)-WIN55,212-2, and antagonist/inverse agonists of cannabinoid receptors, AM251, and cannabidiol were examined in pig brain mitochondria. Different effects of these cannabinoids on mitochondrial respiratory chain complexes and citrate synthase were found. Citrate synthase activity was decreased only by Δ(9)-tetrahydrocannabinol and AM251. Significant increase in the complex I activity was induced by anandamide. At micromolar concentration, all the tested cannabinoids inhibited the activity of electron transport chain complexes II/III and IV. Stimulatory effect of anandamide on activity of complex I may participate on distinct physiological effects of endocannabinoids compared to phytocannabinoids or synthetic cannabinoids. Common inhibitory effect of cannabinoids on activity of complex II/III and IV confirmed a non-receptor-mediated mechanism of cannabinoid action on individual components of system of oxidative phosphorylation.

  17. Sodium glucose co-transporter inhibitors for the management of diabetes mellitus: an opinion paper from the Endocrine and Metabolism Practice and Research Network of the American College of Clinical Pharmacy.

    PubMed

    Clements, Jennifer N; Whitley, Heather P; D'Souza, Jennifer J; Gross, Benjamin; Hess, Rick; Reece, Sara; Gentry, Chad; Shealy, Kayce

    2015-01-01

    Type 2 diabetes mellitus (T2DM) carries a high prevalence in the United States and worldwide. Therefore, the number of medication classes being developed and studied has grown. The individualized management of diabetes is accomplished by evaluating a medication's efficacy, safety, and cost, along with the patient's preference and tolerance to the medication. Sodium glucose co-transporter 2 inhibitors are a new therapeutic class indicated for the treatment of diabetes and have a unique mechanism of action, independent of beta-cell function. The first agent approved by the Food and Drug Administration (FDA) was canagliflozin in March 2013. Two agents - dapagliflozin and empagliflozin - were FDA-approved in January and July 2014, respectively. A clear understanding of the new class is needed to identify its appropriate use in clinical practice. Members of the American College of Clinical Pharmacy Endocrine and Metabolism Practice and Research Network reviewed available literature regarding this therapeutic class. The article addresses the advantages, disadvantages, emerging role, and patient education for sodium glucose co-transporter 2 inhibitors. Key limitations for this article include limited access to clinical trial data not published by the pharmaceutical company and limited data on products produced outside the United States.

  18. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM.

  19. Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH).

    PubMed

    Min, Xiaoshan; Thibault, Stephen T; Porter, Amy C; Gustin, Darin J; Carlson, Timothy J; Xu, Haoda; Lindstrom, Michelle; Xu, Guifen; Uyeda, Craig; Ma, Zhihua; Li, Yihong; Kayser, Frank; Walker, Nigel P C; Wang, Zhulun

    2011-05-03

    Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.

  20. Sodium-glucose co-transporter-2 inhibitor use and dietary carbohydrate intake in Japanese individuals with type 2 diabetes: A randomized, open-label, 3-arm parallel comparative, exploratory study.

    PubMed

    Yabe, Daisuke; Iwasaki, Masahiro; Kuwata, Hitoshi; Haraguchi, Takuya; Hamamoto, Yoshiyuki; Kurose, Takeshi; Sumita, Kiminobu; Yamazato, Hitoshi; Kanada, Shigeto; Seino, Yutaka

    2016-12-19

    This study investigated the safety and efficacy of the sodium-glucose co-transporter-2 (SGLT2) inhibitor luseogliflozin with differing carbohydrate intakes in Japanese individuals with type 2 diabetes (T2D). Participants were randomly assigned to 3 carbohydrate-adjusted meals for 14 days (days 1-14; a high carbohydrate [HC; 55% total energy carbohydrate] and high glycaemic index [HGI] meal; an HC [55% total energy carbohydrate] and low glycaemic index [LGI] meal; or a low carbohydrate [LC; 40% total energy carbohydrate] and HGI meal). All participants received luseogliflozin for the last 7 days (days 8-14), continuous glucose monitoring (CGM) before and after luseogliflozin treatment (days 5-8 and days 12-15) and blood tests on days 1, 8 and 15. Luseogliflozin significantly decreased the area under the curve and mean of CGM values in all 3 groups similarly. Fasting plasma glucose, insulin and glucagon were similar at all time points. Ketone bodies on day 15 were significantly higher in the LC-HGI group compared with the HC-HGI and HC-LGI groups. In conclusion, luseogliflozin has similar efficacy and safety in Japanese people with T2D when meals contain 40% to 55% total energy carbohydrate, but a strict LC diet on this class of drug should be avoided to prevent SGLT2 inhibitor-associated diabetic ketoacidosis.

  1. Physiologically based pharmacokinetic-pharmacodynamic modeling to predict concentrations and actions of sodium-dependent glucose transporter 2 inhibitor canagliflozin in human intestines and renal tubules.

    PubMed

    Mori, Kazumi; Saito, Ryuta; Nakamaru, Yoshinobu; Shimizu, Makiko; Yamazaki, Hiroshi

    2016-11-01

    Canagliflozin is a recently developed sodium-glucose cotransporter (SGLT) 2 inhibitor that promotes renal glucose excretion and is considered to inhibit renal SGLT2 from the luminal side of proximal tubules. Canagliflozin reportedly inhibits SGLT1 weakly and suppresses postprandial plasma glucose, suggesting that it also inhibits intestinal SGLT1. However, it is difficult to measure the drug concentrations of these assumed sites of action directly. The pharmacokinetic-pharmacodynamic (PK/PD) relationships of canagliflozin remain poorly characterized. Therefore, a physiologically based pharmacokinetic (PBPK) model of canagliflozin was developed based on clinical data from healthy volunteers and it was used to simulate luminal concentrations in intestines and renal tubules. In small intestine simulations, the inhibition ratios for SGLT1 were predicted to be 40%-60% after the oral administration of clinical doses (100-300 mg/day). In contrast, inhibition ratios of canagliflozin for renal SGLT2 and SGLT1 were predicted to be approximately 100% and 0.2%-0.4%, respectively. These analyses suggest that canagliflozin only inhibits SGLT2 in the kidney. Using the simulated proximal tubule luminal concentrations of canagliflozin, the urinary glucose excretion rates in canagliflozin-treated diabetic patients were accurately predicted using the renal glucose reabsorption model as a PD model. Because the simulation of canagliflozin pharmacokinetics was successful, this PBPK methodology was further validated by successfully simulating the pharmacokinetics of dapagliflozin, another SGLT2 inhibitor. The present results suggest the utility of this PBPK/PD model for predicting canagliflozin concentrations at target sites and help to elucidate the pharmacological effects of SGLT1/2 inhibition in humans. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Differential effects of the substrate inhibitor l-trans-pyrrolidine-2,4-dicarboxylate (PDC) and the non-substrate inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) of glutamate transporters on neuronal damage and extracellular amino acid levels in rat brain in vivo.

    PubMed

    Montiel, T; Camacho, A; Estrada-Sánchez, A M; Massieu, L

    2005-01-01

    The extracellular concentration of glutamate is highly regulated by transporter proteins, due to its neurotoxic properties. Dysfunction or reverse activation of these transporters is related to the extracellular accumulation of excitatory amino acids and neuronal damage associated with ischemia and hypoglycemia. We have investigated by microdialysis the effects of the substrate and the non-substrate inhibitors of glutamate transporters, l-trans-2,4-pyrrolidine dicarboxylate (PDC) and DL-threo-beta-benzyloxyaspartate (DL-TBOA), respectively, on the extracellular levels of amino acids in the rat hippocampus in vivo. In addition, we have studied the effect of both inhibitors on neuronal damage after direct administration into the hippocampus and striatum. Electroencephalographic activity was recorded after the intrahippocampal infusion of DL-TBOA or PDC. Microdialysis administration of 500 microM DL-TBOA into the hippocampus increased 3.4- and nine-fold the extracellular levels of aspartate and glutamate, respectively. Upon stereotaxic administration it induced neuronal damage dose-dependently in CA1 and dentate gyrus, and convulsive behavior. Electroencephalographic recording showed the appearance of limbic seizures in the hippocampus after DL-TBOA infusion. In the striatum it also induced dose-dependent neuronal damage. These effects were prevented by the i.p. administration of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-iminemaleate and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline. In contrast to dl-TBOA, PDC (500 microM) induced a more discrete elevation of excitatory amino acids levels (2.6- and three-fold in aspartate and glutamate, respectively), no neuronal damage or behavioral changes, and no alterations in electroencephalographic activity. The differential results obtained with DL-TBOA and PDC might be attributed to their distinct effects on the extracellular concentration of amino acids. Results

  3. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  4. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  5. Invokana (Canagliflozin) as a dual inhibitor of acetylcholinesterase and sodium glucose co-transporter 2: advancement in Alzheimer's disease- diabetes type 2 linkage via an enzoinformatics study.

    PubMed

    Rizvi, Syed M D; Shakil, Shahnawaz; Biswas, Deboshree; Shakil, Shazi; Shaikh, Sibhghatulla; Bagga, Paramdeep; Kamal, Mohammad A

    2014-04-01

    Acetylcholinesterase (AChE) is a primary target for Alzheimer's therapy while recently sodium glucose cotransporter 2 (SGLT2) has gained importance as a potential target for Type 2 Diabetes Mellitus (T2DM) therapy. The present study emphasizes the molecular interactions between a new Food and Drug Administration (FDA) approved antidiabetic drug 'Invokana' (chemically known as Canagliflozin) with AChE and SGLT2 to establish a link between the treatment of T2DM and Alzheimer's Disease (AD). Docking study was performed using 'Autodock4.2'. Both hydrophobic and π-π interactions play an important role in the correct positioning of Canagliflozin within SGLT2 and catalytic site (CAS) of AChE to permit docking. Free energy of binding (ΔG) for 'Canagliflozin-SGLT2' interaction and 'Canagliflozin - CAS domain of AChE' interaction were found to be -10.03 kcal/mol and -9.40 kcal/mol, respectively. During 'Canagliflozin-SGLT2' interaction, Canagliflozin was found to interact with the most important amino acid residue Q457 of SGLT2. This residue is known for its interaction with glucose during reabsorption in kidney. However, 'Canagliflozin-CAS domain of AChE' interaction revealed that out of the three amino acids constituting the catalytic triad (S203, H447 and E334), two amino acid residues (S203 and H447) interact with Canagliflozin. Hence, Invokana (Canagliflozin) might act as a potent dual inhibitor of AChE and SGLT2. However, scope still remains in the determination of the three-dimensional structure of SGLT2-Canagliflozin and AChE-Canagliflozin complexes by X-ray crystallography to validate the described data. Since the development of diabetes is associated with AD, the design of new AChE inhibitors based on antidiabetic drug scaffolds would be particularly beneficial. Moreover, the present computational study reveals that Invokana (Canagliflozin) is expected to form the basis of a future dual therapy against diabetes associated neurological disorders.

  6. Anandamide enhances IL-10 production in activated microglia by targeting CB(2) receptors: roles of ERK1/2, JNK, and NF-kappaB.

    PubMed

    Correa, Fernando; Hernangómez, Miriam; Mestre, Leyre; Loría, Frida; Spagnolo, Alessandra; Docagne, Fabian; Di Marzo, Vicenzo; Guaza, Carmen

    2010-01-15

    The endocannabinoid system exhibits anti-inflammatory properties by regulating cytokine production. Anandamide (AEA) down-regulates proinflammatory cytokines in a viral model of multiple sclerosis (MS). However, little is known about the mechanisms by which AEA exerts these effects. Microglial cells are the main source of cytokines within the brain and the first barrier of defense against pathogens by acting as antigen presenting cells. IL-10 is a key physiological negative regulator of microglial activation. In this study we show that AEA enhances LPS/IFNgamma-induced IL-10 production in microglia by targeting CB(2) receptors through the activation of ERK1/2 and JNK MAPKs. AEA also inhibits NF-kappaB activation by interfering with the phosphorylation of IkappaBalpha, which may result in an increase of IL-10 production. Moreover, endogenously produced IL-10 negatively regulates IL-12 and IL-23 cytokines, which in its turn modify the pattern of expression of transcription factors involved in Th commitment of splenocytes. This suggests that by altering the cytokine network, AEA could indirectly modify the type of immune responses within the central nervous system (CNS). Accordingly, pharmacological modulation of AEA uptake and degradation might be a useful tool for treating neuroinflammatory diseases.

  7. Vaccenic acid suppresses intestinal inflammation by increasing anandamide and related N-acylethanolamines in the JCR:LA-cp rat[S

    PubMed Central

    Jacome-Sosa, Miriam; Vacca, Claudia; Mangat, Rabban; Diane, Abdoulaye; Nelson, Randy C.; Reaney, Martin J.; Shen, Jianheng; Curtis, Jonathan M.; Vine, Donna F.; Field, Catherine J.; Igarashi, Miki; Piomelli, Daniele; Banni, Sebastiano; Proctor, Spencer D.

    2016-01-01

    Vaccenic acid (VA), the predominant ruminant-derived trans fat in the food chain, ameliorates hyperlipidemia, yet mechanisms remain elusive. We investigated whether VA could influence tissue endocannabinoids (ECs) by altering the availability of their biosynthetic precursor, arachidonic acid (AA), in membrane phospholipids (PLs). JCR:LA-cp rats were assigned to a control diet with or without VA (1% w/w), cis-9, trans-11 conjugated linoleic acid (CLA) (1% w/w) or VA+CLA (1% + 0.5% w/w) for 8 weeks. VA reduced the EC, 2-arachidonoylglycerol (2-AG), in the liver and visceral adipose tissue (VAT) relative to control diet (P < 0.001), but did not change AA in tissue PLs. There was no additive effect of combining VA+CLA on 2-AG relative to VA alone (P > 0.05). Interestingly, VA increased jejunal concentrations of anandamide and those of the noncannabinoid signaling molecules, oleoylethanolamide and palmitoylethanolamide, relative to control diet (P < 0.05). This was consistent with a lower jejunal protein abundance (but not activity) of their degrading enzyme, fatty acid amide hydrolase, as well as the mRNA expression of TNFα and interleukin 1β (P < 0.05). The ability of VA to reduce 2-AG in the liver and VAT provides a potential mechanistic explanation to alleviate ectopic lipid accumulation. The opposing regulation of ECs and other noncannabinoid lipid signaling molecules by VA suggests an activation of benefit via the EC system in the intestine. PMID:26891736

  8. [SGLT2 inhibitor].

    PubMed

    Kubota, Naoto; Kadowaki, Takashi

    2015-12-01

    SGLT2 is a glucose transporter which plays an important role for reabsorption of urinary glucose depending on the sodium concentration gradient. SGLT2 is mainly present in apical site of S1 segment of renal proximal tubule and accounts for approximately 90% of total urinary glucose reabsorption. SLC5a2, which codes SGLT2, is also known as the causative gene of familial renal glucosuria. SGLT2 inhibitors are attracting attention as newly developed oral anti-diabetic agents which improve glucose intolerance and also have an anti-obese effect by promoting urinary glucose excretion (UGE), which is a different pharmacological effect from other conventional anti-diabetic agents. In this review, we will discuss the effect of SGLT2 inhibitor on the regulation of glucose and lipid metabolism in type 2 diabetes.

  9. Triton WR1339, an inhibitor of lipoprotein lipase, decreases vitamin E concentration in some tissues of rats by inhibiting its transport to liver.

    PubMed

    Abe, Chisato; Ikeda, Saiko; Uchida, Tomono; Yamashita, Kanae; Ichikawa, Tomio

    2007-02-01

    The aim of this experiment was to clarify the contribution of the alpha-tocopherol transfer activity of lipoprotein lipase (LPL) to vitamin E transport to tissues in vivo. We studied the effect of Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoproteins by LPL on vitamin E distribution in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were injected with Triton WR1339 and administered by oral gavage an emulsion containing 10 mg of alpha-tocopherol, 10 mg of gamma-tocopherol, or 29.5 mg of a tocotrienol mixture with 200 mg of sodium taurocholate, 200 mg of triolein, and 50 mg of albumin. alpha-Tocopherol was detected in the serum and other tissues of the vitamin E-deficient rats, but gamma-tocopherol, alpha- and gamma-tocotrienol were not detected. Triton WR1339 injection elevated (P<0.05) the serum alpha-tocopherol concentration and inhibited (P<0.05) the elevation of alpha-tocopherol concentration in the liver, adrenal gland, and spleen due to the oral administration of alpha-tocopherol. Neither alpha-tocopherol administration nor Triton WR1339 injection affected (P>or=0.05) the alpha-tocopherol concentration in the perirenal adipose tissue, epididymal fat, and soleus muscle despite a high expression of LPL in the adipose tissue and muscle. These data show that alpha-tocopherol transfer activity of LPL in adipose tissue and muscle is not important for alpha-tocopherol transport to the tissue after alpha-tocopherol intake or that the amount transferred is small relative to the tissue concentration. Furthermore, Triton WR1339 injection tended to elevate the serum gamma-tocopherol (P=0.071) and alpha-tocotrienol (P=0.053) concentrations and lowered them (P<0.05) in the liver and adrenal gland of rats administered gamma-tocopherol or alpha-tocotrienol. These data suggest that lipolysis of triacylglycerol-rich chylomicron by LPL is necessary for postprandial vitamin E transport to the liver and subsequent transport to the

  10. Fatty acid amide hydrolase inhibitors confer anti-invasive and antimetastatic effects on lung cancer cells

    PubMed Central

    Winkler, Katrin; Ramer, Robert; Dithmer, Sophie; Ivanov, Igor; Merkord, Jutta; Hinz, Burkhard

    2016-01-01

    Inhibition of endocannabinoid degradation has been suggested as tool for activation of endogenous tumor defense. One of these strategies lies in blockade of fatty acid amide hydrolase (FAAH) which catalyzes the degradation of endocannabinoids (anandamide [AEA], 2-arachidonoylglycerol [2-AG]) and endocannabinoid-like substances (N-oleoylethanolamine [OEA], N-palmitoylethanolamine [PEA]). This study addressed the impact of two FAAH inhibitors (arachidonoyl serotonin [AA-5HT], URB597) on A549 lung cancer cell metastasis and invasion. LC-MS analyses revealed increased levels of FAAH substrates (AEA, 2-AG, OEA, PEA) in cells incubated with either FAAH inhibitor. In athymic nude mice FAAH inhibitors were shown to elicit a dose-dependent antimetastatic action yielding a 67% and 62% inhibition of metastatic lung nodules following repeated administration of 15 mg/kg AA-5HT and 5 mg/kg URB597, respectively. In vitro, a concentration-dependent anti-invasive action of either FAAH inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). Using siRNA approaches, a causal link between the TIMP-1-upregulating and anti-invasive action of FAAH inhibitors was confirmed. Moreover, knockdown of FAAH by siRNA was shown to confer decreased cancer cell invasiveness and increased TIMP-1 expression. Inhibitor experiments point toward a role of CB2 and transient receptor potential vanilloid 1 in conferring anti-invasive effects of FAAH inhibitors and FAAH siRNA. Finally, antimetastatic and anti-invasive effects were confirmed for all FAAH substrates with AEA and OEA causing a TIMP-1-dependent anti-invasive action. Collectively, the present study provides first-time proof for an antimetastatic action of FAAH inhibitors. As mechanism of its anti-invasive properties an upregulation of TIMP-1 was identified. PMID:26930716

  11. Gram-scale solution-phase synthesis of selective sodium bicarbonate co-transport inhibitor S0859: in vitro efficacy studies in breast cancer cells.

    PubMed

    Larsen, Ann M; Krogsgaard-Larsen, Niels; Lauritzen, Gitte; Olesen, Christina W; Honoré Hansen, Steen; Boedtkjer, Ebbe; Pedersen, Stine F; Bunch, Lennart

    2012-10-01

    Na(+)-coupled HCO(3)(-) transporters (NBCs) mediate the transport of bicarbonate ions across cell membranes and are thus ubiquitous regulators of intracellular pH. NBC dysregulation is associated with a range of diseases; for instance, NBCn1 is strongly up-regulated in a model of ErbB2-dependent breast cancer, a malignant and widespread cancer with no targeted treatment options, and single-nucleotide polymorphisms in NBCn1 genetically link to breast cancer development and hypertension. The N-cyanosulfonamide S0859 has been shown to selectively inhibit NBCs, and its availability on the gram scale is therefore of significant interest to the scientific community. Herein we describe a short and efficient synthesis of S0859 with an overall yield of 45 % from commercially available starting materials. The inhibitory effect of S0859 on recovery of intracellular pH after an acid load was verified in human and murine cancer cell lines in Ringer solutions. However, S0859 binds very strongly to components in plasma, and accordingly, measurements on isolated murine tissues showed no effect of S0859 at concentrations up to 50 μM.

  12. Dapagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice

    PubMed Central

    Chiba, Yumiko; Yamada, Tetsuya; Tsukita, Sohei; Takahashi, Kei; Munakata, Yuichiro; Shirai, Yuta; Kodama, Shinjiro; Asai, Yoichiro; Sugisawa, Takashi; Uno, Kenji; Sawada, Shojiro; Imai, Junta; Nakamura, Kazuhiro; Katagiri, Hideki

    2016-01-01

    Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves. PMID:26963613

  13. 5'-azido-N-1-naphthylphthalamic acid, a photolabile analog of the auxin transport inhibitor, N-1-naphthylphthalamic acid: synthesis and binding properties

    SciTech Connect

    Voet, J.G.; Howley, K.; Shumsky, J.S.

    1987-05-01

    The polar transport of the plant growth regulator, auxin (indole-3-acetic acid, IAAH), is thought to involve the participation of several proteins in the plasma membrane, including a specific, saturable, voltage independent H/sup +//IAA/sup -/ efflux carrier located preferentially at the basal end of each cell. Auxin transport is specifically inhibited by the herbicide, N-1-naphthylphthalamic acid (NPA), which binds specifically to a protein in the plasma membrane, thought to be either the IAA/sup -/ efflux carrier or an allosteric effector protein. They have synthesized and characterized a photolabile analog of NPA, 5'-azido-N-1-naphthylphthalamic acid (Az-NPA). This potential photoaffinity label for the NPA binding protein competes with /sup 3/H-NPA for binding sites on Curcurbita pepo L. (zucchini) stem cell membranes with K/sub j/ = 1.5 x 10/sup -7/ M. The K/sub i/ for NPA under these conditions is 2 x 10/sup -8/M, indicating that the affinity of Az-NPA for the membranes is only 7.5 fold lower than NPA. While the binding of 4.6 x 10/sup -6/ M Az-NPA to NPA binding sites is reversible in the dark, exposure to light results in a 30% loss in /sup 3/H-NPA binding ability. Pretreatment with 10/sup -4/ M NPA protects the membranes against photodestruction of /sup 3/H-NPA binding sites by Az-NPA, supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.

  14. Effects of triclabendazole on secretion of danofloxacin and moxidectin into the milk of sheep: role of triclabendazole metabolites as inhibitors of the ruminant ABCG2 transporter.

    PubMed

    Barrera, Borja; González-Lobato, Lucía; Otero, Jon A; Real, Rebeca; Prieto, Julio G; Álvarez, Ana I; Merino, Gracia

    2013-11-01

    ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug-drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0-48 h) was 2.99±1.41 μg h/mL in the group treated with TCBZ and moxidectin, and 7.75±3.58 μg h/mL in the group treated with moxidectin alone. The AUC (0-48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34±1.51) than in the group treated with moxidectin alone (11.68±3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

  15. Investigating the selectivity of metalloenzyme inhibitors.

    PubMed

    Day, Joshua A; Cohen, Seth M

    2013-10-24

    The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.

  16. Metabolic and Cardiovascular Benefits and Risks of EMD386088—A 5-HT6 Receptor Partial Agonist and Dopamine Transporter Inhibitor

    PubMed Central

    Kotańska, Magdalena; Śniecikowska, Joanna; Jastrzębska-Więsek, Magdalena; Kołaczkowski, Marcin; Pytka, Karolina

    2017-01-01

    Since 5-HT6 receptors play role in controlling feeding and satiety and dopamine is essential for normal feeding behavior, we evaluated the ability of EMD 386088—5-HT6 receptor partial agonist and dopamine transporter inhibitor—to reduce body weight in obese rats, as well as its anorectic properties (calorie intake reduction) in rat model of excessive eating and the influence on metabolism (plasma glucose and glycerol levels). We also determined the effect of the studied compound on pica behavior in rats and its influence on blood pressure after single administration. EMD 386088 reduced body weight in obese rats fed high-fat diet and decreased calorie intake in both models applied (rat model of obesity and of excessive eating). In both models EMD 386088 regulated plasma glucose and increased plasma glycerol levels. The latter proves that the compound reduced body fat. We think that it might have increased lipolysis, but this requires further studies. The reduction in glucose levels is the first symptom of metabolic disorders compensation. EMD 386088 did not cause pica behavior in rats but increased blood pressure after single administration. We think that partial 5-HT6 agonists might have potential in the treatment of obesity. Thus, EMD 386088 requires extended studies. PMID:28228713

  17. Inhibition of the Serotonin Transporter Is Altered by Metabolites of Selective Serotonin and Norepinephrine Reuptake Inhibitors and Represents a Caution to Acute or Chronic Treatment Paradigms.

    PubMed

    Krout, Danielle; Rodriquez, Meghan; Brose, Stephen A; Golovko, Mikhail Y; Henry, L Keith; Thompson, Brent J

    2016-12-28

    Previous studies of transgenic mice carrying a single isoleucine to methionine substitution (I172M) in the serotonin transporter (SERT) demonstrated a loss of sensitivity to multiple antidepressants (ADs) at SERT. However, the ability of AD metabolites to antagonize SERT was not assessed. Here, we evaluated the selectivity and potency of these metabolites for inhibition of SERT in mouse brain-derived synaptosomes and blood platelets from wild-type (I172 mSERT) and the antidepressant-insensitive mouse M172 mSERT. The metabolites norfluoxetine and desmethylsertraline lost the selectivity demonstrated by the parent compounds for inhibition of wild-type mSERT over M172 mSERT, whereas desvenlafaxine and desmethylcitalopram retained selectivity. Furthermore, we show that the metabolite desmethylcitalopram accumulates in the brain and that the metabolites desmethylcitalopram, norfluoxetine, and desvenlafaxine inhibit serotonin uptake in wild-type mSERT at potencies similar to those of their parent compounds, suggesting that metabolites may play a role in effects observed following AD administration in wild-type and M172 mice.

  18. Structure-guided inhibitor design for human FAAH by interspecies active site conversion.

    PubMed

    Mileni, Mauro; Johnson, Douglas S; Wang, Zhigang; Everdeen, Daniel S; Liimatta, Marya; Pabst, Brandon; Bhattacharya, Keshab; Nugent, Richard A; Kamtekar, Satwik; Cravatt, Benjamin F; Ahn, Kay; Stevens, Raymond C

    2008-09-02

    The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

  19. The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms

    PubMed Central

    Bosier, Barbara; Muccioli, Giulio G; Lambert, Didier M

    2013-01-01

    Background Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between ‘on demand’ generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. Experimental Approach We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. Key Results Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. Conclusions While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when

  20. Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters.

    PubMed

    Takagi, Kazutaka; Dexheimer, Thomas S; Redon, Christophe; Sordet, Olivier; Agama, Keli; Lavielle, Gilbert; Pierré, Alain; Bates, Susan E; Pommier, Yves

    2007-12-01

    Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their alpha-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone gamma-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone gamma-H2AX could be used as a biomarker for the upcoming clinical trials of S39625.

  1. Dietary linoleic acid elevates the endocannabinoids 2-AG and anandamide and promotes weight gain in mice fed a low fat diet.

    PubMed

    Alvheim, Anita Røyneberg; Torstensen, Bente E; Lin, Yu Hong; Lillefosse, Haldis Haukås; Lock, Erik-Jan; Madsen, Lise; Frøyland, Livar; Hibbeln, Joseph R; Malde, Marian Kjellevold

    2014-01-01

    Dietary intake of linoleic acid (LNA, 18:2n-6) has increased dramatically during the 20th century and is associated with greater prevalence of obesity. The endocannabinoid system is involved in regulation of energy balance and a sustained hyperactivity of the endocannabinoid system may contribute to obesity. Arachidonic acid (ARA, 20:4n-6) is the precursor for 2-AG and anandamide (AEA), and we sought to determine if low fat diets (LFD) could be made obesogenic by increasing the endocannabinoid precursor pool of ARA, causing excessive endocannabinoid signaling leading to weight gain and a metabolic profile associated with obesity. Mice (C57BL/6j, 6 weeks of age) were fed 1 en% LNA and 8 en% LNA in low fat (12.5 en%) and medium fat diets (MFD, 35 en%) for 16 weeks. We found that increasing dietary LNA from 1 to 8 en% in LFD and MFD significantly increased ARA in phospholipids (ARA-PL), elevated 2-AG and AEA in liver, elevated plasma leptin, and resulted in larger adipocytes and more macrophage infiltration in adipose tissue. In LFD, dietary LNA of 8 en% increased feed efficiency and caused greater weight gain than in an isocaloric reduction to 1 en% LNA. Increasing dietary LNA from 1 to 8 en% elevates liver endocannabinoid levels and increases the risk of developing obesity. Thus a high dietary content of LNA (8 en%) increases the adipogenic properties of a low fat diet.

  2. Anandamide-induced endoplasmic reticulum stress and apoptosis are mediated by oxidative stress in non-melanoma skin cancer: Receptor-independent endocannabinoid signaling.

    PubMed

    Soliman, Eman; Van Dross, Rukiyah

    2016-11-01

    Endocannabinoids are neuromodulatory lipids that regulate central and peripheral physiological functions. Endocannabinoids have emerged as effective antitumor drugs due to their ability to induce apoptosis in various cancer studies. The G-protein coupled cannabinoid receptors (CB1 and CB2) and the TRPV1 ion channel were reported to mediate the antiproliferative activity of endocannabinoids. However, receptor-independent effects also account for their activity. Our previous studies showed that the antiproliferative activity of anandamide (AEA) was regulated by cyclooxygenase-2 (COX-2) via induction of endoplasmic reticulum (ER) stress. We also determined that AEA induced oxidative stress. However, the role of oxidative stress, the cannabinoid receptors, and TRPV1 in AEA-induced ER stress-apoptosis was unclear. Therefore, the current study examines the role of oxidative stress in ER stress-apoptosis and investigates whether this effect is modulated by CB1, CB2, or TRPV1. In non-melanoma skin cancer (NMSC) cells, AEA reduced the total intracellular level of glutathione and induced oxidative stress. To evaluate the importance of oxidative stress in AEA-induced cell death, the antioxidants, N-acetylcysteine (NAC) and Trolox, were utilized. Each antioxidant ameliorated the antiproliferative effect of AEA. Furthermore, Trolox inhibited AEA-induced CHOP10 expression and caspase 3 activity, indicating that oxidative stress was required for AEA-induced ER stress-apoptosis. On the other hand, selective blockade of CB1, CB2, and TRPV1 did not inhibit AEA-induced oxidative stress or ER stress-apoptosis. These findings suggest that AEA-induced ER stress-apoptosis in NMSC cells is mediated by oxidative stress through a receptor-independent mechanism. Hence, receptor-independent AEA signaling pathways may be targeted to eliminate NMSC. © 2015 Wiley Periodicals, Inc.

  3. CB1 cannabinoid receptor-mediated anandamide signalling reduces the defensive behaviour evoked through GABAA receptor blockade in the dorsomedial division of the ventromedial hypothalamus.

    PubMed

    Dos Anjos-Garcia, Tayllon; Ullah, Farhad; Falconi-Sobrinho, Luiz Luciano; Coimbra, Norberto Cysne

    2017-02-01

    The effects of cannabinoids in brain areas expressing cannabinoid receptors, such as hypothalamic nuclei, are not yet well known. Several studies have demonstrated the role of hypothalamic nuclei in the organisation of behavioural responses induced through innate fear and panic attacks. Panic-prone states are experimentally induced in laboratory animals through a reduction in the GABAergic activity. The aim of the present study was to examine panic-like elaborated defensive behaviour evoked by GABAA receptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus (VMHdm). We also aimed to characterise the involvement of endocannabinoids and the CB1 cannabinoid receptor in the modulation of elaborated defence behavioural responses organised with the VMHdm. The guide-cannula was stereotaxicaly implanted in VMHdm and the animals were treated with anandamide (AEA) at different doses, and the effective dose was used after the pre-treatment with the CB1 receptor antagonist AM251, followed by GABAA receptor blockade in VMHdm. The results showed that the intra-hypothalamic administration of AEA at an intermediate dose (5 pmol) attenuated defence responses induced through the intra-VMHdm microinjection of bicuculline (40 ng). This effect, however, was inhibited when applied central microinjection of the CB1 receptor antagonist AM251 in the VMHdm. Moreover, AM251 potentiates de non-oriented escape induced by bicuculline, effect blocked by pre-treatment with the TRPV1 channel antagonist 6-I-CPS. These results indicate that AEA modulates the pro-aversive effects of intra-VMHdm-bicuculline treatment, recruiting CB1 cannabinoid receptors and the TRPV1 channel is involved in the AM251-related potentiation of bicuculline effects on non-oriented escape behaviour.

  4. A column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry method to determine anandamide and 2-arachidonoylglycerol in plasma samples.

    PubMed

    Marchioni, Camila; de Souza, Israel Donizeti; Grecco, Caroline Fernandes; Crippa, José Alexandre; Tumas, Vitor; Queiroz, Maria Eugênia Costa

    2017-03-23

    This study reports a fast, sensitive, and selective column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG) in plasma samples. This bidimensional system used a restricted access media column (RP-8 ADS, 25 mm × 4 mm × 25 μM) in the first dimension and a core-shell Kinetex C18 (100 mm × 2, 1.7 mm × 1 μM) column in the second dimension, followed by detection in a mass spectrometer triple quadrupole (multiple reactions monitoring mode) operating in the positive mode. RP-8 ADS was used for trace enrichment of eCBs (reverse phase partitioning) and macromolecular matrix size exclusion; the core-shell column was used for the chromatographic separation. The column switching UHPLC-MS/MS method presented a linear range spanning from 0.1 ng mL(-1) (LOQ) to 6 ng mL(-1) for AEA and from 0.04 ng mL(-1) (LOQ) to 10 ng mL(-1) for 2-AG. Excluding the LLOQ values, the precision assays provided coefficients of variation lower than 8% and accuracy with relative standard error values lower than 14%. Neither carryover nor matrix effects were detected. This high-throughput column switching method compared to conventional methods is time saving as it involves fewer steps, consumes less solvent, and presents lower LLOQ. The column switching UHPLC-MS/MS method was successfully applied to determine AEA and 2-AG in plasma samples obtained from Alzheimer's disease patients. Graphical abstract A column switching ultra high-performance liquid chromatography-tandem mass spectrometry method using RP-8 ADS column and core shell column to determine endocannabinoids in plasma samples.

  5. Anandamide and 2-AG Are Endogenously Present within the Laterodorsal Tegmental Nucleus: Functional Implications for a role of eCBs in arousal.

    PubMed

    Soni, Neeraj; Prabhala, Bala Krishna; Mehta, Ved; Mirza, Osman; Kohlmeier, Kristi Anne

    2017-04-09

    Previously, we presented electrophysiological evidence for presence in mice brain slices of functional cannabinoid type I receptors (CB1Rs) within the laterodorsal tegmentum (LDT), a brain stem nucleus critical in control of arousal and rapid eye movement (REM) sleep. Further, using pharmacological agents, we provided data suggestive of the endogenous presence of cannabinoids (CBs) acting at LDT CB1Rs. However, in those studies, we were unable to identify the type(s) of CB ligands endogenously present in the LDT, and this information has not been provided elsewhere. Accordingly, we used the highly-sensitive liquid chromatography/mass spectrometry (LC-MS) method to determine whether N-arachidonoylethanolamide (Anandamide or AEA) and 2-arachidonyl glycerol (2-AG), which are both endogenous CB ligands acting at CB1Rs, are present in the LDT. Mice brain tissue samples of the LDT were assayed using ion trap LC-MS in selected ion monitoring mode. Chromatographic analysis and product-ion MS scans identified presence of the CBs, AEA and 2-AG, from LDT mouse tissue. Data using the LC-MS method show that AEA and 2-AG are endogenously present within the LDT and when coupled with our electrophysiological findings, lead to the suggestion that AEA and 2-AG act at electropharmacologically-demonstrated CB1Rs in this nucleus. Accordingly, AEA and 2-AG likely play a role in processes governed by the LDT, including control of states of cortical gamma band activity seen in alert, aroused states, as well as cortical and motor activity characteristic of REM sleep.

  6. Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells.

    PubMed Central

    Di Marzo, V; Melck, D; Orlando, P; Bisogno, T; Zagoory, O; Bifulco, M; Vogel, Z; De Petrocellis, L

    2001-01-01

    Palmitoylethanolamide (PEA) has been shown to act in synergy with anandamide (arachidonoylethanolamide; AEA), an endogenous agonist of cannabinoid receptor type 1 (CB(1)). This synergistic effect was reduced by the CB(2) cannabinoid receptor antagonist SR144528, although PEA does not activate either CB(1) or CB(2) receptors. Here we show that PEA potently enhances the anti-proliferative effects of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of fatty acid amide hydrolase (FAAH), the major enzyme catalysing AEA degradation. PEA (1-10 microM) enhanced in a dose-related manner the inhibitory effect of AEA on both basal and nerve growth factor (NGF)-induced HBCC proliferation, without inducing any cytostatic effect by itself. PEA (5 microM) decreased the IC(50) values for AEA inhibitory effects by 3-6-fold. This effect was not blocked by the CB(2) receptor antagonist SR144528, and was not mimicked by a selective agonist of CB(2) receptors. PEA enhanced AEA-evoked inhibition of the expression of NGF Trk receptors, which underlies the anti-proliferative effect of the endocannabinoid on NGF-stimulated MCF-7 cells. The effect of PEA was due in part to inhibition of AEA degradation, since treatment of MCF-7 cells with 5 microM PEA caused a approximately 30-40% down-regulation of FAAH expression and activity. However, PEA also enhanced the cytostatic effect of the cannabinoid receptor agonist HU-210, although less potently than with AEA. PEA did not modify the affinity of ligands for CB(1) or CB(2) receptors, and neither did it alter the CB(1)/CB(2)-mediated inhibitory effect of AEA on adenylate cyclase type V, nor the expression of CB(1) and CB(2) receptors in MCF-7 cells. We suggest that long-term PEA treatment of cells may positively affect the pharmacological activity of AEA, in part by inhibiting FAAH expression. PMID:11485574

  7. Cannabinoid CB1 receptor recognition of endocannabinoids via the lipid bilayer: molecular dynamics simulations of CB1 transmembrane helix 6 and anandamide in a phospholipid bilayer

    NASA Astrophysics Data System (ADS)

    Lynch, Diane L.; Reggio, Patricia H.

    2006-08-01

    The phospholipid bilayer plays a central role in the lifecycle of the endogenous cannabinoid, N-arachidonoylethanolamine (anandamide, AEA). Therefore, the orientation and location of AEA in the phospholipid bilayer with respect to key membrane associated proteins, is a central issue in understanding the mechanism of endocannabinoid signaling. In this paper, we report a test of the hypothesis that a βXX β motif (formed by beta branching amino acids, V6.43 and I6.46) on the lipid face of the cannabinoid CB1 receptor in its inactive state may serve as an initial CB1 interaction site for AEA. Eight 6 ns NAMD2 molecular dynamics simulations of AEA were conducted in a model system composed of CB1 transmembrane helix 6 (TMH6) in a 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) bilayer. In addition, eight 6 ns NAMD2 molecular dynamics simulations of a low CB1 affinity (20:2, n-6) analog of AEA were conducted in the same model system. AEA was found to exhibit a higher incidence of V6.43/I6.46 groove insertion than did the (20:2, n-6) analog. In certain cases, AEA established a high energy of interaction with TMH6 by first associating with the V6.43/I6.46 groove and then molding itself to the lipid face of TMH6 to establish a hydrogen bonding interaction with the exposed backbone carbonyl of P6.50. Based upon these results, we propose that the formation of this hydrogen bonded AEA/TMH6 complex may be the initial step in CB1 recognition of AEA in the lipid bilayer.

  8. Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid, on ion currents in NG108-15 neuronal cells.

    PubMed

    Lo, Yuk-Keung; Chiang, Hung-Ting; Wu, Sheng-Nan

    2003-02-15

    The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.

  9. In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

    PubMed

    Yamane, Mizuki; Kawashima, Kosuke; Yamaguchi, Koji; Nagao, Shunsuke; Sato, Mika; Suzuki, Masayuki; Honda, Kiyofumi; Hagita, Hitoshi; Kuhlmann, Olaf; Poirier, Agnes; Fowler, Stephen; Funk, Christoph; Simon, Sandrine; Aso, Yoshinori; Ikeda, Sachiya; Ishigai, Masaki

    2015-03-01

    Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

  10. Polymorphism of rs3813034 in Serotonin Transporter Gene SLC6A4 Is Associated With the Selective Serotonin and Serotonin-Norepinephrine Reuptake Inhibitor Response in Depressive Disorder: Sequencing Analysis of SLC6A4.

    PubMed

    Nonen, Shinpei; Kato, Masaki; Takekita, Yoshiteru; Wakeno, Masataka; Sakai, Shiho; Serretti, Alessandro; Kinoshita, Toshihiko

    2016-02-01

    Selective serotonin and serotonin-norepinephrine reuptake inhibitors (SSRI/SNRI) are commonly used for treating major depression. Regretfully, significant heterogeneity exists regarding the benefits of SSRI/SNRI in individual cases. We previously reported that a polymorphism located in the serotonin transporter linked promoter region (5-HTT LPR) is associated with an interindividual difference in SSRI treatment efficacy. However, this explains only a small part of the variation of this complex phenotype. Other 5-HTT variants in the coding regions, 3' untranslated region (3' UTR), and introns adjacent to each exon could also contribute to treatment response. Therefore, we performed a sequencing analysis of the SLC6A4 gene (coding for 5-HTT) and investigated the association between variants detected in this study and the antidepressant response to SSRI/SNRI in 201 Japanese depressive patients. Seventeen novel mutations were identified by sequencing analysis. We found that the polymorphism G2563T (rs3813034) as a tag single-nucleotide polymorphism of IVS9 A-90G (rs140701), G2356T (rs1042173), and A3641C (rs7224199) is associated with interindividual variability of SSRI/SNRI efficacy at week 6, independent from clinical variables and effect of 5-HTT LPR (P < 0.001 by multiple regression analysis). This polymorphism could help determine individualized SSRI/SNRI treatments for depressive patients in combination with 5-HTT LPR.

  11. Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of the angiotensin I-converting enzyme-inhibitory peptides in Pacific hake (Merluccius productus) fillet hydrolysate.

    PubMed

    Cinq-Mars, Crystal D; Hu, Chun; Kitts, David D; Li-Chan, Eunice C Y

    2008-01-23

    Fish protein hydrolysate (FPH) produced by incubation of Pacific hake fillet with 3.00% Protamex at pH 6.5 and 40 degrees C for 125 min demonstrated in vitro ACE-inhibitory activity (IC50 = 165 microg/mL), which was enhanced by ultrafiltration through a 10 kDa molecular weight cutoff membrane (IC50 = 44 microg/mL). However, after simulated gastrointestinal digestion, FPH and ultrafiltrate had similar ACE-inhibitory activity (IC 50 = 90 microg/mL), indicating that FPH peptides act as "pro-drug type" inhibitors and that enrichment by ultrafiltration may be unnecessary. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed that the molecular weights of major peaks were <1 kDa regardless of ultrafiltration. ACE-inhibitory activities of digested hydrolysates were not significantly affected by preincubation with ACE ( P > 0.05) and exhibited a competitive inhibitory mode. A permeability assay using fully differentiated colorectal adenocarcinoma (Caco-2) cells showed an apical to basolateral transport of peptides that ranged from approximately 2 to 20% after 2 h at 37 degrees C. Pacific hake fillet hydrolysates are a potentially bioavailable source of ACE-inhibitory peptides awaiting further in vivo study.

  12. Dendrite inhibitor

    DOEpatents

    Miller, William E.

    1989-01-01

    An apparatus for removing dendrites or other crystalline matter from the surface of a liquid in a matter transport process, and an electrolytic cell including such an apparatus. A notch may be provided to allow continuous exposure of the liquid surface, and a bore may be further provided to permit access to the liquid.

  13. Dendrite inhibitor

    DOEpatents

    Miller, W.E.

    1988-06-07

    An apparatus for removing dendrites or other crystalline matter from the surface of a liquid in a matter transport process, and an electrolytic cell including such an apparatus. A notch may be provided to allow continuous exposure of the liquid surface, and a bore may be further provided to permit access to the liquid. 2 figs.

  14. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  15. Pharmacology of iron transport.

    PubMed

    Byrne, Shaina L; Krishnamurthy, Divya; Wessling-Resnick, Marianne

    2013-01-01

    Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes. In particular, iron chelators can serve as potential pharmacological tools to alleviate diseases of iron overload. This review focuses on the pharmacology of iron transport, introducing iron transport membrane proteins and known inhibitors.

  16. Pharmacology of Iron Transport

    PubMed Central

    Byrne, Shaina L.; Krishnamurthy, Divya; Wessling-Resnick, Marianne

    2013-01-01

    Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes. In particular, iron chelators can serve as potential pharmacological tools to alleviate diseases of iron overload. This review focuses on the pharmacology of iron transport, introducing iron transport membrane proteins and known inhibitors. PMID:23020294

  17. A validated LC-MS/MS method for the determination of canagliflozin, a sodium-glucose co-transporter 2 (SGLT-2) inhibitor, in a lower volume of rat plasma: application to pharmacokinetic studies in rats.

    PubMed

    Kobuchi, Shinji; Yano, Kyoka; Ito, Yukako; Sakaeda, Toshiyuki

    2016-10-01

    Canagliflozin is a novel, orally selective inhibitor of sodium-dependent glucose co-transporter-2 (SGLT2) for the treatment of patients with type 2 diabetes mellitus. In this study, a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of canagliflozin in a lower volume of rat plasma (0.1 mL) was established and applied to a pharmacokinetic study in rats. Following liquid-liquid extraction by tert-butyl methyl ether, chromatographic separation of canagliflozin was performed on a Quicksorb ODS (2.1 mm i.d. × 150 mm, 5 µm size) using acetonitrile-0.1% formic acid (90:10, v/v) as the mobile phase at a flow rate of 0.2 mL/min. The detection was carried out using an API 3200 triple-quadrupole mass spectrometer operating in the positive electrospray ionization mode. Selected ion monitoring transitions of m/z = 462.0 [M + NH4 ](+)  → 191.0 for canagliflozin and m/z = 451.2 [M + H](+)  → 71.0 for empagliflozin (internal standard) were obtained. The validation of the method was investigated, and it was found to be of sufficient specificity, accuracy and precision. Canagliflozin in rat plasma was stable under the analytical conditions used. This validated method was successfully applied to assess the pharmacokinetics of canagliflozin in rats using 0.1 mL rat plasma. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Evaluation of Rifampin's Transporter Inhibitory and CYP3A Inductive Effects on the Pharmacokinetics of Venetoclax, a BCL-2 Inhibitor: Results of a Single- and Multiple-Dose Study.

    PubMed

    Agarwal, Suresh K; Hu, Beibei; Chien, David; Wong, Shekman L; Salem, Ahmed Hamed

    2016-11-01

    Venetoclax is a selective, potent, first-in-class B-cell lymphoma-2 inhibitor that has demonstrated clinical efficacy in a variety of hematological malignancies. A single-dose and multiple-dose rifampin study was conducted to evaluate the effect of CYP3A induction and transporter inhibition on the pharmacokinetics of venetoclax. Subjects received a single dose of venetoclax 200 mg on day 1 of period 1 and days 1 and 14 of period 2, a single dose of rifampin 600 mg on day 1 of period 2, and rifampin 600 mg once daily on days 5 through 17 of period 2. Blood samples were collected up to 96 hours after each venetoclax dose on day 1 of period 1 and days 1 and 14 of period 2. Compared with venetoclax alone, coadministration with a single dose of rifampin increased venetoclax Cmax and AUC∞ by 106% (90%CI, 73%-145%) and 78% (90%CI, 50%-111%), respectively, whereas coadministration with multiple doses of rifampin decreased venetoclax Cmax and AUC∞ by 42% (90%CI, 31%-52%) and 71% (90%CI, 66%-76%), respectively. It was possible to isolate the net effect of chronic CYP3A induction from acute P-glycoprotein (P-gp) inhibition by comparing venetoclax exposures following coadministration with multiple doses of rifampin versus a single dose of rifampin, which showed that CYP3A induction decreased venetoclax Cmax and AUC by 72% and 84%, respectively. These results are consistent with venetoclax being a P-gp substrate and indicate that CYP3A plays a major role in venetoclax metabolism. Prescribers should consider agents with little or no CYP3A induction during treatment with venetoclax.

  19. Monoamine Reuptake Inhibitors in Parkinson's Disease

    PubMed Central

    Huot, Philippe; Fox, Susan H.; Brotchie, Jonathan M.

    2015-01-01

    The motor manifestations of Parkinson's disease (PD) are secondary to a dopamine deficiency in the striatum. However, the degenerative process in PD is not limited to the dopaminergic system and also affects serotonergic and noradrenergic neurons. Because they can increase monoamine levels throughout the brain, monoamine reuptake inhibitors (MAUIs) represent potential therapeutic agents in PD. However, they are seldom used in clinical practice other than as antidepressants and wake-promoting agents. This review article summarises all of the available literature on use of 50 MAUIs in PD. The compounds are divided according to their relative potency for each of the monoamine transporters. Despite wide discrepancy in the methodology of the studies reviewed, the following conclusions can be drawn: (1) selective serotonin transporter (SERT), selective noradrenaline transporter (NET), and dual SERT/NET inhibitors are effective against PD depression; (2) selective dopamine transporter (DAT) and dual DAT/NET inhibitors exert an anti-Parkinsonian effect when administered as monotherapy but do not enhance the anti-Parkinsonian actions of L-3,4-dihydroxyphenylalanine (L-DOPA); (3) dual DAT/SERT inhibitors might enhance the anti-Parkinsonian actions of L-DOPA without worsening dyskinesia; (4) triple DAT/NET/SERT inhibitors might exert an anti-Parkinsonian action as monotherapy and might enhance the anti-Parkinsonian effects of L-DOPA, though at the expense of worsening dyskinesia. PMID:25810948

  20. Effects of a sodium glucose co-transporter 2 selective inhibitor, ipragliflozin, on the diurnal profile of plasma glucose in patients with type 2 diabetes: A study using continuous glucose monitoring

    PubMed Central

    Yamada, Kentaro; Nakayama, Hitomi; Yoshinobu, Satoko; Kawano, Seiko; Tsuruta, Munehisa; Nohara, Masayuki; Hasuo, Rika; Akasu, Shoko; Tokubuchi, Ichiro; Wada, Nobuhiko; Hirao, Saori; Iwata, Shinpei; Kaku, Hiroo; Tajiri, Yuji

    2015-01-01

    Aims/Introduction To assess the effects of sodium glucose co-transporter 2 inhibitor therapy on the pathophysiology of type 2 diabetes. Materials and Methods We administered ipragliflozin to 21 inpatients with type 2 diabetes for 7 days, and analyzed the diurnal profiles of plasma glucose and 3-hydroxybutyrate. A total of 21 age-, sex- and body mass index-matched diabetic patients served as controls. Results Continuous glucose monitoring showed that the 24-h glucose curve was shifted downward without hypoglycemia by the administration of ipragliflozin. The average glucose level was reduced from 182 ± 54 mg/dL to 141 ± 33 mg/dL (P < 0.0001). The magnitude of the reduction was highly correlated with the baseline average glucose level. Homeostasis model assessment of insulin resistance was decreased, and homeostasis model assessment of β-cell function was increased during the treatment. Urinary glucose excretion was correlated with the average glucose level both on day 0 and on day 7, although the regression line was steeper and shifted leftward on day 7. The ipragliflozin-treated patients lost more weight than the control patients (1.4 ± 0.5 vs 0.5 ± 0.6 kg, P < 0.0001). Plasma levels of 3-hydroxybutyrate were significantly increased with peaks before breakfast and before dinner. Patient age and bodyweight loss were negatively and positively correlated with the peak levels of 3-hydroxybutyrate on day 7, respectively. Conclusions The ipragliflozin treatment improved the 24-h glucose curve without causing hypoglycemia. The close correlation between the magnitude of glucose reduction and the baseline plasma glucose concentration suggests that the risk of hypoglycemia is likely low. It might be prudent to monitor ketone body levels in younger patients and in patients with rapid weight loss. PMID:26543545

  1. Potent multitarget FAAH-COX inhibitors: Design and structure-activity relationship studies.

    PubMed

    Migliore, Marco; Habrant, Damien; Sasso, Oscar; Albani, Clara; Bertozzi, Sine Mandrup; Armirotti, Andrea; Piomelli, Daniele; Scarpelli, Rita

    2016-02-15

    Non-steroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by inhibiting cyclooxygenase (COX)-1 and COX-2. Though widely prescribed for pain and inflammation, these agents have limited utility in chronic diseases due to serious mechanism-based adverse events such as gastrointestinal damage. Concomitant blockade of fatty acid amide hydrolase (FAAH) enhances the therapeutic effects of the NSAIDs while attenuating their propensity to cause gastrointestinal injury. This favorable interaction is attributed to the accumulation of protective FAAH substrates, such as the endocannabinoid anandamide, and suggests that agents simultaneously targeting COX and FAAH might provide an innovative strategy to combat pain and inflammation with reduced side effects. Here, we describe the rational design and structure-active relationship (SAR) properties of the first class of potent multitarget FAAH-COX inhibitors. A focused SAR exploration around the prototype 10r (ARN2508) led to the identification of achiral (18b) as well as racemic (29a-c and 29e) analogs. Absolute configurational assignment and pharmacological evaluation of single enantiomers of 10r are also presented. (S)-(+)-10r is the first highly potent and selective chiral inhibitor of FAAH-COX with marked in vivo activity, and represents a promising lead to discover novel analgesics and anti-inflammatory drugs.

  2. The dual FAAH/MAGL inhibitor JZL195 has enhanced effects on endocannabinoid transmission and motor behavior in rats as compared to those of the MAGL inhibitor JZL184.

    PubMed

    Seillier, Alexandre; Dominguez Aguilar, David; Giuffrida, Andrea

    2014-09-01

    The biological actions of the endocannabinoids anandamide and 2-arachidonoyl glycerol (2-AG) are terminated by enzymatic hydrolysis of these lipids via fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. While several selective FAAH inhibitors have been developed and characterized in vitro and in vivo, none of the initial MAGL blockers have shown adequate potency and specificity for in vivo applications. More recently, a selective MAGL inhibitor, JZL184, has been shown to produce a long-lasting elevation of brain 2-AG, as well as cannabinoid-like behavioral responses in mice. However, its effectiveness in rats remains controversial. Indeed, although JZL184 can elicit behavioral responses that are mediated, at least in part, via activation of cannabinoid CB1 receptors, several reports indicate that this compound does not alter 2-AG levels in this species. In this study we compared the behavioral and neurochemical effects of JZL 184 with those of the dual FAAH/MAGL inhibitor JZL195, and showed that systemic administration of the former can selectively elevate brain 2-AG in rats and produce motor suppression through a CB1-independent mechanism. These findings indicate that, despite its lower potency against rat MAGL, JZL184 can be used to enhance 2-AG transmission and elicit behavioral responses in rodents.

  3. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  4. Δ9-tetrahydrocannabinol and endocannabinoid degradative enzyme inhibitors attenuate intracranial self-stimulation in mice.

    PubMed

    Wiebelhaus, Jason M; Grim, Travis W; Owens, Robert A; Lazenka, Matthew F; Sim-Selley, Laura J; Abdullah, Rehab A; Niphakis, Micah J; Vann, Robert E; Cravatt, Benjamin F; Wiley, Jenny L; Negus, S Stevens; Lichtman, Aron H

    2015-02-01

    A growing body of evidence implicates endogenous cannabinoids as modulators of the mesolimbic dopamine system and motivated behavior. Paradoxically, the reinforcing effects of Δ(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis, have been difficult to detect in preclinical rodent models. In this study, we investigated the impact of THC and inhibitors of the endocannabinoid hydrolytic enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) on operant responding for electrical stimulation of the medial forebrain bundle [intracranial self-stimulation (ICSS)], which is known to activate the mesolimbic dopamine system. These drugs were also tested in assays of operant responding for food reinforcement and spontaneous locomotor activity. THC and the MAGL inhibitor JZL184 (4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) attenuated operant responding for ICSS and food, and also reduced spontaneous locomotor activity. In contrast, the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) was largely without effect in these assays. Consistent with previous studies showing that combined inhibition of FAAH and MAGL produces a substantially greater cannabimimetic profile than single enzyme inhibition, the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1-piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) produced a similar magnitude of ICSS depression as that produced by THC. ICSS attenuation by JZL184 was associated with increased brain levels of 2-arachidonoylglycerol (2-AG), whereas peak effects of SA-57 were associated with increased levels of both N-arachidonoylethanolamine (anandamide) and 2-AG. The cannabinoid receptor type 1 receptor antagonist rimonabant, but not the cannabinoid receptor type 2 receptor antagonist SR144528, blocked the attenuating effects of THC, JZL184, and SA-57 on

  5. Selective Monoacylglycerol Lipase Inhibitors: Antinociceptive versus Cannabimimetic Effects in Mice

    PubMed Central

    Wilkerson, Jenny L.; Mustafa, Mohammed; Abdullah, Rehab; Niphakis, Micah; Wiley, Jenny L.; Cravatt, Benjamin F.; Lichtman, Aron H.

    2015-01-01

    The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) plays an important role in a variety of physiologic processes, but its rapid breakdown by monoacylglycerol lipase (MAGL) results in short-lived actions. Initial MAGL inhibitors were limited by poor selectivity and low potency. In this study, we tested JZL184 [4-nitrophenyl 4-[bis(2H-1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate] and MJN110 [2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate], MAGL inhibitors that possess increased selectivity and potency, in mouse behavioral assays of neuropathic pain [chronic constriction injury (CCI) of the sciatic nerve], interoceptive cannabimimetic effects (drug-discrimination paradigm), and locomotor activity in an open field test. MJN110 (1.25 and 2.5 mg/kg) and JZL184 (16 and 40 mg/kg) significantly elevated 2-AG and decreased arachidonic acid but did not affect anandamide in whole brains. Both MAGL inhibitors significantly reduced CCI-induced mechanical allodynia with the following potencies [ED50 (95% confidence limit [CL]) values in mg/kg: MJN110 (0.43 [0.30–0.63]) > JZL184 (17.8 [11.6–27.4])] and also substituted for the potent cannabinoid receptor agonist CP55,940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol] in the drug-discrimination paradigm [ED50 (95% CL) values in mg/kg: MJN110 (0.84 [0.69–1.02]) > JZL184 (24.9 [14.6–42.5])]; however, these compounds elicited differential effects on locomotor behavior. Similar to cannabinoid 1 (CB1) receptor agonists, JZL184 produced hypomotility, whereas MJN110 increased locomotor behavior and did not produce catalepsy or hypothermia. Although both drugs substituted for CP55,940 in the drug discrimination assay, MJN110 was more potent in reversing allodynia in the CCI model than in producing CP55,940-like effects. Overall, these results suggest that MAGL inhibition may alleviate neuropathic pain, while displaying limited

  6. (4-Phenoxyphenyl)tetrazolecarboxamides and related compounds as dual inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL).

    PubMed

    Holtfrerich, Angela; Hanekamp, Walburga; Lehr, Matthias

    2013-05-01

    Inhibitors of the enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principle enzymes involved in the degradation of endogenous cannabinoids like anandamide and 2-arachidonoylglycerol, have potential utility in the treatment of several disorders including pain, inflammation and anxiety. In the present study, the effectivity and selectivity of eight known FAAH and MAGL inhibitors for inhibition of the appropriate enzyme were measured applying in vitro assays, which work under comparable conditions. Because many of the known FAAH and MAGL inhibitors simply consist of a lipophilic scaffold to which a heterocyclic system is bound, furthermore, different heterocyclic structures were evaluated for their contribution to enzyme inhibition by attaching them to the same lipophilic backbone, namely 4-phenoxybenzene. One of the most active compound synthesized during this investigation was N,N-dimethyl-5-(4-phenoxyphenyl)-2H-tetrazole-2-carboxamide (16) (IC50 FAAH: 0.012 μM; IC50 MAGL: 0.028 μM). This inhibitor was systematically modified in the lipophilic 4-phenoxyphenyl region. Structure-activity relationship studies revealed that the inhibitory potency against FAAH and MAGL, respectively, could still be increased by replacement of the phenoxy residue of 16 by 3-chlorophenoxy (45) or pyrrol-1-yl groups (49). Finally, the tetrazolecarboxamide 16 and some related compounds were tested for metabolic stability with rat liver S9 fractions showing that these kind of FAAH/MAGL inhibitors are readily inactivated by cleavage of the bond between the tetrazole ring and its carboxamide substituent.

  7. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  8. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  9. Synthesis and QSAR of Fatty Acid Amide Hydrolase Inhibitors: Modulation at the N-Portion of Biphenyl-3-yl Alkylcarbamates

    PubMed Central

    Mor, Marco; Lodola, Alessio; Rivara, Silvia; Vacondio, Federica; Duranti, Andrea; Tontini, Andrea; Sanchini, Silvano; Piersanti, Giovanni; Clapper, Jason R.; King, Alvin R.; Tarzia, Giorgio; Piomelli, Daniele

    2013-01-01

    Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (IC50, for FAAH = 63 nM) with a selected set of substituents of different size, shape, flexibility and lipophilicity. Docking experiments and Linear Interaction Energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the β-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3′-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors. PMID:18507372

  10. Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor.

    PubMed

    Johnson, Douglas S; Stiff, Cory; Lazerwith, Scott E; Kesten, Suzanne R; Fay, Lorraine K; Morris, Mark; Beidler, David; Liimatta, Marya B; Smith, Sarah E; Dudley, David T; Sadagopan, Nalini; Bhattachar, Shobha N; Kesten, Stephen J; Nomanbhoy, Tyzoon K; Cravatt, Benjamin F; Ahn, Kay

    2011-02-10

    Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.

  11. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  12. PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

    PubMed

    Zestos, Alexander G; Mikelman, Sarah R; Kennedy, Robert T; Gnegy, Margaret E

    2016-06-15

    Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.

  13. CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR

    PubMed Central

    Kunitz, M.

    1947-01-01

    A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496

  14. Radiation Transport

    SciTech Connect

    Urbatsch, Todd James

    2015-06-15

    We present an overview of radiation transport, covering terminology, blackbody raditation, opacities, Boltzmann transport theory, approximations to the transport equation. Next we introduce several transport methods. We present a section on Caseology, observing transport boundary layers. We briefly broach topics of software development, including verification and validation, and we close with a section on high energy-density experiments that highlight and support radiation transport.

  15. 2D- and 3D-QSAR studies of a series of benzopyranes and benzopyrano[3,4b][1,4]-oxazines as inhibitors of the multidrug transporter P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Jabeen, Ishrat; Wetwitayaklung, Penpun; Chiba, Peter; Pastor, Manuel; Ecker, Gerhard F.

    2013-02-01

    The ATP-binding cassette efflux transporter P-glycoprotein (P-gp) is notorious for contributing to multidrug resistance in antitumor therapy. Due to its expression in many blood-organ barriers, it also influences the pharmacokinetics of drugs and drug candidates and is involved in drug/drug- and drug/nutrient interactions. However, due to lack of structural information the molecular basis of ligand/transporter interaction still needs to be elucidated. Towards this goal, a series of Benzopyranes and Benzopyrano[3,4b][1,4]oxazines have been synthesized and pharmacologically tested for their ability to inhibit P-gp mediated daunomycin efflux. Both quantitative structure-activity relationship (QSAR) models using simple physicochemical and novel GRID-independent molecular descriptors (GRIND) were established to shed light on the structural requirements for high P-gp inhibitory activity. The results from 2D-QSAR showed a linear correlation of vdW surface area (Å2) of hydrophobic atoms with the pharmacological activity. GRIND (3D-QSAR) studies allowed to identify important mutual distances between pharmacophoric features, which include one H-bond donor, two H-bond acceptors and two hydrophobic groups as well as their distances from different steric hot spots of the molecules. Activity of the compounds particularly increases with increase of the distance of an H-bond donor or a hydrophobic feature from a particular steric hot spot of the benzopyrane analogs.

  16. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  17. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  18. [Mechanisms and efficacy of SGLT2 inhibitors].

    PubMed

    Shiba, Teruo

    2015-03-01

    SGLT2 is a low affinity, high capacity glucose co-transporter, almost exclusively expressed in the kidney cortex. Inhibition of SGLT2 has been shown to increase the daily 50g or more urinary glucose excretion, as compared to placebo, leading to a reduction in blood glucose levels and indicated only for the treatment of type 2 diabetes. In Japan 6 species of SGLT2 inhibitors have already been sold and reported to results in a decrease of FPG by 14.4 to 45.8 (mg/dL), in a reduction of HbA1c by 0.35 to 1.24% and in loss of body weight by 1.29 to 2.50(kg). There is less effect of the SGLT2 inhibitor in diabetic subjects with renal impairment and the reduction in HbA1c and FPG will be approximately half of the average in those with 30 ≤ eGFR ≤ 59. The position of SGLT2 inhibitors would be considered as the drug administered in combination or add-on therapy when the young obese type 2 diabetics without renal impairment has not yet reached to the glycemic target with other drugs although in AACE consensus statement of 2013, it has been shelved for inexperienced use with respect to the positioning of the SGLT2 inhibitors.

  19. Fatty acid amide hydrolase (FAAH) inhibitors exert pharmacological effects, but lack antinociceptive efficacy in rats with neuropathic spinal cord injury pain.

    PubMed

    Hama, Aldric T; Germano, Peter; Varghese, Matthew S; Cravatt, Benjamin F; Milne, G Todd; Pearson, James P; Sagen, Jacqueline

    2014-01-01

    Amelioration of neuropathic spinal cord injury (SCI) pain is a clinical challenge. Increasing the endocannabinoid anandamide and other fatty acid amides (FAA) by blocking fatty acid amide hydrolase (FAAH) has been shown to be antinociceptive in a number of animal models of chronic pain. However, an antinociceptive effect of blocking FAAH has yet to be demonstrated in a rat model of neuropathic SCI pain. Four weeks following a SCI, rats developed significantly decreased hind paw withdrawal thresholds, indicative of below-level cutaneous hypersensitivity. A group of SCI rats were systemically treated (i.p.) with either the selective FAAH inhibitor URB597 or vehicle twice daily for seven days. A separate group of SCI rats received a single dose (p.o.) of either the selective FAAH inhibitor PF-3845 or vehicle. Following behavioral testing, levels of the FAA N-arachidonoylethanolamide, N-oleoyl ethanolamide and N-palmitoyl ethanolamide were quantified in brain and spinal cord from SCI rats. Four weeks following SCI, FAA levels were markedly reduced in spinal cord tissue. Although systemic treatment with URB597 significantly increased CNS FAA levels, no antinociceptive effect was observed. A significant elevation of CNS FAA levels was also observed following oral PF-3845 treatment, but only a modest antinociceptive effect was observed. Increasing CNS FAA levels alone does not lead to robust amelioration of below-level neuropathic SCI pain. Perhaps utilizing FAAH inhibition in conjunction with other analgesic mechanisms could be an effective analgesic therapy.

  20. Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid TumorsS⃞

    PubMed Central

    Desmoulin, Sita Kugel; Wang, Yiqiang; Wu, Jianmei; Stout, Mark; Hou, Zhanjun; Fulterer, Andreas; Chang, Min-Hwang; Romero, Michael F.; Cherian, Christina; Gangjee, Aleem

    2010-01-01

    The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the microenvironments of solid tumors. We tested 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-{4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl}-l-glutamic acid (compound 2) and N-{4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl}*-l-glutamic acid (compound 3), respectively) were inhibitory, with 2 ≫ 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCFT transport in PCFT-expressing CHO (R2/hPCFT4) cells from pH 5.5 to 7.2; inhibition increased with decreasing pH. In Xenopus laevis oocytes microinjected with PCFT cRNA, uptake of 2, like that of Pmx, was electrogenic. Cytotoxicity of 2 toward R2/hPCFT4 cells was abolished in the presence of adenosine or 5-amino-4-imidazolecarboxamide, suggesting that glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis was the primary target. Compound 2 decreased GTP and ATP pools by ∼50 and 75%, respectively. By an in situ GARFTase assay, 2 was ∼20-fold more inhibitory toward intracellular GARFTase than toward cell growth or colony formation. Compound 2 irreversibly inhibited clonogenicity, although this required at least 4 h of exposure. Our results document the potent antiproliferative activity of compound 2, attributable to its efficient cellular uptake by PCFT, resulting in inhibition of GARFTase and de novo purine biosynthesis. Furthermore, they establish the feasibility of selective chemotherapy drug delivery via PCFT over RFC, a process that takes advantage of a unique biological feature of solid tumors. PMID

  1. Thrombin inhibitor design.

    PubMed

    Sanderson, P E; Naylor-Olsen, A M

    1998-08-01

    Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

  2. When transporters fail to be transported: how to rescue folding-deficient SLC6 transporters

    PubMed Central

    Sucic, Sonja; Kasture, Ameya; Mazhar Asjad, H. M.; Kern, Carina; El-Kasaby, Ali; Freissmuth, Michael

    2017-01-01

    The human dopamine transporter (hDAT) belongs to the solute carrier 6 (SLC6) gene family. Point mutations in hDAT (SLC6A3) have been linked to a syndrome of dopamine transporter deficiency or infantile dystonia/parkinsonism. The mutations impair DAT folding, causing retention of variant DATs in the endoplasmic reticulum and subsequently impair transport activity. The folding trajectory of DAT itself is not understood, though many insights have been gained from studies of folding-deficient mutants of the closely related serotonin transporter (SERT); i.e. their functional rescue by pharmacochaperoning with (nor)ibogaine or heat-shock protein inhibitors. We recently provided a proof-of-principle that folding-deficits in DAT are amenable to rescue in vitro and in vivo. As a model we used the Drosophila melanogaster DAT mutant dDAT-G108Q, which phenocopies the fumin/sleepless DAT-knockout. Treatment with noribogaine and/or HSP70 inhibitor pifithrin-μ restored folding of, and dopamine transport by, dDAT-G108Q, its axonal delivery and normal sleep time in mutant flies. The possibility of functional rescue of misfolded DATs in living flies by pharmacochaperoning grants new therapeutic prospects in the remedy of folding diseases, not only in hDAT, but also in other SLC6 transporters, in particular mutants of the creatine transporter-1, which give rise to X-linked mental retardation.

  3. Drug-drug interactions between HMG-CoA reductase inhibitors (statins) and antiviral protease inhibitors.

    PubMed

    Chauvin, Benoit; Drouot, Sylvain; Barrail-Tran, Aurélie; Taburet, Anne-Marie

    2013-10-01

    The HMG-CoA reductase inhibitors are a class of drugs also known as statins. These drugs are effective and widely prescribed for the treatment of hypercholesterolemia and prevention of cardiovascular morbidity and mortality. Seven statins are currently available: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin. Although these drugs are generally well tolerated, skeletal muscle abnormalities from myalgia to severe lethal rhabdomyolysis can occur. Factors that increase statin concentrations such as drug-drug interactions can increase the risk of these adverse events. Drug-drug interactions are dependent on statins' pharmacokinetic profile: simvastatin, lovastatin and atorvastatin are metabolized through cytochrome P450 (CYP) 3A, while the metabolism of the other statins is independent of this CYP. All statins are substrate of organic anion transporter polypeptide 1B1, an uptake transporter expressed in hepatocyte membrane that may also explain some drug-drug interactions. Many HIV-infected patients have dyslipidemia and comorbidities that may require statin treatment. HIV-protease inhibitors (HIV PIs) are part of recommended antiretroviral treatment in combination with two reverse transcriptase inhibitors. All HIV PIs except nelfinavir are coadministered with a low dose of ritonavir, a potent CYP3A inhibitor to improve their pharmacokinetic properties. Cobicistat is a new potent CYP3A inhibitor that is combined with elvitegravir and will be combined with HIV-PIs in the future. The HCV-PIs boceprevir and telaprevir are both, to different extents, inhibitors of CYP3A. This review summarizes the pharmacokinetic properties of statins and PIs with emphasis on their metabolic pathways explaining clinically important drug-drug interactions. Simvastatin and lovastatin metabolized through CYP3A have the highest potency for drug-drug interaction with potent CYP3A inhibitors such as ritonavir- or cobicistat-boosted HIV-PI or the

  4. Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

    PubMed

    Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F

    2015-03-01

    We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted.

  5. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  6. Chitosan nanoparticles conjugate with trypsin and trypsin inhibitor.

    PubMed

    Chanphai, P; Tajmir-Riahi, H A

    2016-06-25

    Chitosan-protein conjugates are widely used in therapeutic drug delivery. We report the bindings of chitosan nanoparticles with trypsin (try) and trypsin inhibitor (tryi), using thermodynamic analysis and multiple spectroscopic methods. Thermodynamic parameters ΔS, ΔH and ΔG showed chitosan-protein bindings occur mainly via H-bonding and van der Waals contacts with trypsin inhibitor forming more stable conjugate than trypsin. As chitosan size increased more stable polymer-protein conjugate was formed. Chitosan complexation induces more perturbations of trypsin inhibitor structure than trypsin with reduction of protein alpha-helix and major increase of random structure. The negative value of ΔG indicates spontaneous protein-chitosan complexation at room temperature. Chitosan nanoparticles can be used to transport trypsin and trypsin inhibitor.

  7. Synergistic Interactions of MmpL3 Inhibitors with Antitubercular Compounds In Vitro

    PubMed Central

    Li, Wei; Sanchez-Hidalgo, Andrea; Jones, Victoria; de Moura, Vinicius Calado Nogueira; North, E. Jeffrey

    2017-01-01

    ABSTRACT A number of inhibitors of the essential Mycobacterium tuberculosis mycolic acid transporter, MmpL3, are currently under development as potential novel antituberculosis agents. Using the checkerboard method to study the interaction profiles of various antituberculosis drugs or experimental compounds with two different chemotypes inhibiting this transporter (indolcarboxamides and adamantyl ureas), we showed that MmpL3 inhibitors act synergistically with rifampin, bedaquiline, clofazimine, and β-lactams. PMID:28115355

  8. Transport Experiments

    NASA Technical Reports Server (NTRS)

    Hall, Timothy M.; Wuebbles, Donald J.; Boering, Kristie A.; Eckman, Richard S.; Lerner, Jean; Plumb, R. Alan; Rind, David H.; Rinsland, Curtis P.; Waugh, Darryn W.; Wei, Chu-Feng

    1999-01-01

    MM II defined a series of experiments to better understand and characterize model transport and to assess the realism of this transport by comparison to observations. Measurements from aircraft, balloon, and satellite, not yet available at the time of MM I [Prather and Remsberg, 1993], provide new and stringent constraints on model transport, and address the limits of our transport modeling abilities. Simulations of the idealized tracers the age spectrum, and propagating boundary conditions, and conserved HSCT-like emissions probe the relative roles of different model transport mechanisms, while simulations of SF6 and C02 make the connection to observations. Some of the tracers are related, and transport diagnostics such as the mean age can be derived from more than one of the experiments for comparison to observations. The goals of the transport experiments are: (1) To isolate the effects of transport in models from other processes; (2) To assess model transport for realistic tracers (such as SF6 and C02) for comparison to observations; (3) To use certain idealized tracers to isolate model mechanisms and relationships to atmospheric chemical perturbations; (4) To identify strengths and weaknesses of the treatment of transport processes in the models; (5) To relate evaluated shortcomings to aspects of model formulation. The following section are included:Executive Summary, Introduction, Age Spectrum, Observation, Tropical Transport in Models, Global Mean Age in Models, Source-Transport Covariance, HSCT "ANOY" Tracer Distributions, and Summary and Conclusions.

  9. Protease inhibitors and proteolytic signalling cascades in insects.

    PubMed

    Gubb, David; Sanz-Parra, Arantza; Barcena, Laura; Troxler, Laurent; Fullaondo, Ane

    2010-12-01

    Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.

  10. Development of scale inhibitors

    SciTech Connect

    Gill, J.S.

    1996-12-01

    During the last fifty years, scale inhibition has gone from an art to a science. Scale inhibition has changed from simple pH adjustment to the use of optimized dose of designer polymers from multiple monomers. The water-treatment industry faces many challenges due to the need to conserve water, availability of only low quality water, increasing environmental regulations of the water discharge, and concern for human safety when using acid. Natural materials such as starch, lignin, tannin, etc., have been replaced with hydrolytically stable organic phosphates and synthetic polymers. Most progress in scale inhibition has come from the use of synergistic mixtures and copolymerizing different functionalities to achieve specific goals. Development of scale inhibitors requires an understanding of the mechanism of crystal growth and its inhibition. This paper discusses the historic perspective of scale inhibition and the development of new inhibitors based on the understanding of the mechanism of crystal growth and the use of powerful tools like molecular modeling to visualize crystal-inhibitor interactions.

  11. 1-[4-[4[(4R,5R)-3,3-Dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2]octane methanesulfonate (SC-435), an ileal apical sodium-codependent bile acid transporter inhibitor alters hepatic cholesterol metabolism and lowers plasma low-density lipoprotein-cholesterol concentrations in guinea pigs.

    PubMed

    West, Kristy L; Ramjiganesh, Tripurasundari; Roy, Suheeta; Keller, Bradley T; Fernandez, Maria Luz

    2002-10-01

    Male Hartley guinea pigs (10/group) were assigned either to a control diet (no drug treatment) or to diets containing 0.4, 2.2, or 7.3 mg/day of an ileal apical sodium-codependent bile acid transporter (ASBT) inhibitor, 1-[4-[4[(4R,5R)-3,3-dibutyl-7-(dimethylamino)-2,3,4,5-tetrahydro-4-hydroxy-1,1-dioxido-1-benzothiepin-5-yl]phenoxy]butyl]-4-aza-1-azoniabicyclo[2.2.2] octane methanesulfonate (SC-435). Based on food consumption, guinea pigs received 0, 0.8, 3.7, or 13.4 mg/kg/day of the ASBT inhibitor. The amount of cholesterol in the four diets was maintained at 0.17%, equivalent to 1200 mg/day in the human situation. Guinea pigs treated with 13.4 mg/kg/day SC-435 had 41% lower total cholesterol and 44% lower low-density lipoprotein (LDL)-cholesterol concentrations compared with control (P < 0.01), whereas no significant differences were observed with either of the lower doses of SC-435. Hepatic cholesterol esters were significantly reduced by 43, 56, and 70% in guinea pigs fed 0.8, 3.7, and 13.4 mg/kg/day of the ASBT inhibitor, respectively (P < 0.01). In addition, the highest dose of the inhibitor resulted in a 42% increase in the number of very low-density lipoprotein (VLDL) triacylglycerol molecules and a larger VLDL diameter compared with controls (P < 0.05). Acyl-CoA cholesterol/acyltransferase activity was 30% lower with the highest dose treatment, whereas cholesterol 7alpha-hydroxylase, the regulatory enzyme of bile acid synthesis, was 30% higher with the highest ASBT inhibitor dose (P < 0.05). Furthermore, bile acid excretion increased 2-fold with the highest dose of SC-435 compared with the control group (P < 0.05). These results suggest that the reduction in total and LDL-cholesterol concentrations by the ASBT inhibitor is a result of alterations in hepatic cholesterol metabolism due to modifications in the enterohepatic circulation of bile acids.

  12. Potential Therapeutic Value of a Novel FAAH Inhibitor for the Treatment of Anxiety

    PubMed Central

    Marco, Eva M.; Rapino, Cinzia; Caprioli, Antonio; Borsini, Franco; Laviola, Giovanni; Maccarrone, Mauro

    2015-01-01

    Anxiety disorders are among the most prevalent psychiatric diseases with high personal costs and a remarkable socio-economic burden. However, current treatment of anxiety is far from satisfactory. Novel pharmacological targets have emerged in the recent years, and attention has focused on the endocannabinoid (eCB) system, given the increasing evidence that supports its central role in emotion, coping with stress and anxiety. In the management of anxiety disorders, drug development strategies have left apart the direct activation of type-1 cannabinoid receptors to indirectly enhance eCB signalling through the inhibition of eCB deactivation, that is, the inhibition of the fatty acid amide hydrolase (FAAH) enzyme. In the present study, we provide evidence for the anxiolytic-like properties of a novel, potent and selective reversible inhibitor of FAAH, ST4070, orally administered to rodents. ST4070 (3 to 30 mg/kg per os) administered to CD1 male mice induced an increase of time spent in the exploration of the open arms of the elevated-plus maze. A partial reduction of anxiety-related behaviour by ST4070 was also obtained in Wistar male rats, which moderately intensified the time spent in the illuminated compartment of the light-dark box. ST4070 clearly inhibited FAAH activity and augmented the levels of two of its substrates, N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine, in anxiety-relevant brain regions. Altogether, ST4070 offers a promising anxiolytic-like profile in preclinical studies, although further studies are warranted to clearly demonstrate its efficacy in the clinic management of anxiety disorders. PMID:26360704

  13. Activation of TRPA1 channels by the fatty acid amide hydrolase inhibitor 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).

    PubMed

    Niforatos, Wende; Zhang, Xu-Feng; Lake, Marc R; Walter, Karl A; Neelands, Torben; Holzman, Thomas F; Scott, Victoria E; Faltynek, Connie R; Moreland, Robert B; Chen, Jun

    2007-05-01

    As a member of the transient receptor potential (TRP) ion channel superfamily, the ligand-gated ion channel TRPA1 has been implicated in nociceptive function and pain states. The endogenous ligands that activate TRPA1 remain unknown. However, various agonists have been identified, including environmental irritants (e.g., acrolein) and ingredients of pungent natural products [e.g., allyl isothiocyanate (ITC), cinnamaldehyde, allicin, and gingerol]. In general, these agents are either highly reactive, nonselective, or not potent or efficacious, significantly limiting their utilities in the study of TRPA1 channel properties and biological functions. In a search for novel TRPA1 agonists, we identified 3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597), a potent and systemically active inhibitor of fatty acid amide hydrolase (FAAH). This enzyme is responsible for anandamide degradation and therefore has been pursued as an antinociceptive and antiepileptic drug target. Using Ca(2+) influx assays and patch-clamp techniques, we demonstrated that URB597 could activate heterologously expressed human and rat TRPA1 channels, whereas two other FAAH inhibitors (i.e., URB532 and Compound 7) had no effect. When applied to inside-out membrane patches expressing rat TRPA1, URB597 elicited single-channel activities with a unitary conductance of 40 pS. Furthermore, URB597 activated TRPA1 channels endogenously expressed in a population of rat dorsal root ganglion neurons that also responded to ITC. In contrast to its effect on TRPA1, URB597 inhibited TRPM8 and had no effects on TRPV1 or TRPV4. Thus, we conclude that URB597 is a novel agonist of TRPA1 and probably activates the channel through a direct gating mechanism.

  14. Vortex shedding from solid rocket propellant inhibitors

    NASA Technical Reports Server (NTRS)

    Shu, P. H.; Sforzini, R. H.; Foster, W. A., Jr.

    1986-01-01

    Vortex shedding frequency caused by the protrusion of inhibitors into the flow field of a solid rocket motor is investigated by experimental and mathematical models. The time dependent Navier-Stokes equations are solved using a finite difference technique assuming incompressible, two-dimensional flow under both laminar and turbulent flow conditions. For laminar flow, explicit solutions are obtained using a vorticity-transport equation in place of the Navier-Stokes equations. For turbulent flow, a two-equation (k-epsilon) model is used for turbulent modeling and the SIMPLE algorithm is employed as the computational scheme. Cold flow tests were conducted to confirm the basic flow structure and to determine the vortex shedding frequency under both laminar and turbulent flow conditions. The vortex shedding frequencies were determined using a stroboscope to measure the oscillating frequency of yarn tufts which were fastened to one inhibitor in the models. A hot-film anemometer established the velocity history behind the inhibitor. Good agreement between the theoretical results and measurements of the vortex shedding frequencies is demonstrated.

  15. Vortex shedding from solid rocket propellant inhibitors

    NASA Astrophysics Data System (ADS)

    Shu, P. H.; Sforzini, R. H.; Foster, W. A., Jr.

    1986-06-01

    Vortex shedding frequency caused by the protrusion of inhibitors into the flow field of a solid rocket motor is investigated by experimental and mathematical models. The time dependent Navier-Stokes equations are solved using a finite difference technique assuming incompressible, two-dimensional flow under both laminar and turbulent flow conditions. For laminar flow, explicit solutions are obtained using a vorticity-transport equation in place of the Navier-Stokes equations. For turbulent flow, a two-equation (k-epsilon) model is used for turbulent modeling and the SIMPLE algorithm is employed as the computational scheme. Cold flow tests were conducted to confirm the basic flow structure and to determine the vortex shedding frequency under both laminar and turbulent flow conditions. The vortex shedding frequencies were determined using a stroboscope to measure the oscillating frequency of yarn tufts which were fastened to one inhibitor in the models. A hot-film anemometer established the velocity history behind the inhibitor. Good agreement between the theoretical results and measurements of the vortex shedding frequencies is demonstrated.

  16. Quantum Transport.

    DTIC Science & Technology

    1994-08-15

    Notre Dame was concerned with a variety of quantum transport in mesoscopic structures. This research was funded by the Air Force Office of Scientific...Research under Grant No. AFOSR-91-0211. The major issues examined included quantum transport in high magnetic fields and modulated channels, Coulomb...lifetimes in quasi-1D structures, quantum transport experiments in metals, the mesoscopic photovoltaic effect, and new techniques for fabricating quantum structures in semiconductors.

  17. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  18. Mechanism of ochratoxin A transport in kidney

    SciTech Connect

    Sokol, P.P.; Ripich, G.; Holohan, P.D.; Ross, C.R.

    1988-08-01

    The effect of the fungal metabolite (mycotoxin) Ochratoxin A (OTA) on the transport of p-amino(/sup 3/H)hippurate (PAH), a prototypic organic anion, was examined in renal brush border (BBMV) and basolateral membrane vesicles (BLMV). OTA was as effective an inhibitor of PAH uptake in both membranes as probenecid. The dose response curves for OTA in BBMV and BLMV gave IC50 values of 20 +/- 6 and 32 +/- 7 microM, respectively. The effect was specific since the transport of the organic cation N1-methylnicotinamide was not affected. The phenomenon of counterflow was studied to establish that OTA is translocated. OTA produced trans stimulation of PAH transport in both BBMV and BLMV, demonstrating that OTA is transported across both these membranes. The data suggest that OTA interacts with the PAH transport system in both BBMV and BLMV. We conclude that OTA transport in the kidney is mediated via the renal organic anion transport system.

  19. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  20. Screening for Inhibitors of Essential Leishmania Glucose Transporters

    DTIC Science & Technology

    2013-07-01

    be used effectively in an automated high-throughput format (Assay Guidance Manual , http://www.ncgc.nih.gov/guidance/manual_toc.html). For initial...quality employing control samples. The details of recommended quality control tests are provided by the online Assay Guidance Manual published by Eli...values for this assay are well within the range of the ‘acceptance criteria’, as defined in the Assay Guidance Manual . Table 1. Summary of

  1. Screening for Inhibitors of Essential Leishmania Glucose Transporters

    DTIC Science & Technology

    2012-07-01

    high-throughput format (Assay Guidance Manual , http://www.ncgc.nih.gov/guidance/manual_toc.html). For initial optimization of the cell growth assay...The details of recommended quality control tests are provided by the online Assay Guidance Manual published by Eli Lilly and company and the NIH...within the range of the ‘acceptance criteria’, as defined in the Assay Guidance Manual . 6 Table 1. Summary of statistical variables calculated for

  2. School Transportation.

    ERIC Educational Resources Information Center

    Executive Educator, 1989

    1989-01-01

    A special report on school transportation covers the following topics: (1) a school bus safety update; (2) equipping school buses with motion detectors; (3) state training requirements for school bus drivers; (4) recruiting and retaining drivers; (5) regulations covering underground fuel-storage tanks; and (6) a transportation directory. (MLF)

  3. Transport Phenomena.

    ERIC Educational Resources Information Center

    McCready, Mark J.; Leighton, David T.

    1987-01-01

    Discusses the problems created in graduate chemical engineering programs when students enter with a wide diversity of understandings of transport phenomena. Describes a two-semester graduate transport course sequence at the University of Notre Dame which focuses on fluid mechanics and heat and mass transfer. (TW)

  4. Multidrug Efflux Pumps Attenuate the Effect of MGMT Inhibitors.

    PubMed

    Tomaszowski, Karl-Heinz; Schirrmacher, Ralf; Kaina, Bernd

    2015-11-02

    Various mechanisms of drug resistance attenuate the effectiveness of cancer therapeutics, including drug transport and DNA repair. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key factor determining the resistance against alkylating anticancer drugs inducing the genotoxic DNA lesions O(6)-methylguanine and O(6)-chloroethylguanine, and MGMT inactivation or depletion renders cells more susceptible to treatment with methylating and chloroethylating agents. Highly specific and efficient inhibitors of the repair protein MGMT were designed, including O(6)-benzylguanine (O(6)BG) and O(6)-(4-bromothenyl)guanine (O(6)BTG) that are nontoxic on their own. Unfortunately, these inhibitors do not select between MGMT in normal and cancer cells, causing nontarget effects in the healthy tissue. Therefore, a targeting strategy for MGMT inhibitors is required. Here, we used O(6)BG and O(6)BTG conjugated to β-d-glucose (O(6)BG-Glu and O(6)BTG-Glu, respectively) in order to selectively inhibit MGMT in tumors, harnessing their high demand for glucose. Both glucose conjugates efficiently inhibited MGMT in several cancer cell lines, but with different extents of sensitization to DNA alkylating agents, with lomustine being more effective than temozolomide. We further show that the glucose conjugates are subject to ATP-binding cassette (ABC) transporter mediated efflux, involving P-glycoprotein, MRP1, and BCRP, which impacts the efficiency of MGMT inhibition. Surprisingly, also O(6)BG and O(6)BTG were subject to an active transport out of the cell. We also show that pharmacological inhibition of efflux transporters increases the induction of cell death following treatment with these MGMT inhibitors and temozolomide. We conclude that strategies of attenuating the efflux by ABC transporters are required for achieving successful MGMT targeting.

  5. Sequencing of aromatase inhibitors

    PubMed Central

    Bertelli, G

    2005-01-01

    Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. PMID:16100523

  6. Sirtuin activators and inhibitors

    PubMed Central

    Villalba, José M.; Alcaín, Francisco J.

    2012-01-01

    Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD+ for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activity. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex®, SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. PMID:22730114

  7. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  8. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  9. Reversal of Acetylcholinesterase Inhibitor Toxicity In Vivo by Inhibitors of Choline Transport.

    DTIC Science & Technology

    1983-10-31

    neostigmine on acetylcholine levels in either the heart or brain. Thus, hemicholinium-3 may be exerting a different effect. The protecting effect of...hemicholinium-3 (HC-3) on the acetylcholine 27 (ACh) concentration of mouse brzin and heart . Figure 10 Effect of hemicbolinium-3 (HC-3) on the choline (Ch) 28...concentration of mouse brain and heart . S (V) . . List of Tables Table 1. Effect of hemicholinium-3 (HC-3) on the time to death of mice treated with

  10. Transport Phenomena.

    ERIC Educational Resources Information Center

    Shah, D. B.

    1984-01-01

    Describes a course designed to achieve a balance between exposing students to (1) advanced topics in transport phenomena, pointing out similarities and differences between three transfer processes and (2) common methods of solving differential equations. (JN)

  11. Harnessing Solute Carrier Transporters for Precision Oncology.

    PubMed

    Nyquist, Michael D; Prasad, Bhagwat; Mostaghel, Elahe A

    2017-03-28

    Solute Carrier (SLC) transporters are a large superfamily of transmembrane carriers involved in the regulated transport of metabolites, nutrients, ions and drugs across cellular membranes. A subset of these solute carriers play a significant role in the cellular uptake of many cancer therapeutics, ranging from chemotherapeutics such as antimetabolites, topoisomerase inhibitors, platinum-based drugs and taxanes to targeted therapies such as tyrosine kinase inhibitors. SLC transporters are co-expressed in groups and patterns across normal tissues, suggesting they may comprise a coordinated regulatory circuit serving to mediate normal tissue functions. In cancer however, there are dramatic changes in expression patterns of SLC transporters. This frequently serves to feed the increased metabolic demands of the tumor cell for amino acids, nucleotides and other metabolites, but also presents a therapeutic opportunity, as increased transporter expression may serve to increase intracellular concentrations of substrate drugs. In this review, we examine the regulation of drug transporters in cancer and how this impacts therapy response, and discuss novel approaches to targeting therapies to specific cancers via tumor-specific aberrations in transporter expression. We propose that among the oncogenic changes in SLC transporter expression there exist emergent vulnerabilities that can be exploited therapeutically, extending the application of precision medicine from tumor-specific drug targets to tumor-specific determinants of drug uptake.

  12. Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: Effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment

    PubMed Central

    Guindon, Josée; Lai, Yvonne; Takacs, Sara M.; Bradshaw, Heather B.; Hohmann, Andrea G.

    2012-01-01

    SUMMARY Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and cold allodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB1 and CB2) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB1 (AM251 3 mg/kg), CB2 (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB1, CB2, TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the neuropathy while amitriptyline, administered acutely, was ineffective. CB1 or CB2 antagonist

  13. Advances in plant-based inhibitors of P-glycoprotein.

    PubMed

    Yu, Jun; Zhou, Peng; Asenso, James; Yang, Xiao-Dan; Wang, Chun; Wei, Wei

    2016-12-01

    Multidrug resistance (MDR) has emerged as the main problem in anti-cancer therapy. Although MDR involves complex factors and processes, the main pivot is the expression of multidrug efflux pumps. P-glycoprotein (P-gp) belongs to the family of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. It functions in cellular detoxification, pumping a wide range of xenobiotic compounds out of the cell. An attractive therapeutic strategy for overcoming MDR is to inhibit the transport function of P-gp and thus, increase intracellular concentration of drugs. Recently, various types of P-gp inhibitors have been found and used in experiments. However, none of them has passed clinical trials due to their high side-effects. Hence, the search for alternatives, such as plant-based P-gp inhibitors have gained attention recently. Therefore, we give an overview of the source, function, structure and mechanism of plant-based P-gp inhibitors and give more attention to cancer-related studies. These products could be the future potential drug candidates for further research as P-gp inhibitors.

  14. Auxin influx inhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics in tobacco cells

    PubMed Central

    Laňková, Martina; Smith, Richard S.; Pešek, Bedřich; Kubeš, Martin; Zažímalová, Eva; Petrášek, Jan; Hoyerová, Klára

    2010-01-01

    The phytohormone auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar transport machinery. This machinery consists of a balanced system of