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Sample records for 1a2 3a4 2c9

  1. Effects of mace and nutmeg on human cytochrome P450 3A4 and 2C9 activity.

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Hatano, Tsutomu

    2010-01-01

    Pharmacokinetic or pharmacodynamic interactions between herbal medicines or food constituents and drugs have been studied as crucial factors determining therapeutic efficacy and outcome. Most of these interactions are attributed to inhibition or induction of activity of cytochrome P450 (CYP) metabolic enzymes. Inhibition or induction of CYP enzymes by beverages, including grapefruit, pomegranate, or cranberry juice, has been well documented. Because spices are a common daily dietary component, other studies have reported inhibition of CYP activity by spices or their constituents/derivatives. However, a systematic evaluation of various spices has not been performed. In this study, we investigated effects of 55 spices on CYP3A4 and CYP2C9 activity. Cinnamon, black or white pepper, ginger, mace, and nutmeg significantly inhibited CYP3A4 or CYP2C9 activity. Furthermore, bioassay-guided fractionation of mace (Myristica fragrans) led to isolation and structural characterization of a new furan derivative (1) along with other 16 known compounds, including an acylphenol, neolignans, and phenylpropanoids. Among these isolates, (1S,2R)-1-acetoxy-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane (9) exhibited the most potent CYP2C9 inhibitory activity with an IC₅₀ value comparable to that of sulfaphenazole, a CYP2C9 inhibitor. Compound 9 competitively inhibited CYP2C9-mediated 4'-hydroxylation of diclofenac. The inhibitory constant (K(i)) of 9 was determined to be 0.037 µM. Compound 9 was found to be 14-fold more potent than was sulfaphenazole. PMID:21139236

  2. In Silico Prediction of Cytochrome P450-Drug Interaction: QSARs for CYP3A4 and CYP2C9

    PubMed Central

    Nembri, Serena; Grisoni, Francesca; Consonni, Viviana; Todeschini, Roberto

    2016-01-01

    Cytochromes P450 (CYP) are the main actors in the oxidation of xenobiotics and play a crucial role in drug safety, persistence, bioactivation, and drug-drug/food-drug interaction. This work aims to develop Quantitative Structure-Activity Relationship (QSAR) models to predict the drug interaction with two of the most important CYP isoforms, namely 2C9 and 3A4. The presented models are calibrated on 9122 drug-like compounds, using three different modelling approaches and two types of molecular description (classical molecular descriptors and binary fingerprints). For each isoform, three classification models are presented, based on a different approach and with different advantages: (1) a very simple and interpretable classification tree; (2) a local (k-Nearest Neighbor) model based classical descriptors and; (3) a model based on a recently proposed local classifier (N-Nearest Neighbor) on binary fingerprints. The salient features of the work are (1) the thorough model validation and the applicability domain assessment; (2) the descriptor interpretation, which highlighted the crucial aspects of P450-drug interaction; and (3) the consensus aggregation of models, which largely increased the prediction accuracy. PMID:27294921

  3. In Silico Prediction of Cytochrome P450-Drug Interaction: QSARs for CYP3A4 and CYP2C9.

    PubMed

    Nembri, Serena; Grisoni, Francesca; Consonni, Viviana; Todeschini, Roberto

    2016-01-01

    Cytochromes P450 (CYP) are the main actors in the oxidation of xenobiotics and play a crucial role in drug safety, persistence, bioactivation, and drug-drug/food-drug interaction. This work aims to develop Quantitative Structure-Activity Relationship (QSAR) models to predict the drug interaction with two of the most important CYP isoforms, namely 2C9 and 3A4. The presented models are calibrated on 9122 drug-like compounds, using three different modelling approaches and two types of molecular description (classical molecular descriptors and binary fingerprints). For each isoform, three classification models are presented, based on a different approach and with different advantages: (1) a very simple and interpretable classification tree; (2) a local (k-Nearest Neighbor) model based classical descriptors and; (3) a model based on a recently proposed local classifier (N-Nearest Neighbor) on binary fingerprints. The salient features of the work are (1) the thorough model validation and the applicability domain assessment; (2) the descriptor interpretation, which highlighted the crucial aspects of P450-drug interaction; and (3) the consensus aggregation of models, which largely increased the prediction accuracy. PMID:27294921

  4. Potent inhibition of human cytochrome P450 3A4, 2D6, and 2C9 isoenzymes by grapefruit juice and its furocoumarins.

    PubMed

    Girennavar, B; Jayaprakasha, G K; Patil, B S

    2007-10-01

    The cytochrome P450 enzyme family is the most abundant and responsible for the metabolism of more than 60% of currently marketed drugs and is considered central in many clinically important drug interactions. Seven different grapefruit and pummelo juices as well as 5 furocoumarins isolated from grapefruit juice were evaluated at different concentration on cytochrome P450 3A4 (CYP3A4), cytochrome P450 2C9 (CYP2C9), and cytochrome P450 2D6 (CYP2D6) isoenzyme activity. Grapefruit and pummelo juices were found to be potent inhibitors of cytochrome CYP3A4 and CYP2C9 isoenzymes at 25% concentration, while CYP2D6 is inhibited significantly low at all the tested concentration of juices (P < 0.05). Among the 5 furocoumarins tested, the inhibitory potency was in the order of paradisin A > dihydroxybergamottin > bergamottin > bergaptol > geranylcoumarin at 0.1 microM to 0.1 mM concentrations. The IC(50) value was lowest for paradisin A for CYP3A4 with 0.11 microM followed by DHB for CYP2C9 with 1.58 microM. PMID:17995595

  5. Transcriptional Regulation of CYP3A4/2B6/2C9 Mediated via Nuclear Receptor PXR by Helicid and Its Metabolites

    PubMed Central

    Chen, Qun; Xie, Hai-tang; Li, Yan; Wang, Guo; Xu, Zhe; Pu, Zhi-chen; Hu, Hua

    2015-01-01

    Objective. This study aims at establishing and validating an in vitro system to screen drug inducers of CYPs mediated via hPXR, as well as studying transcriptional regulation of CYPs mediated via hPXR by helicid and its two metabolites. Methods. Cloning the nuclear receptor hPXR and the promoters of CYP3A4, CYP2B6, CYP2C9, and inserting the trans-element to the upstream of firefly luciferase reporter gene of the pGL4.17 vectors, then cotransfecting the report vectors and hPXR expression plasmid to HepG2 cell line. After 24 hours, the transfected cells were treated with helicid (0.004, 0.04, and 0.4 μmol/L) and its metabolite I and metabolite II (0.0004, 0.004, and 0.04 μmol/L) for 48 h, while rifampin (10 μmol/L) was included as the positive control and 0.1% DMSO as the negative control group. Cells were lysized and luciferase activity was determined using a dual luciferase reporter assay kit. Results. Helicid and its metabolites did not significantly increase promoter activities of CYP3A4, CYP2B6, and CYP2C9 in HepG2 cells transfected with PXR expression plasmid (P > 0.05). Conclusion. PXR-expressed CYP3A4, CYP2B6, and CYP2C9 dual luciferase reporter gene platforms were successfully established, and helicid and its metabolites I, II do not significantly induce the transcription of CYP3A4, CYP2B6, and CYP2C9. PMID:25977700

  6. Pregnane X receptor dependent up-regulation of CYP2C9 and CYP3A4 in tumor cells by antitumor acridine agents, C-1748 and C-1305, selectively diminished under hypoxia.

    PubMed

    Niemira, Magdalena; Dastych, Jarosław; Mazerska, Zofia

    2013-07-15

    Induction of proteins involved in drug metabolism and in drug delivery has a significant impact on drug-drug interactions and on the final therapeutic effects. Two antitumor acridine derivatives selected for present studies, C-1748 (9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine) and C-1305 (5-dimethylaminopropylamino-8-hydroxy-triazoloacridinone), expressed high and low susceptibility to metabolic transformations with liver microsomes, respectively. In the current study, we examined the influence of these compounds on cytochrome P450 3A4 (CYP3A4) and 2C9 (CYP2C9) enzymatic activity and gene expression in HepG2 tumor cells. Luminescence and HPLC examination, real-time RT-PCR and western blot analyses along with transfection of pregnane X receptor (PXR) siRNA and CYP3A4 reporter gene assays were applied. We found that both compounds strongly induced CYP3A4 and CYP2C9 activity and expression as well as expression of UGT1A1 and MDR1 in a concentration- and time-dependent manner. C-1748-mediated CYP3A4 and CYP2C9 mRNA induction equal to rifampicin occurred at extremely low concentrations (0.001 and 0.01μM), whereas 10μM C-1305 induced three-times higher CYP3A4 and CYP2C9 mRNA levels than rifampicin did. CYP3A4 and CYP2C9 expressions were shown to be PXR-dependent; however, neither compound influenced PXR expression. Thus, the observed drug-mediated induction of isoenzymes occurs on a PXR-mediated regulatory level. Furthermore, C-1748 and C-1305 were demonstrated to be selective PXR agonists. These effects are hypoxia-inhibited only in the case of C-1748, which is sensitive to P450 metabolism. In summary, PXR was found to be a new target of the studied compounds. Thus, possible combinations of these compounds with other therapeutics might lead to the PXR-dependent enzyme-mediated drug-drug interactions. PMID:23688499

  7. Pharmacogenetics in American Indian Populations: Analysis of CYP2D6, CYP3A4, CYP3A5, and CYP2C9 in the Confederated Salish and Kootenai Tribes

    PubMed Central

    Fohner, Alison; Muzquiz, LeeAnna I.; Austin, Melissa A.; Gaedigk, Andrea; Gordon, Adam; Thornton, Timothy; Rieder, Mark J.; Pershouse, Mark A.; Putnam, Elizabeth A.; Howlett, Kevin; Beatty, Patrick; Thummel, Kenneth E.; Woodahl, Erica L.

    2014-01-01

    Objectives Cytochrome P450 enzymes play a dominant role in drug elimination and variation in these genes is a major source of interindividual differences in drug response. Little is known, however, about pharmacogenetic variation in American Indian and Alaska Native (AI/AN) populations. We have developed a partnership with the Confederated Salish and Kootenai Tribes (CSKT) in northwestern Montana to address this knowledge gap. Methods We resequenced CYP2D6 in 187 CSKT subjects and CYP3A4, CYP3A5, and CYP2C9 in 94 CSKT subjects. Results We identified 67 variants in CYP2D6, 15 in CYP3A4, 10 in CYP3A5, and 41 in CYP2C9. The most common CYP2D6 alleles were CYP2D6*4 and *41 (20.86 and 11.23%, respectively). CYP2D6*3, *5, *6, *9, *10, *17, *28, *33, *35, *49, *1xN, *2xN, and *4xN frequencies were less than 2%. CYP3A5*3, CYP3A4*1G, and *1B were detected with frequencies of 92.47, 26.81, and 2.20%, respectively. Allelic variation in CYP2C9 was low: CYP2C9*2 (5.17%) and *3 (2.69%). In general, allele frequencies in CYP2D6, CYP2C9 and CYP3A5 were similar to those observed in European Americans. There was, however, a marked divergence in CYP3A4 for the CYP3A4*1G allele. We also observed low levels of linkage between CYP3A4*1G and CYP3A5*1 in the CSKT. The combination of nonfunctional CYP3A5*3 and putative reduced function CYP3A4*1G alleles may predict diminished clearance of CYP3A substrates. Conclusions These results highlight the importance of conducting pharmacogenomic research in AI/AN populations and demonstrate that extrapolation from other populations is not appropriate. This information could help to optimize drug therapy for the CSKT population. PMID:23778323

  8. Generation of in-silico cytochrome P450 1A2, 2C9, 2C19, 2D6, and 3A4 inhibition QSAR models.

    PubMed

    Gleeson, M Paul; Davis, Andrew M; Chohan, Kamaldeep K; Paine, Stuart W; Boyer, Scott; Gavaghan, Claire L; Arnby, Catrin Hasselgren; Kankkonen, Cecilia; Albertson, Nan

    2007-01-01

    In-silico models were generated to predict the extent of inhibition of cytochrome P450 isoenzymes using a set of relatively interpretable descriptors in conjunction with partial least squares (PLS) and regression trees (RT). The former was chosen due to the conservative nature of the resultant models built and the latter to more effectively account for any non-linearity between dependent and independent variables. All models are statistically significant and agree with the known SAR and they could be used as a guide to P450 liability through a classification based on the continuous pIC50 prediction given by the model. A compound is classified as having either a high or low P450 liability if the predicted pIC(50) is at least one root mean square error (RMSE) from the high/low pIC(50) cut-off of 5. If predicted within an RMSE of the cut-off we cannot be confident a compound will be experimentally low or high so an indeterminate classification is given. Hybrid models using bulk descriptors and fragmental descriptors do significantly better in modeling CYP450 inhibition, than bulk property QSAR descriptors alone. PMID:18034311

  9. Effects of salvianolic acid B and tanshinone IIA on the pharmacokinetics of losartan in rats by regulating the activities and expression of CYP3A4 and CYP2C9.

    PubMed

    Wang, Rong; Zhang, Hai; Wang, Yujie; Yu, Xiaoyan; Yuan, Yongfang

    2016-03-01

    Losartan (LST) is a common chemical drug used to treat high blood pressure and reduce the risk of stroke in certain people with heart disease. Danshen, prepared from the dried root and rhizome of Salvia miltiorrhiza Bunge, has been widely used for prevention and treatment of various cardiovascular and cerebrovascular diseases. There are more than 35 formulations containing Danshen indexed in the 2010 Chinese Pharmacopoeia, which are often combined with LST to treat cardiovascular and cerebrovascular diseases in the clinic. The effects of the two major components of Danshen, salvianolic acid B (SA-B) and tanshinone IIA (Tan IIA), on the pharmacokinetics of losartan and its metabolite, EXP3174, in rats were investigated by liquid chromatography coupled with mass spectrometry (LC-MS). Male Sprague-Dawley rats were randomly assigned to 3 groups: LST, LST+SA-B and LST+Tan IIA, and the main pharmacokinetic parameters were estimated after oral administration of LST, LST+SA-B and LST+Tan IIA. It was found that there are significant differences in the pharmacokinetic parameters among the three groups: Cmax, t1/2, AUC, AUMC in the LST+SA-B group was smaller than those in group LST, while larger in group LST+Tan IIA. Further, the effects of SA-B and Tan IIA on the metabolism of losartan was also investigated using rat liver microsomes in vitro. The results indicated that SA-B can induce the metabolism of LST, while Tan IIA can inhibit the metabolism of LST in rat liver microsomes in vitro by regulating activities of CYP450 enzymes. In addition, the effect of SA-B and Tan IIA on CYP3A4 and CYP2C9 expression was studied in Chang liver cells by western-blotting and Real-time PCR. It was concluded that the two components of Danshen, SA-B and Tan IIA have different influences on the metabolism of LST: SA-B can obviously speed up the metabolism of LST by inducing CYP3A4/CYP2C9 activities and expression, however, Tan IIA can slow down the metabolism of LST by inhibiting CYP3A4/CYP2C

  10. [In vivo evaluation of the metabolic ratio of CYP2C9 and CYP1A2 drug markers after administration of afobazole in comparison to standard inducers and inhibitors of cytochromes].

    PubMed

    Novitskaia, Ia G; Gribakina, O G; Kolyvanov, G B; Zherdev, V P; Smirnov, V V; Seredenin, S B

    2013-01-01

    The effect of subchronic peroral administration in effective doses of afobazole (5 mg/kg), and cytochrome P450 inductors (rifampicin, 13.4 mg/kg; phenytoin, 10.4 mg/kg) and inhibitors (fluconazole, 35.7 mg/kg; ciprofloxacin, 44.0 mg/kg) on the metabolic ratio (MR) of drugs-markers of CYP2C9 and CYP1A2 activity was studied in rats. Afobazole did not change the MR of compounds metabolized by the P450 isoforms studied. After peroral administration of standard P450 inductors and inhibitors, statistically significant bidirectional effects were identified, which demonstrated the expedience of administering a complex of selected compounds, markers, and CYP2C9 and CYP1A2 activity modificators for comparative evaluation of the effects of new drugs in rats. It is recommended to evaluate the activity of CYP1A2 by determining the MR for one of two caffeine metabolites, paraxanthine or theobromine, and the activity of CYP2C9 by determining the MR of metabolite Exp-3174 to losartan. PMID:24555232

  11. The effects of H2S on the activities of CYP2B6, CYP2D6, CYP3A4, CYP2C19 and CYP2C9 in vivo in rat.

    PubMed

    Wang, Xianqin; Han, Anyue; Wen, Congcong; Chen, Mengchun; Chen, Xinxin; Yang, Xuezhi; Ma, Jianshe; Lin, Guanyang

    2013-01-01

    Hydrogen sulfide (H2S) is a colorless, flammable, extremely hazardous gas with a "rotten egg" smell. The human body produces small amounts of H2S and uses it as a signaling molecule. The cocktail method was used to evaluate the influence of H2S on the activities of CYP450 in rats, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: bupropion, metroprolol, midazolam, omeprazole and tolbutamide, respectively. The rats were randomly divided into two groups, control group and H2S group. The H2S group rats were given 5 mg/kg NaHS by oral administration once a day for seven days. The mixture of five probes was given to rats through oral administration and the blood samples were obtained at a series of time-points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. In comparing the H2S group with the control group, there was a statistically pharmacokinetics difference for midazolam and tolbutamide; the area under the plasma concentration-time curve (AUC) was decreased for midazolam (p < 0.05) and increased for tolbutamide (p < 0.05); while there was no statistical pharmacokinetics difference for bupropion, metroprolol and omeprazole. H2S could not influence the activities of CYP2B6, CYP2D6 and CYP2C19 in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in rats. PMID:24336065

  12. The Effects of H2S on the Activities of CYP2B6, CYP2D6, CYP3A4, CYP2C19 and CYP2C9 in Vivo in Rat

    PubMed Central

    Wang, Xianqin; Han, Anyue; Wen, Congcong; Chen, Mengchun; Chen, Xinxin; Yang, Xuezhi; Ma, Jianshe; Lin, Guanyang

    2013-01-01

    Hydrogen sulfide (H2S) is a colorless, flammable, extremely hazardous gas with a “rotten egg” smell. The human body produces small amounts of H2S and uses it as a signaling molecule. The cocktail method was used to evaluate the influence of H2S on the activities of CYP450 in rats, which were reflected by the changes of pharmacokinetic parameters of five specific probe drugs: bupropion, metroprolol, midazolam, omeprazole and tolbutamide, respectively. The rats were randomly divided into two groups, control group and H2S group. The H2S group rats were given 5 mg/kg NaHS by oral administration once a day for seven days. The mixture of five probes was given to rats through oral administration and the blood samples were obtained at a series of time-points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. In comparing the H2S group with the control group, there was a statistically pharmacokinetics difference for midazolam and tolbutamide; the area under the plasma concentration-time curve (AUC) was decreased for midazolam (p < 0.05) and increased for tolbutamide (p < 0.05); while there was no statistical pharmacokinetics difference for bupropion, metroprolol and omeprazole. H2S could not influence the activities of CYP2B6, CYP2D6 and CYP2C19 in rats, while H2S could induce the activity of CYP3A4 and inhibit the activity of CYP2C9 in rats. PMID:24336065

  13. Complex Drug Interactions of HIV Protease Inhibitors 2: In Vivo Induction and In Vitro to In Vivo Correlation of Induction of Cytochrome P450 1A2, 2B6, and 2C9 by Ritonavir or Nelfinavir

    PubMed Central

    Kirby, Brian J.; Collier, Ann C.; Kharasch, Evan D.; Dixit, Vaishali; Desai, Pankaj; Whittington, Dale; Thummel, Kenneth E.

    2011-01-01

    Drug-drug interactions (DDIs) with the HIV protease inhibitors (PIs) are complex, paradoxical (e.g., ritonavir/alprazolam), and involve multiple mechanisms. As part of a larger study to better understand these DDIs and to devise a framework for in vitro to in vivo prediction of these DDIs, we determined the inductive effect of ∼2 weeks of administration of two prototypic PIs, nelfinavir (NFV), ritonavir (RTV), and rifampin (RIF; induction positive control) on the cytochrome P450 enzymes CYP1A2, CYP2B6, CYP2C9, and CYP2D6 and the inductive or inductive plus inhibitory effect of NFV, RTV, or RIF on CYP3A and P-glycoprotein in healthy human volunteers. Statistically significant induction of CYP1A2 (2.1-, 2.9-, and 2.2-fold), CYP2B6 (1.8-, 2.4-, and 4-fold), and CYP2C9 (1.3-, 1.8-, and 2.6-fold) was observed after NFV, RTV, or RIF treatment, respectively (as expected, CYP2D6 was not induced). Moreover, we accurately predicted the in vivo induction of these enzymes by quantifying their induction by the PIs in human hepatocytes and by using RIF as an in vitro to in vivo scalar. On the basis of the modest in vivo induction of CYP1A2, CYP2B6, or CYP2C9, the in vivo paradoxical DDIs with the PIs are likely explained by mechanisms other than induction of these enzymes such as induction of other metabolic enzymes, transporters, or both. PMID:21930825

  14. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  15. In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.

    NASA Astrophysics Data System (ADS)

    Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

    2009-03-01

    Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

  16. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  17. In vitro inhibition of human CYP1A2, CYP2D6, and CYP3A4 by six herbs commonly used in pregnancy.

    PubMed

    Langhammer, Astrid Jordet; Nilsen, Odd Georg

    2014-04-01

    Black elderberry, cranberry, fennel, ginger, horsetail, and raspberry leaf, herbs frequently used in pregnancy, were investigated for their in vitro CYP1A2, 2D6, and 3A4 inhibitory potential. Aqueous or ethanolic extracts were made from commercially available herbal products, and incubations were performed with recombinant cDNA-expressed human CYP enzymes in the presence of positive inhibitory controls. Metabolite formation was determined by validated LCMS/MS or HPLC methodologies. IC50 inhibition constants were estimated from CYP activity inhibition plots using non-linear regression. The most potent inhibition was shown for fennel towards CYP2D6 and 3A4 with respective IC50 constants of 23 ± 2 and 40 ± 4 µg/ml, horsetail towards CYP1A2 with an IC50 constant of 27 ± 1 µg/ml, and raspberry leaf towards CYP1A2, 2D6, and 3A4 with IC50 constants of 44 ± 2, 47 ± 8, and 81 ± 11 µg/ml, respectively. Based on the recommended dosing of the different commercial herbal products, clinically relevant systemic CYP inhibitions could be possible for fennel, horsetail, and raspberry leaf. In addition, fennel and raspberry leaf might cause a clinically relevant inhibition of intestinal CYP3A4. The in vivo inhibitory potential of these herbs towards specific CYP enzymes should be further investigated. PMID:23843424

  18. Effects of CYP2C9 genetic polymorphisms on the pharmacokinetics of zafirlukast.

    PubMed

    Lee, Hyun-Jee; Kim, Young-Hoon; Kim, Se-Hyung; Lee, Choong-Min; Yang, Ae-Yun; Jang, Choon-Gon; Lee, Seok-Yong; Bae, Jung-Woo; Choi, Chang-Ik

    2016-07-01

    Zafirlukast, a cysteinyl leukotriene receptor antagonist, is indicated for the treatment of patients with mild to moderate asthma. Zafirlukast is metabolized mainly by CYP3A4 and CYP2C9. We investigated the effects of the major CYP2C9 variant alleles in Asian populations, CYP2C9*3 and CYP2C9*13, on the pharmacokinetics of zafirlukast in healthy Korean subjects. A single 20-mg oral dose of zafirlukast was given to 23 Korean male subjects divided into two genotype groups according to CYP2C9 genotypes, CYP2C9EM (n = 11; CYP2C9*1/*1) and CYP2C9IM (n = 12; 9 and 3 carriers of CYP2C9*1/*3 and *1/*13, respectively). Zafirlukast concentrations were determined using a validated HPLC-MS/MS analytical method in plasma samples collected after the drug intake. Compared with the CYP2C9EM group, the Cmax and AUCinf of zafirlukast in the CYP2C9IM group were 1.44- and 1.70-fold higher, respectively (p < 0.01 and p < 0.0001). The CL/F of zafirlukast was 42.8 % lower in the CYP2C9IM group compared with the CYP2C9EM group (p < 0.001). Slightly higher Cmax and AUC, and lower CL/F of zafirlukast were observed in subjects with the CYP2C9*1/*13 genotype compared with the CYP2C9*1/*3 genotype subjects. CYP2C9*3 and CYP2C9*13 alleles significantly affected the plasma concentrations of zafirlukast. PMID:27377818

  19. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

    PubMed Central

    Bethke, Lara; Webb, Emily; Sellick, Gabrielle; Rudd, Matthew; Penegar, Stephen; Withey, Laura; Qureshi, Mobshra; Houlston, Richard

    2007-01-01

    Background Cytochrome P450 (CYP) enzymes have the potential to affect colorectal cancer (CRC) risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs) that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively). Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility. PMID:17615053

  20. Differential effects of 2C9*3 and 2C9*2 variants of cytochrome P-450 CYP2C9 on sensitivity to acenocoumarol.

    PubMed

    Hermida, José; Zarza, José; Alberca, Ignacio; Montes, Ramón; López, María Luz; Molina, Eva; Rocha, Eduardo

    2002-06-01

    The 2C9*3 and 2C9*2 polymorphisms of cytochrome P-450 CYP2C9 are associated with hypersensitivity to warfarin and bleeding. The effect of these polymorphisms on sensitivity to acenocoumarol is unknown. Three groups of patients, with low, medium, or high acenocoumarol-dose requirements, were studied. Age influenced the acenocoumarol sensitivity. Bearing the 2C9*3 allele was associated with the need for a lower acenocoumarol dose (odds ratio [OR], 6.02; 95% confidence interval [CI], 1.50-24.18); 80% of carriers of the 2C9*3 allele required a low dose. The 2C9*2 allele was associated with a lower acenocoumarol-dose requirement (OR, 2.70; 95% CI, 1.11-6.58) because of a reduced risk of the need for a high acenocoumarol dose (4.8% of the patients in the high-dose group carried the 2C9*2 allele versus 34.1% and 30.2%, respectively, in the medium-dose and low-dose groups). Therefore, carriers of 2C9*3 may need a low initial loading dose of acenocoumarol. Because acenocoumarol sensitivity with the 2C9*2 variant does not seem to be clinically relevant, the drug could be an alternative to warfarin in 2C9*2 carriers. PMID:12010835

  1. Hydroxywarfarin metabolites potently inhibit CYP2C9 metabolism of S-warfarin.

    PubMed

    Jones, Drew R; Kim, So-Young; Guderyon, Michael; Yun, Chul-Ho; Moran, Jeffery H; Miller, Grover P

    2010-05-17

    Coumadin (R/S-warfarin) anticoagulant therapy poses a risk to over 50 million Americans, in part due to interpersonal variation in drug metabolism. Consequently, it is important to understand how metabolic capacity is influenced among patients. Cytochrome P450s (P450 or CYP for a specific isoform) catalyze the first major step in warfarin metabolism to generate five hydroxywarfarins for each drug enantiomer. These primary metabolites are thought to reach at least 5-fold higher levels in plasma than warfarin. We hypothesized that hydroxywarfarins inhibit the hydroxylation of warfarin by CYP2C9, thereby limiting enzymatic capacity toward S-warfarin. To test this hypothesis, we investigated the ability of all five racemic hydroxywarfarins to block CYP2C9 activity toward S-warfarin using recombinant enzyme and human liver microsomes. We initially screened for the inhibition of CYP2C9 by hydroxywarfarins using a P450-Glo assay to determine IC(50) values for each hydroxywarfarin. Compared to the substrate, CYP2C9 bound its hydroxywarfarin products with less affinity but retained high affinity for 10- and 4'-hydroxywarfarins, products from CYP3A4 reactions. S-Warfarin steady-state inhibition studies with recombinant CYP2C9 and pooled human liver microsomes confirmed that hydroxywarfarin products from CYP reactions possess the capacity to competitively inhibit CYP2C9 with biologically relevant inhibition constants. Inhibition of CYP2C9 by 7-hydroxywarfarin may be significant given its abundance in human plasma, despite its weak affinity for the enzyme. 10-Hydroxywarfarin, which has been reported as the second most abundant plasma metabolite, was the most potent inhibitor of CYP2C9, displaying approximately 3-fold higher affinity than S-warfarin. These results indicate that hydroxywarfarin metabolites produced by CYP2C9 and other CYPs may limit metabolic capacity toward S-warfarin through competitive inhibition. Subsequent processing of hydroxywarfarins to secondary

  2. Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells

    PubMed Central

    Du, Huihui; Wei, Zhiyun; Yan, Yucai; Xiong, Yuyu; Zhang, Xiaoqing; Shen, Lu; Ruan, Yunfeng; Wu, Xi; Xu, Qingqing; He, Lin; Qin, Shengying

    2016-01-01

    Variability in activity of CYP2C9, which is involved in the metabolism of approximately 15% of current therapeutic drugs, is an important contributor to interindividual differences in drug response. To evaluate the functional alternations of CYP2C9*2, CYP2C9*3, CYP2C9*8, CYP2C9*11 and CYP2C9*31, identified in our previous study in Chinese Han population, allelic variants as well as the wild-type CYP2C9 were transiently expressed in COS-7 cells. Kinetic parameters (Km, Vmax, and Clint) for S-warfarin 7-hydroxylation by these recombinant CYP2C9s were determined. Relative to CYP2C9.1, recombinant CYP2C9.3 and CYP2C9.11 exhibited significantly higher Km values, and all allelic variants showed significantly decreased Vmax and Clint values. Among all allelic variants, catalytic activity of CYP2C9.3 and CYP2C9.11 reduced the most (8.2% and 9.8% of Clint ratio, respectively; P < 0.001). These findings should be useful for predicting the phenotype profiles of CYP2C9 in Chinese Han population, comparing the functional results of these alleles accurately, and finally optimizing pharmacotherapy of drug treatment. PMID:27199745

  3. CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48.

    PubMed

    Holm, Niels Bjerre; Nielsen, Line Marie; Linnet, Kristian

    2015-09-01

    Synthetic cannabinoid designer drugs have emerged as drugs of abuse during the last decade, and acute intoxication cases are documented in the scientific literature. Synthetic cannabinoids are extensively metabolized, but our knowledge of the involved enzymes is limited. Here, we investigated the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared the formed metabolites to human liver microsomal (HLM) incubations with specific inhibitors against CYP2D6, 2C19, and 3A4, respectively. The data reported here demonstrate CYP3A4 to be the major CYP enzyme responsible for the oxidative metabolism of AKB-48, preferentially performing the oxidation on the adamantyl moiety. Genetic polymorphisms are likely not important with regard to toxicity given the major involvement of CYP3A4. Adverse drug-drug interactions (DDIs) could potentially occur in cases with co-intake of strong CYP3A4 inhibitors, e.g., HIV antivirals and azole antifungal agents. PMID:26002511

  4. Genetic Polymorphism of Cytochrome p450 (2C9) Enzyme in Iranian Baluch Ethnic Group

    PubMed Central

    Tabari, Mojdeh Ghiyas; Naseri, Fatemeh; Ataby, Maryam Agh; Marjani, Abdoljalal

    2015-01-01

    The aim of the present study is to assess and compare the frequencies of the cytochrome P450 CYP2C9 variations in the Baluch ethnic group (n=110) with other ethnic groups. The allele frequencies of CYP2C9*1, CYP2C9*2 and CYP2C9*3 were 80.90%, 11.82% and 7.27%, respectively. 70.90%, 11.82%, 8.18%, 4.55%, 2.73% and 1.82% of subjects were with CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes, respectively. Different mutants may effect on prediction of drug dose requirements in different ethnic groups. Thus, CYP2C9 variants to be determined for findings high risk groups use optimal dosage of drugs metabolized by this polymorphic enzyme. PMID:26464589

  5. Genetic variation in the 3′-UTR of CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2, and UGT2B7: potential effects on regulation by microRNA and pharmacogenomics relevance

    PubMed Central

    Swart, Marelize; Dandara, Collet

    2014-01-01

    Introduction: Pharmacogenomics research has concentrated on variation in genes coding for drug metabolizing enzymes, transporters and nuclear receptors. However, variation affecting microRNA could also play a role in drug response. This project set out to investigate potential microRNA target sites in 11 genes and the extent of variation in the 3′-UTR of six selected genes; CYP1A2, CYP2B6, CYP2D6, CYP3A4, NR1I2, and UGT2B7. Methods: Fifteen microRNA target prediction algorithms were used to identify microRNAs predicted to regulate 11 genes. The 3′-UTR of the 6 genes which topped the list of potential microRNA targets was sequenced in 30 black South Africans. In addition, genetic variants within these genes were investigated for interference with mRNA-microRNA interactions. Potential effects of observed variants were determined using in silico prediction tools. Results: The 11 genes coding for DMEs, transporters and nuclear receptors were predicted to be targets of microRNAs with CYP2B6, NR1I2 (PXR), CYP3A4, and CYP1A2, interacting with the most microRNAs. The majority of identified genetic variants were predicted to interfere with microRNA regulation. For example, the variant, rs1054190C in NR1I2 was predicted to result in the presence of a binding site for the microRNA miR-1250-5p, while the variant rs1054191G was predicted to result in the absence of a recognition site for miR-371b-3p, miR-4258 and miR-4707-3p. Fifteen of the seventeen, novel variants occurred within microRNA target sequences. Conclusion: The 3′-UTR harbors variation that is likely to influence regulation of specific genes by microRNA. In silico prediction followed by functional validation could aid in decoding the contribution of variation in the 3′-UTR, to some unexplained heritability that affects drug response. Understanding the specific role of each microRNA may lead to identification of markers for targeted therapy and therefore improve personalized drug treatment. PMID:24926315

  6. Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9.

    PubMed

    Kim, Sun Joo; Kim, Su Jin; Hong, Miri; Choi, Hyun Gyu; Kim, Jeong Ah; Lee, Sangkyu

    2016-10-01

    1. Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated. 2. We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4'-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC50) 1.3 and 16.1 μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively. 3. Glycyrol decreased CYP2C9-catalyzed diclofenac 4'-hydroxylation activity with IC50 values of 0.67 μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs. PMID:26750984

  7. Inhibitory effect of salvianolate on human cytochrome P450 3A4 in vitro involving a noncompetitive manner

    PubMed Central

    Qin, Chong-Zhen; Ren, Xian; Zhou, Hong-Hao; Mao, Xiao-Yuan; Liu, Zhao-Qian

    2015-01-01

    Salvianolic acid B (Sal B), which is purified from Danshen, is a popular herb extract. Sal B has anti-oxidative, anti-inflammatory, anti-hypoxic, anti-arteriosclerotic and anti-apoptotic properties. This substance can also ameliorate brain injury or neurodegenerative diseases. The listed drug Salvianolate, which contains a substantial amount of Sal B, has been used for the treatment of coronary heart disease. Our present work aimed to evaluate the inhibitory effect of salvianolate on seven cytochrome P450 isoforms (CYP450), namely, CYP1A2, CYP2A6, CYP2E1, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, in human liver microsomes (HLMs) and recombinant enzymes through high-performance liquid chromatography (HPLC) assay. Salvianolate have a potent inhibitory effect on CYP3A4 activity with IC50 values of 1.438 (HLMs) and 3.582 (recombinant cDNA-expressed CYP3A4) mg/L, respectively. Salvianolate strongly dose, but not time-dependently decreased CYP3A4 activity in HLMs. The typical Lineweaver-Burk plots showed that Salvianolate inhibited CYP3A4 activity noncompetitively, with a Ki value of 2.27 mg/L in HLMs. Other CYP450 isoforms are not markedly affected by Salvianolate. These findings indicate that salvianolate may be involved in potential drug interactions when co-administrated with CYP3A4 substrates. PMID:26629047

  8. The role of CYP2C9 genetic polymorphism in carvedilol O-desmethylation in vitro.

    PubMed

    Pan, Pei-Pei; Weng, Qing-Hua; Zhou, Chen-Jian; Wei, Yan-Li; Wang, Li; Dai, Da-Peng; Cai, Jian-Ping; Hu, Guo-Xin

    2016-02-01

    We aimed at investigating the role of CYP2C9 in carvedilol O-desmethylation and identifying the effect of 35 CYP2C9 allelic variants we found in Chinese Han population on the in vitro metabolism of carvedilol. Recombinant CYP2C9 and CYP2D6 microsomes of the wild type were used to test and verify the enzymes involved in carvedilol O-desmethylation. Recombinant CYP2C9 microsomes of distinguished genotypes were used to characterize the corresponding enzyme activity toward carvedilol. 2-100 μM carvedilol was incubated for 30 min at 37 °C. The products were detected using high-performance liquid chromatography. CYP2C9 plays a certain role in carvedilol metabolism. Compared with wild-type CYP2C9*1, the intrinsic clearance (V max/K m) values of all variants toward carvedilol O-desmethylation were significantly altered. The variants exhibited significantly decreased values (from 30 to 99.8 %) due to increased K m and/or decreased V max values. We conclude that recombinant system could be used to investigate the enzymes involved in drug metabolism and these findings complement the database where CYP2C9 polymorphism interacts with biotransformation of exogenous substances like drugs and toxins. PMID:25476996

  9. Is cytochrome P450 2C9 genotype associated with NSAID gastric ulceration?

    PubMed Central

    Martin, Jennifer H; Begg, Evan J; Kennedy, Martin A; Roberts, Rebecca; Barclay, Murray L

    2001-01-01

    Aims The aim of this study was to explore whether genetic variation of cytochrome P450 2C9 (CYP2C9) contributes to NSAID-associated gastric ulceration. The hypothesis tested was that CYP2C9 poor metabolizer genotype would predict higher risk of gastric ulceration in patients on NSAIDs that are metabolized by CYP2C9, due to higher plasma NSAID concentrations. Methods Peripheral blood DNA samples from 23 people with a history of gastric ulceration attributed to NSAIDs metabolized by CYP2C9, and from 32 people on NSAIDs without gastropathy, were analysed to determine CYP2C9 genotype. Results The following genotypes were found: *1/*1 (wild type) in 70% of cases and 58% of controls, *1/*2 in 17% of cases and 29% of controls, *1/*3 in 13% of cases and 13% of controls. The difference between case and control nonwild-type genotype frequency was 11.5% (95% CI −14,37%), with the direction of the difference being against the hypothesis. No individuals with homozygote poor metaboliser genotype were identified. The differences in genotype frequencies between the two groups were not significant and the frequencies were similar to those in a large published population study. Ninety-five percent binomial confidence interval analysis confirms that there is no apparent clinically significant relationship between CYP2C9 genotype and risk of gastric ulceration although a small difference in risk in poor metabolizers cannot be excluded. Conclusions These results do not support the hypothesis that gastric ulceration resulting from NSAID usage is linked to the poor metabolizing genotypes of CYP2C9. PMID:11422024

  10. Influence of CYP2C9 Genotype on warfarin dose among African American and European Americans.

    PubMed

    Limdi, Na; Goldstein, Ja; Blaisdell, Ja; Beasley, Tm; Rivers, Ca; Acton, Rt

    2007-05-01

    BACKGROUND: Cytochrome P4502C9 (CYP2C9) plays a vital role in drug metabolism. There has been an increased effort to identify polymorphisms within the gene and determine their clinical consequences. However, most of these efforts have focused on populations of European descent. Herein we report the influence of CYP2C9 genotype on warfarin dose among European American and African American patients. We also identify two new mutations; one in the coding region and one in the non-coding region of the CYP2C9 gene. METHODS: Patients (≥20 years of age) are enrolled after obtaining medical, lifestyle and concomitant medication history. Changes in International Normalized Ratio (INR), warfarin dose, co-medications, diet, physical activity and the occurrence of complications are documented. CYP2C9 genotype was determined using PCR-RFLP and pyrosequencing. Differences in genotype frequencies and HWE assumptions were assessed using χ(2) statistics and exact tests. The genotype dose association was evaluated using multivariable linear regression. RESULTS: This report includes 490 patients (mean age 60.6 ± 15.6, 51.3% men). African American patients comprise 48.9% of the cohort with mean follow-up of 13.5 (±10.6) months. Both the CYP2C9 *2 and *3 allele were more frequent in European Americans (11.24%, 5.1%) compared to African Americans (1.1% and 1.8%). CYP2C9 *5 (0.9%), *6 (0.4%), and *11 (1.1%) variants were only observed in African Americans. The variant genotype is more frequent among European Americans compared to African Americans (29.8% vs. 9.73%, p<0.0001). Warfarin dose was significantly related to CYP2C9 genotype (p<0.0001) both in univariate and multivariate analyses. Multivariable race-specific analyses highlight the contribution of CYP2C9 genotype among European American but not among African American patients. CONCLUSION: The variant CYP2C9 genotype is more frequent among European Americans compared to African Americans. Among African Americans the variant

  11. Contribution of CYP2C9 to variability in vitamin K antagonist metabolism.

    PubMed

    Daly, Ann K; King, Barry P

    2006-02-01

    CYP2C9 is the third most important cytochrome P450 (CYP) in terms of number of drugs metabolised. A considerable amount of information on this isoform is now available with respect to its structural biology, the mechanisms by which it can be induced and the existence of a range of variant alleles, which are often functionally significant. CYP2C9 makes a very important contribution to metabolism of vitamin K antagonist anticoagulants, and is the main oxidising enzyme for S-warfarin and S-acenocoumarol as well as contributing to phenprocoumon metabolism. A large number of studies have now shown that CYP2C9 genotype predicts dose requirement for both warfarin and acenocoumarol, with a possible contribution for phenprocoumon. Patients with variant alleles are likely to require a lower dose and may be at risk of overcoagulation and resultant bleeding, especially during the induction phase of therapy. Although CYP2C9 genotype is clearly a predictor of vitamin K antagonist dose requirement, especially in Caucasian populations in whom variant alleles are common, a number of recent studies have shown that age, genotype for the gene encoding the target gene vitamin K epoxide reductase and concomitant drugs are equally important factors in determining dose. There is a need for prospective studies to assess the value of predicting dose requirement on the basis of all these factors, including the CYP2C9 genotype. PMID:16863464

  12. Genetic polymorphisms analysis of drug-metabolizing enzyme CYP2C9 in the Uyghur population.

    PubMed

    Jin, Tianbo; Xun, Xiaojie; Du, Shuli; Geng, Tingting; Wang, Hong; Feng, Tian; Chen, Chen; Yuan, Dongya; Kang, Longli

    2016-08-01

    Genetic variations in cytochrome P450 2C9 are known to contribute to interindividual and interethnic variability in response to clinical drugs, but little is known about the genetic variation of CYP2C9 in the Uyghur population. We directly sequenced the whole CYP2C9 gene in 96 unrelated, healthy Uyghur from Xinjiang Uygur Autonomous Region of China and screened for genetic variants in the promoter, exons, introns and 3'-UTR. Thirty five previously reported alleles and six genotypes were detected in this study. The allele frequencies of CYP2C9*1, *2, *11, *12, *29 and *33 were 89.58, 7.81, 0.52, 0.52, 1.04 and 0.52%, respectively. We detected one non-synonymous novel variant at position 329 from Arg to Cys and this mutation is predicted to be intolerant by SIFT. Our results provide basic information about CYP2C9 alleles in Uyghur, which may help to optimize pharmacotherapy effectiveness by providing personalized medicine to this ethnic group. PMID:26610168

  13. Assessment of arginine 97 and lysine 72 as determinants of substrate specificity in cytochrome P450 2C9 (CYP2C9).

    PubMed

    Davies, Carwyn; Witham, Katey; Scott, Justin R; Pearson, Andrew; DeVoss, James J; Graham, Sandra E; Gillam, Elizabeth M J

    2004-04-01

    CYP2C9 is distinguished by a preference for substrates bearing a negative charge at physiological pH. Previous studies have suggested that CYP2C9 residues R97 and K72 may play roles in determining preference for anionic substrates by interaction at the active site or in the access channel. The aim of the present study was to assess the role of these two residues in determining substrate selectivity. R97 and K72 were substituted with negative, uncharged polar and hydrophobic residues using a degenerate polymerase chain reaction-directed strategy. Wild-type and mutant enzymes were expressed in bicistronic format with human cytochrome P450 reductase in Escherichia coli. Mutation of R97 led to a loss of holoenzyme expression for R97A, R97V, R97L, R97T, and R97E mutants. Low levels of hemoprotein were detected for R97Q, R97K, R97I, and R97P mutants. Significant apoenzyme was observed, suggesting that heme insertion or protein stability was compromised in R97 mutants. These observations are consistent with a structural role for R97 in addition to any role in substrate binding. By contrast, all K72 mutants examined (K72E, K72Q, K72V, and K72L) could be expressed as hemoprotein at levels comparable to wild-type. Type I binding spectra were obtained with wild-type and K72 mutants using diclofenac and ibuprofen. Mutation of K72 had little or no effect on the interaction with these substrates, arguing against a critical role in determining substrate specificity. Thus, neither residue appears to play a role in determining substrate specificity, but a structural role for R97 can be proposed consistent with recently published crystallographic data for CYP2C9 and CYP2C5. PMID:15039296

  14. Enzyme Source Effects on CYP2C9 Kinetics and Inhibition

    PubMed Central

    Kumar, Vikas; Rock, Dan A.; Warren, Chad J.; Tracy, Timothy S.; Wahlstrom, Jan L.

    2008-01-01

    When choosing a recombinant P450 enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. While literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes™, Baculosomes®, RECO® system, and in-house purified, reconstituted enzyme) to determine if the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher Km values, reduced substrate affinity, and lower calculated intrinsic clearance values compared to baculovirus microsomal preparations. Six to twenty five-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. Additionally, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of twelve inhibitors, decreased inhibition potency was observed across eleven of those inhibitors in the RECO® purified, reconstituted enzyme as compared to the Supersomes™ baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9. PMID:16928789

  15. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.; Wade, Rebecca C.

    2011-08-11

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

  16. 1-Aminobenzotriazole coincubated with (S)-warfarin results in potent inactivation of CYP2C9.

    PubMed

    Sodhi, Jasleen K; Ford, Kevin A; Mukadam, Sophie; Wong, Susan; Hop, Cornelis E C A; Khojasteh, S Cyrus; Halladay, Jason S

    2014-05-01

    1-Aminobenzotriazole (ABT) is a nonselective, mechanism-based inactivator of cytochrome P450 (P450) and a useful tool compound to discern P450- from non-P450-mediated metabolism. ABT effectively inactivates major human P450 isoforms, with the notable exception of CYP2C9. Here we propose that ABT preferentially binds to the warfarin-binding pocket in the CYP2C9 active-site cavity; thus, ABT bioactivation and subsequent inactivation is not favored. Therefore, coincubation with (S)-warfarin would result in displacement of ABT from the warfarin-binding pocket and subsequent binding to the active site, converting ABT into a potent inactivator of CYP2C9. To test this hypothesis, in vitro studies were conducted using various coincubation combinations of ABT and (S)-warfarin or diclofenac to modulate the effectiveness of CYP2C9 inactivation by ABT. Coincubation of ABT with (S)-warfarin (diclofenac probe substrate) resulted in potent inactivation, whereas weak inactivation was observed following coincubation of ABT with diclofenac [(S)-warfarin probe substrate]. The kinetic parameters of time-dependent inhibition of ABT for CYP2C9 in the absence and presence of (S)-warfarin (20 μM) were 0.0826 and 0.273 min(-1) for kinact and 3.49 and 0.157 mM for KI, respectively. In addition, a 73.4-fold shift was observed in the in vitro potency (kinact/KI ratio), with an increase from 23.7 ml/min/mmol (ABT alone) to 1740 ml/min/mmol [ABT with (S)-warfarin (20 μM)]. These findings were supported by in silico structural modeling, which showed ABT preferentially binding to the warfarin-binding pocket and the displacement of ABT to the active site in the presence of (S)-warfarin. PMID:24550229

  17. The Effect of Genetic Polymorphism on the Inhibition of Azole Antifungal Agents Against CYP2C9-Mediated Metabolism.

    PubMed

    Niwa, Toshiro; Hata, Tomomi

    2016-03-01

    We investigated the effect of cytochrome P450 (CYP) 2C9 polymorphism on the inhibition of methylhydroxylation activity of tolbutamide, a typical CYP2C9 substrate, by triazole antifungal agents, fluconazole and voriconazole. Although the Michaelis constants (Km), maximal velocities (Vmax), and Vmax/Km values for CYP2C9.1 (wild type) and CYP2C9.2 (Arg144Cys) were similar and CYP2C9.3 (Ile359Leu) had a higher Km and a lower Vmax than CYP2C9.1 and CYP2C9.2, the inhibition constants of fluconazole and voriconazole against CYP2C9.2 were lower than that against CYP2C9.1 and CYP2C9.3. These results suggest that more careful administration of azole antifungals to patients with the CYP2C9*2 allele might be required because of the strong inhibitory effects. PMID:26886310

  18. Induction of cytochrome P450 3A4 and P-glycoprotein by the isoxazolyl-penicillin antibiotic flucloxacillin.

    PubMed

    Huwyler, Jörg; Wright, Matthew B; Gutmann, Heike; Drewe, Juergen

    2006-02-01

    Clinical findings indicate that co-administration of the isoxazolyl-penicillin flucloxacillin with cyclosporine may reduce the plasma concentrations of cyclosporine. We have explored in the present study if induction of cytochrome P450 3A4 or P-glycoprotein may offer a mechanistic explanation of the observed effects. Flucloxacillin is neither an inhibitor nor a substrate of drug metabolizing cytochrome P450 isoenzymes (CYP3A4, 1A2, 2C9, 2C19 and 2D6) or P-glycoprotein as shown by an in vitro assay for CYP inhibition, a fluorescent indicator assay for P-glycoprotein inhibition and a functional P-glycoprotein ATPase assay. However, incubation of human LS 180 colorectal adenocarcinoma cells with flucloxacillin led to a dose-dependent induction of MDR1 as well as of CYP3A4 mRNA, which was also confirmed in primary human hepatocytes. At high concentrations, flucloxacillin activated the human Pregnane-X-Receptor, PXR, a ligand-dependent transcription factor that is the target of many drugs that induce CYP3A4, with consequences for the metabolism of other drugs. Liver microsomes from control rats or rats, which received for 3 consecutive days 100 mg/kg of oral flucloxacillin, were used to study the metabolism and metabolite pattern of midazolam, a model substrate of CYP 3A4. There was a trend towards a higher intrinsic microsomal clearance of midazolam using microsomes from flucloxacillin treated rats. In addition, there was a significant increase in the formation of the principal midazolam metabolites 1-hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy midazolam as compared to controls. These findings indicate that flucloxacillin has the potential to induce expression of both CYP3A4 as well as P-glycoprotein, most likely through activation of the nuclear hormone receptor PXR. This would offer an explanation for the observed clinical drug-drug interactions between the antibiotic and cyclosporine. PMID:16472102

  19. Warfarin dose requirements in a patient with the CYP2C9*14 allele.

    PubMed

    Lee, Yee Ming; Eggen, Jessica; Soni, Vinay; Drozda, Katarzyna; Nutescu, Edith A; Cavallari, Larisa H

    2014-05-01

    We describe a 64-year-old male of Indian descent with a history of atrial fibrillation who was started on warfarin after hospital admission for acute stroke. He received genotype-guided warfarin dosing as per the standard-of-care at our hospital, with daily dose recommendations provided by the pharmacogenetics service. Genotyping revealed the rare CYP2C9*1/*14 genotype and warfarin insensitive VKORC1 -1639GG and CYP4F2 433Met/Met genotypes. The patient received an initial warfarin loading dose of 4 mg for 2 days, followed by 2-3 mg/day for the following 11 days. He reached a therapeutic international normalized ratio on day 5, which was maintained over the following week. This report adds to the limited data of the effects of the CYP2C9*14 allele on warfarin dose requirements. PMID:24956244

  20. Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase.

    PubMed

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-07-01

    Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans. PMID:25948712

  1. [Polymorphism of the biotransformation gene--cytochrome-450 2C9 in the patients with tuberculosis].

    PubMed

    Antonenko, P B; Kresyun, V I

    2014-01-01

    The goal of this work was to study cytochrome-450 (CYP) 2C9 (CYP2C9) gene polymorphism in patients with tuberculosis (TB) and its meaning for development, progress, and outcome of TB, for the pharmacokinetics of the antituberculosis antibiotic rifampicin on the basis of the southern region of Ukraine. Among the TB patients it was 24.9% less than in carriers of the genotype *1/*1 and than in healthy donors. At the same time, it was 25.0% less than in carriers of the genotypes *1/*2, *1/*3. In the TB patientswith the genotype *2/*3, *3/*3 the level of rifampicin in blood was the lowest. At the beginning of the treatment in carriers of genotype *1/*1 the pulmonary destruction was observed 2.5 times more often than in *1/*2, *1/*3 genotype. According to the cultural method, the carriers of *1/*1 more frequently became smear-negative than the carriers of *1/*2, *1/*3 genotype. PMID:25335408

  2. Quantitative Assessment of the Effect of Cytochrome P450 2C9 Gene Polymorphism and Colorectal Cancer

    PubMed Central

    Zhang, Liang; Wang, Yichao; Ma, Yushui; Zhang, Feng; Fu, Da; Wang, Xiaofeng

    2013-01-01

    CYP2C9 enzyme activity is involved in the metabolism of substances related to colorectal cancer (CRC), and it is functionally linked to a genetic polymorphism. Two allelic variants of the CYP2C9 gene, namely CYP2C9*2 and CYP2C9*3, differ from wild-type CYP2C9*1 by single amino acid substitutions. These mutated alleles encode enzymes with altered properties that are associated with impaired metabolism. In the past decade, a number of case-control studies have been carried out to investigate the relationship between the CYP2C9 polymorphism and CRC susceptibility, but the results were conflicting. To investigate this inconsistency, we performed a meta-analysis of 13 studies involving a total of 20,879 subjects for CYP2C9*2 and *3 polymorphisms to evaluate the effect of CYP2C9 on genetic susceptibility for CRC. Overall, the summary odds ratio of CRC was 0.94 (95%CI: 0.87–1.03, P = 0.18) and 1.00 (95%CI: 0.86–1.16, P = 0.99) for CYP2C9 *2 and *3 carriers, respectively. No significant results were observed in heterozygous and homozygous when compared with wild genotype for these polymorphisms. In the stratified analyses according to ethnicity, sample size, diagnostic criterion, HWE status and sex, no evidence of any gene-disease association was obtained. Our result suggest that the *2, *3 polymorphisms of CYP2C9 gene are not associated with CRC susceptibility. PMID:23577132

  3. Modulation of CYP3A4 activity alters the cytotoxicity of lipophilic phycotoxins in human hepatic HepaRG cells.

    PubMed

    Ferron, P J; Hogeveen, K; De Sousa, G; Rahmani, R; Dubreil, E; Fessard, V; Le Hegarat, L

    2016-06-01

    The aim of this study was to investigate (i) the cytotoxic effects of lipophilic phycotoxins, including okadaic acid (OA) and dinophysistoxin-1 and -2 (DTX-1 and DTX-2), pectenotoxin-2 (PTX-2), yessotoxin (YTX), spirolide (SPX), and azaspiracids-1, -2 and -3 (AZA-1, AZA-2 and AZA-3), in human HepaRG cells using a multiparametric high content analysis approach, (ii) the ability of nine lipophilic phycotoxins to act as PXR agonists in a HepG2-PXR cell line, (iii) their potential to induce CYP450 activity, and (iv) the role of CYP3A4 in cytotoxicity induced by lipophilic phycotoxins. Our results indicate that while OA, DTX-1 and DTX-2 activated PXR-dependent transcriptional activity in HepG2 cells, no increase of CYP450 (1A2, 3A4, 2C9, 2C19) activities were observed in HepaRG cell following a 72h treatment with these toxins. Multiparametric analysis showed that OA, DTX-1, DTX-2, and PTX-2 were highly cytotoxic in HepaRG cells; inducing cell loss, activation of caspase-3 and γ-H2AX formation. However, no toxicity was observed for YTX, SPX, and AZAs. Moreover, we found that inhibition of CYP3A4 activity by ketoconazole enhances the toxic effects of OA, DTX-1, DTX-2, and PTX-2 in HepaRG cells. Taken together, these results suggest that CYP3A4-mediated metabolism of some lipophilic phycotoxins decreases their in vitro toxicity. PMID:26956883

  4. Evaluation of flurbiprofen urinary ratios as in vivo indices for CYP2C9 activity

    PubMed Central

    Zgheib, N K; Frye, R F; Tracy, T S; Romkes, M; Branch, R A

    2007-01-01

    Aims We investigated flurbiprofen pharmacokinetics in 12 volunteers to develop a phenotypic trait measure that correlates with the fractional clearance to 4′-hydroxyflurbiprofen. The effect of the CYP2C9 inhibitor fluconazole on flurbiprofen metabolism was also evaluated. Methods Flurbiprofen pharmacokinetics were evaluated before and after the first and seventh doses of fluconazole. The urinary recovery ratio was calculated as FLRR = 4′-OHF/ [4′-OHF + Ftot] and the urinary metabolic ratio was calculated as FLMR = 4′-OHF/Ftot, where 4′-OHF and Ftot represent total (conjugated and unconjugated) amounts recovered in urine. Results There was a statistically significant relationship between the 4′-OHF formation clearance (4OHCLf) and both the 8-h FLRR and the 8-h FLMR with and without administration of fluconazole. The flurbiprofen apparent oral clearance (CL/F) was decreased by 53% [90% confidence interval (CI) −58, −48] and 64% (90% CI −69, −59), respectively, after administration of one and seven doses of fluconazole when compared with administration of flurbiprofen alone; similarly, the 4OHCLf decreased by 69% (90% CI −74, −64) and 78% (90% CI −83, −73), the 8-h FLRR decreased by 35% (90% CI −41, −29) and 40% (90% CI −46, −35) and the 8-h FLMR decreased by 61% (90% CI −65, −58) and 67% (90% CI −70, −63). The magnitude of decrease in CL/F and 4OHCLf was greater after seven doses compared with after one dose of fluconazole (P < 0.005). Conclusions This study provides strong evidence that both the 8-h FLRR and the 8-h FLMR are suitable phenotypic indices for CYP2C9 activity. PMID:17054666

  5. CYP2C9 variants as a risk modifier of NSAID-related gastrointestinal bleeding: a case–control study

    PubMed Central

    Estany-Gestal, Ana; Aguirre, Carmelo; Ruiz, Borja; Vidal, Xavier; Carvajal, Alfonso; Salado, Inés; Salgado-Barreira, Angel; Rodella, Luca; Moretti, Ugo; Ibáñez, Luisa

    2016-01-01

    Objective The aim of this study was to assess whether the CYP2C9*2 and/or *3 variants might modify the risk for NSAID-related upper gastrointestinal bleeding (UGIB) in NSAID users. Patients and methods We conducted a multicenter, case–control study in which cases were patients aged more than 18 years with a diagnosis of UGIB, and controls were matched (1 : 3) by sex, age, date of admission, and hospital. Exposure was defined as the mean number of defined daily doses (DDDs) of NSAIDs metabolized by CYP2C9 in the week preceding the index date. Three DDD categories were defined (0, ≤0.5, and >0.5). Exposure was constructed taking both NSAID use and CYP2C9 polymorphisms into account. Patients of non-European origin were excluded from the analysis. Results A total of 577 cases and 1343 controls were finally included in the analysis: 103 cases and 89 controls consumed NSAIDs metabolized by CYP2C9, and 88 cases and 177 controls were CYP2C9*3 carriers. The adjusted odds ratios (aORs) of UGIB associated with the CYP2C9*2 and wild-type alleles proved to be similar [OR=8.79 (4.50–17.17) and 10.15 (2.92–35.35), respectively] and lower than those of the CYP2C9*3 allele [aOR=18.07 (6.34–51.53)] for consumers taking more than 0.5 DDDs of NSAIDs metabolized by CYP2C9. Grouping genotypes into carriers and noncarriers of the CYP2C9*3 variant resulted in aORs of 16.92 (4.96–57.59) for carriers and 9.72 (4.55–20.76) for noncarriers, where DDDs were greater than 0.5. Conclusion The presence of the CYP2C9*3 variant increases the risk for UGIB associated with NSAID for DDDs greater than 0.5. The presence of the CYP2C9*2 allele shows no such effect. PMID:26544900

  6. Identification of CYP2C9 and VKORC1 polymorphisms in Iranian patients who are under warfarin therapy

    PubMed Central

    Poopak, Behzad; Rabieipoor, Saghar; Safari, Nazila; Naraghi, Emadedin; Sheikhsofla, Fatemeh; Khosravipoor, Gelareh

    2015-01-01

    Background: Although catalytic properties of different genetic polymorphisms of VKORC1 and CYP2C9 products have been identified, there is limited study available regarding warfarin dose requirement in Iranian patient population. This study investigates the impact of these polymorphisms on 115 patients, referred to Payvand Clinical and Specialty Laboratory for determining the appropriate dose of warfarin. Results of the study may be applicable to individuals who are under warfarin therapy to avoid warfarin resistance or intolerance. Subjects and Methods: PT-INR test was utilized as a screening method. Genotyping were performed for VKORC1 and CYP2C9 using PCR method. Statistical analyses including unpaired t-test or ANOVA and regression were done using SPSS. Results: VKORC1 GA was the most common genotype of VKORC1 allele among the study samples, with a rate of 57.4%. In CYP2C9 variant, 20% and 14.8% of subjects carried CYP2C9*1/*2 and CYP2C9*1/*3 genotyping, respectively. By contrast, the WT *1/*1 genotype was more abundant and dominant. The high frequency of VKORC1 (_1639) GA genotype (57.4%), was significant versus for the rest of the cohort (42.6%). In addition, a significant relationship was found between CYP2C9*1 and drug dose (P>0.021). Conclusion: In this study, samples were characterized by higher frequencies of CYP2C9*1 and VKORC1 G/A, determined as higher warfarin taking doses. The results showed a significant relationship of the VCORC1 and CYP2C9 polymorphisms with warfarin sensitivity and severe side effects. Estimating right doses of warfarin to prescribe can help to reduce the risk of over- or under-anticoagulation and subsequently, the risk of thromboembolism or bleeding. PMID:26865929

  7. In Silico Investigation of Cytochrome P450 2C9 in relation to Aging Using Traditional Chinese Medicine

    PubMed Central

    Hung, Tzu-Chieh; Kuo, Chia-Chen; Chen, Calvin Yu-Chian

    2014-01-01

    Cytochrome P450 2C9 (CYP2C9) metabolizes dehydroepiandrosterone-sulfate (DHEA-S), but in elderly people the amount of DHEA-S remaining after CYP2C9 metabolization may be insufficient for optimal health. A prediction model, molecular docking, and molecular dynamics were used to screen the Traditional Chinese Medicine (TCM) database to determine molecular compounds that may inhibit CYP2C9. The candidate compounds apocynoside(I), 4-methoxymagndialdehyde, and prunasin have higher Dock Scores, and prediction bioactivity than warfarin (the control drug). The interaction between 4-methoxymagndialdehyde and CYP2C9 is more intense than with other TCM compounds, but the simulation is longer. In these compounds, apocynoside(I) and prunasin have a greater number of pathways for their flexible structure, but these structures create weak interactions. These candidate compounds, which are known to have antioxidation and hypolipidemic functions that have an indirect effect on the aging process, can be extracted from traditional Chinese medicines. Thus, these candidate compounds may become CYP2C9 inhibitors and play an important role in providing optimal health in the elderly. PMID:24899908

  8. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  9. Cytochrome P450 2C9 Type II Binding Studies on Quinoline-4-carboxamide Analogs

    PubMed Central

    Peng, Chi-Chi; Cape, Jonathan L.; Rushmore, Tom; Crouch, Gregory J.; Jones, Jeffrey P.

    2009-01-01

    CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient pre-drug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogs to study the factors that determine the structure-activity relationships. The results of this study suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with the ferric heme iron, but this is not seen for the meta or ortho isomers. The π-cation interaction of the naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic, and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination with the heme iron. PMID:19053752

  10. Two Flavonolignans from Milk Thistle (Silybum marianum) Inhibit CYP2C9-Mediated Warfarin Metabolism at Clinically Achievable Concentrations

    PubMed Central

    Brantley, Scott J.; Oberlies, Nicholas H.; Kroll, David J.

    2010-01-01

    Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC50 of 8.2, 18, 74, and >100 μM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9*1 (6.7 versus 12 μM), rCYP2C9*2 (9.3 versus 19 μM), and rCYP2C9*3 (2.4 versus 9.3 μM). Using a matrix of five substrate (1–15 μM) and six inhibitor (1–80 μM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (Ki values of 4.8 and 10 μM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5–75 μM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction. PMID:19934397

  11. Two flavonolignans from milk thistle (Silybum marianum) inhibit CYP2C9-mediated warfarin metabolism at clinically achievable concentrations.

    PubMed

    Brantley, Scott J; Oberlies, Nicholas H; Kroll, David J; Paine, Mary F

    2010-03-01

    Milk thistle (Silybum marianum) is a popular herbal product used for hepatoprotection and chemoprevention. Two commercially available formulations are the crude extract, silymarin, and the semipurified product, silibinin. Silymarin consists of at least seven flavonolignans, of which the most prevalent are the diastereoisomers silybin A and silybin B; silibinin consists only of silybin A and silybin B. Based on a recent clinical study showing an interaction between a silymarin product and the CYP2C9 substrate losartan, the CYP2C9 inhibition properties of silybin A and silybin B and corresponding regioisomers, isosilybin A and isosilybin B, were evaluated using human liver microsomes (HLMs), recombinant CYP2C9 (rCYP2C9) enzymes, and the clinically relevant probe, (S)-warfarin. Silybin B was the most potent inhibitor in HLMs, followed by silybin A, isosilybin B, and isosilybin A (IC(50) of 8.2, 18, 74, and >100 microM, respectively). Next, silybin A and silybin B were selected for further characterization. As with HLMs, silybin B was more potent than silybin A toward rCYP2C9 1 (6.7 versus 12 microM), rCYP2C9 2 (9.3 versus 19 microM), and rCYP2C9 3 (2.4 versus 9.3 microM). Using a matrix of five substrate (1-15 microM) and six inhibitor (1-80 microM) concentrations and HLMs, both diastereoisomers inhibited (S)-warfarin 7-hydroxylation in a manner described best by a mixed-type inhibition model (K(i) values of 4.8 and 10 microM for silybin B and silybin A, respectively). These observations, combined with the high systemic silibinin concentrations (>5-75 microM) achieved in a phase I study involving prostate cancer patients, prompt clinical evaluation of a potential warfarin-milk thistle interaction. PMID:19934397

  12. Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping.

    PubMed

    Makia, Ngome L; Surapureddi, Sailesh; Monostory, Katalin; Prough, Russell A; Goldstein, Joyce A

    2014-08-01

    Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes. PMID:24830941

  13. CYP2C9 Promoter Variable Number Tandem Repeat Polymorphism Regulates mRNA Expression in Human Livers

    PubMed Central

    Sun, Xiaochun; Gong, Yan; Gawronski, Brian E.; Langaee, Taimour Y.; Shahin, Mohamed Hossam A.; Khalifa, Sherief I.; Johnson, Julie A.

    2012-01-01

    CYP2C9 is involved in metabolism of nearly 25% of clinically used drugs. Coding region polymorphisms CYP2C9*2 and *3 contribute to interperson variability in drug dosage and clinical outcomes, whereas the role of a regulatory polymorphism remains uncertain. Measuring allelic RNA expression in 87 human liver samples, combined with genotyping, sequencing, and reporter gene assays, we identified a promoter variable number tandem repeat polymorphism (pVNTR) that fully accounted for allelic CYP2C9 mRNA expression differences. Present in three different variant forms [short (pVNTR-S), medium (pVNTR-M), and long (pVNTR-L)], only the pVNTR-S allele reduced the CYP2C9 mRNA level compared with the pVNTR-M (reference) allele. pVNTR-S is in linkage disequilibrium with *3, with linkage disequilibrium r2 of 0.53 to 0.75 in different populations. In patients who were taking a maintenance dose of warfarin, the mean warfarin dose was associated with the copies of pVNTR-S (p = 0.0001). However, in multivariate regression models that included the CYP2C9*3, pVNTR-S was no longer a significant predictor of the warfarin dose (p = 0.60). These results indicate that although pVNTR-S reduced CYP2C9 mRNA expression, the in vivo effects of pVNTR-S on warfarin metabolism cannot be separated from the effects of *3. Therefore, it is not necessary to consider pVNTR-S as an additional biomarker for warfarin dosing. Larger clinical studies are needed to define whether the pVNTR-S has a minimal effect in vivo, or whether the effect attributed to *3 is really a combination of effects on expression by the pVNTR-S along with effects on catalytic activity from the nonsynonymous *3 variant. PMID:22289258

  14. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP2C9 and HLA-B Genotype and Phenytoin Dosing

    PubMed Central

    Caudle, Kelly E.; Rettie, Allan E.; Whirl-Carrillo, Michelle; Smith, Lisa H.; Mintzer, Scott E.; Lee, Ming Ta Michael; Klein, Teri E.; Callaghan, J. Thomas

    2014-01-01

    Phenytoin is a widely used antiepileptic drug with a narrow therapeutic index and large inter-patient variability partly due to genetic variations in CYP2C9. Furthermore, the variant allele HLA-B*15:02 is associated with an increased risk of Stevens-Johnson syndrome and toxic epidermal necrolysis in response to phenytoin treatment. We summarize evidence from the published literature supporting these associations and provide recommendations for the use of phenytoin based on CYP2C9 and/or HLA-B genotype (also available on PharmGKB: www.pharmgkb.org). PMID:25099164

  15. Phenytoin toxicity in two-month-old Thai infant with CYP2C9 gene polymorphism--A case report.

    PubMed

    Veeravigrom, Montida; Jaroonvanichkul, Vorapol; Netbaramee, Wiracha; Phaisarn, Pichaya; Uyathanarat, Thanita

    2016-01-01

    Phenytoin is one of the most well established and most effective antiepileptic medications for the treatment of focal seizures. In our clinical practice, it has proven difficult to maintain therapeutic phenytoin levels in infants less than three months of age. Incidence of phenytoin toxicity in infants is very rare. The cytochrome P450 super family plays an important role in phenytoin metabolism, especially CYP2C9 and CYP2C19. In this case report, we profiled a two-month-old Thai infant who developed phenytoin toxicity resulting from CYP2C9 gene polymorphism. PMID:25998968

  16. Effects of CYP3A4 polymorphisms on the plasma concentration of voriconazole.

    PubMed

    He, H-R; Sun, J-Y; Ren, X-D; Wang, T-T; Zhai, Y-J; Chen, S-Y; Dong, Y-L; Lu, J

    2015-04-01

    Voriconazole is frequently utilized for the prevention and treatment of invasive fungal infections (IFIs), and is extensively metabolized by the cytochrome P450 (CYP) system. The impact of activity of the genes encoding CYP3A4, CYP3A5, and CYP2C9 on the pharmacokinetics of voriconazole cannot be ignored because, second to CYP2C19, they are the most important enzymes involved in voriconazole metabolism. The influence of genetic polymorphisms in CYP3A4, CYP3A5, and CYP2C9 on the plasma concentrations of voriconazole was evaluated in the present study. The study cohort comprised 158 patients with IFIs in whom 22 single-nucleotide polymorphisms (SNPs) in CYP3A4, CYP3A5, and CYP2C9 were genotyped using the Sequenom MassARRAY RS1000 system, and voriconazole plasma concentrations were measured by high-performance liquid chromatography (HPLC). 40, 91, and 27 patients presented with low (<1 mg/L), normal (1-4 mg/L), and high (>4 mg/L) plasma voriconazole concentrations, respectively. Correlation analysis between polymorphisms and the plasma voriconazole concentration revealed an association between the presence of the rs4646437 T allele and a higher plasma voriconazole concentration [p = 0.033, odds ratio (OR) = 2.832, 95% confidence interval (CI) = 1.086-7.384]. This study has identified a new SNP related to the metabolism of voriconazole, potentially providing novel insight into the influence of CYP3A4 on the pharmacokinetics of this antifungal agent. PMID:25515945

  17. Stereoselective glucuronidation and hydroxylation of etodolac by UGT1A9 and CYP2C9 in man.

    PubMed

    Tougou, K; Gotou, H; Ohno, Y; Nakamura, A

    2004-05-01

    1. In vitro metabolic studies with etodolac were performed. S- and R-etodolac were converted to the acylglucuronide and hydroxylated metabolites by UDP-glucuronosyltransferase (UGT) and cytochrome P450 in microsomes. However, the stereoselectivities of UGT and P450 for the isomers were opposite. S-etodolac was glucuronidated preferentially than R-etodolac by UGT. In contrast, R-etodolac was hydroxylated preferentially than S-etodolac by P450. 2. Of several human P450 enzymes, CYP2C9 had the greatest activity for hydroxylation of R-etodolac. Sulfaphenazole, an inhibitor of CYP2C9, and anti-CYP2C9 antibody inhibited the hydroxylation of R-etodolac in human liver microsomes. CYP2C9 therefore contributes to the stereoselective hydroxylation of R-etodolac. 3. Of several human UGT enzymes, UGT1A9 had the greatest activity for glucuronidation of S-etodolac. Propofol and thyroxine, inhibitors of UGT1A9, inhibited the glucuronidation of S-etodolac in human liver microsomes. Therefore, UGT1A9 is mainly responsible for the stereoselective glucuronidation of S-etodolac. 4. Because S-etodolac was metabolized more rapidly than R-etodolac in human cryopreserved hepatocytes, the stereoselectivities of UGT1A9 for etodolac substantially influenced the overall metabolism of S- and R-etodolac in man. PMID:15370961

  18. Investigating the Role of Plasma Glucose Concentration as a Phenotypic Marker for CYP2C9 Genetic Variants, in the Diabetic Population of Gujarat.

    PubMed

    Bhatt, D; Chauhan, N; Sharma, A; Dhawan, D; Bhatt, R V; Phatak, S; Padh, H

    2014-01-01

    The present study was aimed to investigate the role of plasma glucose concentration as a phenotypic marker and to study the frequency distribution of CYP2C9 genetic variants in Gujarat state diabetic population. One hundred and nine unrelated diabetes mellitus patients treated with sulfonylureas were genotyped for CYP2C9*2 and CYP2C9*3 alleles. Their pre- and posttreatment postprandial blood glucose levels were recorded and mean glucose drop per milligram of drug values were calculated and further used as an index for phenotypic correlation. The frequencies of CYP2C9*1, CYP2C9*2 and CYP2C9*3 alleles in the Gujarat state diabetic population were 0.84, 0.07 and 0.09, respectively. The distribution of CYP2C9*1/*1, CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes were 0.73, 0.08, 0.13, 0.0, 0.06 and 0.0, respectively. Patients with CYP2C9*1/*2 genotype did not show any significant difference in the mean glucose drop per milligram of drug values when compared with wild-type patients in glipizide-treatment group. Patients with CYP2C9*1/*3 genotype showed greater mean glucose drop per milligram of drug values than patients with CYP2C9*1/*1 wild-type genotype for both glipizide and glimepiride while patients with CYP2C9*2/*3 genotype showed greater drop than patients with CYP2C9*1/*1 genotype only in the glipizide-treatment group. The presence of CYP2C9*3 allele significantly affected plasma glucose drop per milligram of drug values in patients taking glipizide and glimepiride, while effects of CYP2C9*2 allele were insignificant. Further studies are needed to confirm the effects of CYP2C9*2 allele on plasma glucose drop per milligram of drug values. However, plasma glucose concentration is a complex physiological marker that cannot be used to establish perfect genotype-phenotype correlation. Hence studies exploring robust phenotypic markers must be initiated. PMID:24799741

  19. Differences in CYP2C9 Genotype and Enzyme Activity Between Swedes and Koreans of Relevance for Personalized Medicine: Role of Ethnicity, Genotype, Smoking, Age, and Sex.

    PubMed

    Hatta, Fazleen H M; Lundblad, Mia; Ramsjo, Margareta; Kang, Ju-Hee; Roh, Hyung-Keun; Bertilsson, Leif; Eliasson, Erik; Aklillu, Eleni

    2015-06-01

    Global personalized medicine demands the characterization of person-to-person and between-population differences in drug pharmacokinetics and pharmacodynamics. CYP2C9 pharmacokinetic pathway is subject to modulation by both genetic and environmental factors. CYP2C9 genotype-based dose recommendations (e.g., for warfarin) is advocated. However, the overall contribution of genotype for variation in enzyme activity may differ between populations. We evaluated the importance of ethnicity, genotype, smoking, body weight, age, and sex for CYP2C9 enzyme activity. CYP2C9 genotype and phenotype was determined in 148 Swedes and 146 Koreans using losartan as a probe. CYP2C9 enzyme activity was assessed using urinary losartan/metabolite E-3174 ratio. The frequency of CYP2C9 defective variant alleles (*2 and *3) was significantly higher in Swedes (10.8% and 12.5%) than in Koreans (0% and 5.8%). In matched genotypes, CYP2C9 enzyme activity was significantly lower in Swedes compared to Koreans (p<0.0001). In a univariate analysis, age, weight, ethnicity, genotype, and smoking were significant predictors of CYP2C9 phenotype. A stepwise multivariate analysis indicated ethnicity, genotype, and smoking remained as significant predictors of CYP2C9 enzyme activity, accounting for 50% of the total variance. In both study populations, CYP2C9 genotype was a significant predictor of CYP2C9 enzyme activity, but its contribution in explaining the total variance was lower in Koreans (26.6%) than Swedes (40%). In conclusion, we report significantly lower CYP2C9 enzyme activity in Swedes compared to Koreans, partly but not exclusively due to CYP2C9 pharmacogenetic variations. Ethnicity and environment factors need to be considered together with genotype for population-specific dose optimization and global personalized medicine. PMID:25977991

  20. Inhibition of Recombinant Cytochrome P450 Isoforms 2D6 and 2C9 by Diverse Drug-like Molecules

    PubMed Central

    McMasters, Daniel R.; Torres, Rhonda A.; Crathern, Susan J.; Dooney, Deborah L.; Nachbar, Robert B.; Sheridan, Robert P.; Korzekwa, Kenneth R.

    2008-01-01

    The affinities of a diverse set of 500 drug-like molecules to cytochrome P450 isoforms 2C9 and 2D6 were measured using recombinant expressed enzyme. The dose–response curve of each compound was fitted with a series of equations representing typical or various types of atypical kinetics. Atypical kinetics was identified where the Akaike Information Criterion, plus other criteria, suggested the kinetics was more complex than expected for a Michaelis–Menten model. Approximately 20% of the compounds were excluded due to poor solubility, and approximately 15% were excluded due to fluorescence interference. Of the remaining compounds, roughly half were observed to bind with an affinity of 200 μM or lower for each of the two isoforms. Atypical kinetics were observed in 18 percent of the compounds that bind to cytochrome 2C9 but less than 2 percent for 2D6. The resulting collection of competitive inhibitors and inactive compounds was analyzed for trends in binding affinity. For CYP2D6, a clear relationship between polar surface area and charge was observed, with the most potent inhibitors having a formal positive charge and a low percent polar surface area. For CYP2C9, no clear trend between activity and physicochemical properties could be seen for the group as a whole; however, certain classes of compounds have altered frequencies of activity and atypical kinetics. PMID:17559204

  1. Association analysis of CYP2C9*3 and phenytoin-induced severe cutaneous adverse reactions (SCARs) in Thai epilepsy children.

    PubMed

    Suvichapanich, Supharat; Jittikoon, Jiraphun; Wichukchinda, Nuanjun; Kamchaisatian, Wasu; Visudtibhan, Anannit; Benjapopitak, Suwat; Nakornchai, Somjai; Manuyakorn, Wiparat; Mahasirimongkol, Surakameth

    2015-08-01

    CYP2C9 is the key enzyme in aromatic antiepileptic drugs (AEDs) metabolism. CYP2C9*3 is a loss of function polymorphism. This study was designed to investigate genetic association between CYP2C9*3 and aromatic AED-induced severe cutaneous adverse reactions (SCARs) in Thai children. The 37 aromatic AED-induced SCARs patients (20 phenobarbital and 17 phenytoin) and 35 tolerances (19 phenobarbital and 16 phenytoin) were enrolled. CYP2C9*3 was genotyped by allele-specific PCRs. The association between CYP2C9*3 with phenytoin-induced SCARs and phenobarbital-induced SCARs were analyzed in comparison with tolerances and healthy samples. Significant association between phenytoin-induced SCARs and CYP2C9*3 was discovered (odds ratio=14.52; 95% confidence interval (CI)=1.18-∞, P-value=0.044). CYP2C9*3 was not associated with phenobarbital-induced SCARs. This study is the first report of CYP2C9*3 association to phenytoin-induced SCARs in Thai epileptic children. The CYP2C9*3 is a reasonable predictive genetic marker to anticipate SCARs from phenytoin. PMID:25994870

  2. Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

    PubMed

    Wang, Zhe; Sun, Wei; Huang, Cheng-Ke; Wang, Li; Xia, Meng-Ming; Cui, Xiao; Hu, Guo-Xin; Wang, Zeng-Shou

    2015-04-01

    Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment. PMID:24517573

  3. Tecarfarin, a novel vitamin K reductase antagonist, is not affected by CYP2C9 and CYP3A4 inhibition following concomitant administration of fluconazole in healthy participants.

    PubMed

    Bavisotto, Linda M; Ellis, David J; Milner, Peter G; Combs, Daniel L; Irwin, Ian; Canafax, Daniel M

    2011-04-01

    Comparative pharmacokinetics of vitamin K epoxide reductase antagonists tecarfarin and warfarin were assessed before and after coadministration for 21 days of the CYP450 inhibitor fluconazole in a randomized, open-label, single-center drug interaction study. Twenty healthy adult participants were randomized 1:1 to receive approximately equipotent single oral doses of tecarfarin (50 mg) or warfarin (17.5 mg). Following 7 days of baseline serial blood level collections, each participant received oral fluconazole 400 mg daily for 21 days. A second identical single oral dose of tecarfarin or warfarin was given 14 days after starting fluconazole with serial pharmacokinetic sampling. Key pharmacokinetic parameters C(max), t(max), AUC(0-168), apparent clearance, and t(1/2) demonstrated no tecarfarin-fluconazole interaction but a strong warfarin-fluconazole interaction. The ratio of log-transformed mean AUC(0-168) with versus without fluconazole for tecarfarin was 91.2% (90% confidence interval [CI]: 83.3-99.8) and for racemic warfarin was 213% (90% CI: 202-226). The 90% CI was entirely within the standard 80% to 125% bioequivalence interval for tecarfarin but well outside the bioequivalence interval for warfarin, confirming a clinically significant pharmacokinetic interaction between warfarin and fluconazole. In contrast, tecarfarin pharmacokinetics were apparently unchanged by fluconazole. PMID:20622200

  4. Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping

    PubMed Central

    Swar, Balkrishna D.; Bendkhale, Shital R.; Rupawala, Abbas; Sridharan, Kannan; Gogtay, Nithya J.; Thatte, Urmila M.; Kshirsagar, Nilima A.

    2016-01-01

    Objectives: Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug. Materials and Methods: A 100 mg FLB capsule was administered to 298 healthy adult participants. Venous blood samples were analyzed at 2 h postdose for the estimation of FLB and 4-hydroxy FLB. Metabolic ratio (MR) was calculated to determine the extent of poor metabolizer (PM) and rapid metabolizer status using probit plot. Genotyping of CYP2C9 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism technique. Results: Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed. Conclusion: To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients. PMID:27298492

  5. Baseline blood flow and bradykinin-induced vasodilator responses in the human forearm are insensitive to the cytochrome P450 2C9 (CYP2C9) inhibitor sulphaphenazole.

    PubMed

    Passauer, Jens; Büssemaker, Eckhart; Lässig, Grit; Pistrosch, Frank; Fauler, Joachim; Gross, Peter; Fleming, Ingrid

    2003-10-01

    A substantial portion of the vasodilator response elicited by bradykinin in the human forearm is unaffected by the combined inhibition of nitric oxide (NO) synthases and cyclo-oxygenases. The cytochrome P450 (CYP) 2C9 inhibitor sulphaphenazole was recently identified as a potent inhibitor of NO- and prostacyclin (PGI2)-independent relaxation in porcine coronary arteries. The aim of the present study was to determine the effect of sulphaphenazole on basal and bradykinin-induced NO/PGI2-independent changes in the forearm blood flow (FBF) of healthy subjects. Eleven healthy male volunteers participated in this placebo-controlled study. Test agents were infused into the brachial artery and FBF was measured by bilateral venous occlusion plethysmography. Sulphaphenazole (0.02-2 mg/min) alone did not affect basal blood flow. Inhibition of the NO synthases by NG-monomethyl-L-arginine (L-NMMA; 4 micromol/min) and cyclo-oxygenases by ibuprofen (1200 mg, orally) reduced FBF to 48 +/- 7% in the absence and 50 +/- 8% in the presence of sulphaphenazole (2 mg/min; P=not significant). After pretreatment with L-NMMA (16 micromol/min) and ibuprofen (1200 mg, orally), sulphaphenazole (6 mg/min) did not substantially inhibit bradykinin-induced vasodilation. We conclude that CYP2C9-derived metabolites (i) are not involved in the regulation of baseline blood flow, and (ii) do not mediate bradykinin-induced NO/PGI2-independent vasorelaxation in the human forearm. However, determining the contribution of this enzyme to regulation of blood flow in pathological conditions associated with endothelial dysfunction requires further studies. PMID:12826020

  6. Calculating the properties of C2H2-C9H16 alkynes, based on the additivity of energy contributions

    NASA Astrophysics Data System (ADS)

    Smolyakov, V. M.; Grebeshkov, V. V.

    2015-05-01

    A ten-constant additive model is obtained for calculating the physicochemical properties of a number of C n H2 n-2 alkynes, based on the group additivity method (with allowance for the initial atomic environment), two topological indices that allow for the second atomic environment, and pairwise non-valence interactions (in implicit form) between three atoms, four atoms, and so forth along the chain of a molecule. Two linear dependences are revealed. The obtained formula is used for numerical calculations of the normal heats of vaporization L NBT and normal boiling temperatures T b of C2H2-C9H16 alkynes, neither of which had been studied experimentally.

  7. Pharmacogenetic evaluation of ABCB1, Cyp2C9, Cyp2C19 and methylene tetrahydrofolate reductase polymorphisms in teratogenicity of anti-epileptic drugs in women with epilepsy

    PubMed Central

    Jose, Manna; Banerjee, Moinak; Mathew, Anila; Bharadwaj, Tashi; Vijayan, Neetha; Thomas, Sanjeev V.

    2014-01-01

    Aim: Pregnancy in women with epilepsy (WWE) who are on anti-epileptic drugs (AEDs) has two- to three-fold increased risk of fetal malformations. AEDs are mostly metabolized by Cyp2C9, Cyp2C19 and Cyp3A4 and transported by ABCB1. Patients on AED therapy can have folate deficiency. We hypothesize that the polymorphisms in ABCB1, Cyp2C9, Cyp2C19 and methylene tetrahydrofolate reductase (MTHFR) might result in differential expression resulting in differential drug transport, drug metabolism and folate metabolism, which in turn may contribute to the teratogenic impact of AEDs. Materials and Methods: The ABCB1, Cyp2C9, Cyp2C19 and MTHFR polymorphisms were genotyped for their role in teratogenic potential and the nature of teratogenecity in response to AED treatment in WWE. The allelic, genotypic associations were tested in 266 WWE comprising of 143 WWE who had given birth to babies with WWE-malformation (WWE-M) and 123 WWE who had normal offsprings (WWE-N). Results: In WWE-M, CC genotype of Ex07 + 139C/T was overrepresented (P = 0.0032) whereas the poor metabolizer allele *2 and *2 *2 genotype of CYP2C219 was significantly higher in comparison to WWE-N group (P = 0.007 and P = 0.005, respectively). All these observations were independent of the nature of malformation (cardiac vs. non cardiac malformations). Conclusion: Our study indicates the possibility that ABCB1 and Cyp2C19 may play a pivotal role in the AED induced teratogenesis, which is independent of nature of malformation. This is one of the first reports indicating the pharmacogenetic role of Cyp2C19 and ABCB1 in teratogenesis of AED in pregnant WWE. PMID:25221392

  8. Comparison in the in vitro inhibitory effects of major phytocannabinoids and polycyclic aromatic hydrocarbons contained in marijuana smoke on cytochrome P450 2C9 activity.

    PubMed

    Yamaori, Satoshi; Koeda, Kyoko; Kushihara, Mika; Hada, Yui; Yamamoto, Ikuo; Watanabe, Kazuhito

    2012-01-01

    Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, and polycyclic aromatic hydrocarbons (PAHs) contained in marijuana smoke on catalytic activity of human cytochrome P450 (CYP) 2C9 were investigated. These phytocannabinoids concentration-dependently inhibited S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of human liver microsomes (HLMs) and recombinant CYP2C9 (rCYP2C9). In contrast, none of the twelve PAHs including benz[a]anthracene and benzo[a]pyrene exerted substantial inhibition (IC₅₀ > 10 µM). The inhibitory potentials of Δ⁹-THC (Ki = 0.937-1.50 µM) and CBN (Ki = 0.882-1.29 µM) were almost equivalent regardless of the enzyme sources used, whereas the inhibitory potency of CBD (Ki > = 0.954-9.88 µM) varied depending on the enzyme sources and substrates used. Δ⁹-THC inhibited both S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of HLMs and rCYP2C9 in a mixed manner. CBD and CBN competitively inhibited the activities of HLMs and rCYP2C9, with the only notable difference being that CBD and CBN exhibited mixed-type inhibitions against diclofenac 4'-hydroxylation and S-warfarin 7-hydroxylation, respectively, by rCYP2C9. None of Δ⁹-THC, CBD, and CBN exerted metabolism-dependent inhibition. These results indicated that the three major phytocannabinoids but not PAHs contained in marijuana smoke potently inhibited CYP2C9 activity and that these cannabinoids can be characterized as direct inhibitors for CYP2C9. PMID:22166891

  9. In Vitro and in Vivo Inhibitory Effects of Glycyrrhetinic Acid in Mice and Human Cytochrome P450 3A4

    PubMed Central

    Lv, Qiao-Li; Wang, Gui-Hua; Chen, Shu-Hui; Hu, Lei; Zhang, Xue; Ying, Guo; Qin, Chong-Zhen; Zhou, Hong-Hao

    2015-01-01

    Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC50 values of 8.195 μM in HLMs and 7.498 μM in the recombinant cDNA-expressed CYP3A4 enzyme system, respectively. It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4. PMID:26712778

  10. Genetic polymorphisms of CYP2C9 and CYP2C19 are not related to drug-induced idiosyncratic liver injury (DILI)

    PubMed Central

    Pachkoria, K; Lucena, M I; Ruiz-Cabello, F; Crespo, E; Cabello, M R; Andrade, R J

    2007-01-01

    Background and purpose: The general view on the pathogenesis of drug-induced idiosyncratic liver injury (DILI) is that parent compounds are rendered hepatotoxic by metabolism, mainly by cytochrome (CYP) 450, although other metabolic pathways can contribute. Anecdotal reports suggest a role of CYP 450 polymorphisms in DILI. We aimed to assess in a series of Spanish DILI patients the prevalence of important allelic variants of CYP2C9 and CYP2C19, known to be involved in the metabolism of several hepatotoxic drugs. Experimental approach: Genotyping of CYP2C9 (*2, *3) and CYP2C19 (*2 and *3), was carried out in a total of 28 and 32 patients with a well established diagnosis of DILI. CYP2C9 and CYP2C19 variants were analysed in genomic DNA by means of PCR-FRET and compared with previous findings in other Caucasian populations. Key results: CYP2C9 and CYP2C19 allele and genotype frequencies were in agreement with Hardy-Weinberg equilibrium. Fourteen patients (50%) were heterozygous and 1(4%) found to be compound heterozygous for the CYP2C9 allele. Seven (22%) were found to carry one and 1(3%) carried two CYP2C19 mutated alleles. No patients were homozygous for *3 allele. The distribution of both CYP2C9 and CYP2C19 allelic variants in DILI patients were similar to those in other Caucasian populations. Patients with variant and those with wild-type alleles did not differ in regard to clinical presentation of DILI, type of injury and outcome. Conclusions and Implications: We find no evidence to support CYP2C9 and CYP2C19 genetic polymorphisms as predictable potential risk factors for DILI. PMID:17279092

  11. The Role of CYP2C8 and CYP2C9 Genotypes in Losartan-Dependent Inhibition of Paclitaxel Metabolism in Human Liver Microsomes.

    PubMed

    Mukai, Yuji; Senda, Asuna; Toda, Takaki; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2016-06-01

    The aim of the present study was to further investigate a previously identified metabolic interaction between losartan and paclitaxel, which is one of the marker substrates of CYP2C8, by using human liver microsomes (HLMs) from donors with different CYP2C8 and CYP2C9 genotypes. Although CYP2C8 and CYP2C9 exhibit genetic linkage, previous studies have yet to determine whether losartan or its active metabolite, EXP-3174 which is specifically generated by CYP2C9, is responsible for CYP2C8 inhibition. Concentrations of 6α-hydroxypaclitaxel and EXP-3174 were measured by high-performance liquid chromatography after incubations with paclitaxel, losartan or EXP-3174 in HLMs from seven donors with different CYP2C8 and CYP2C9 genotypes. The half maximal inhibitory concentration (IC50 ) values were not fully dependent on CYP2C8 genotypes. Although the degree of inhibition was small, losartan significantly inhibited the production of 6α-hydroxypaclitaxel at a concentration of 1 μmol/L in only HL20 with the CYP2C8*3/*3 genotype. HLMs with either CYP2C9*2/*2 or CYP2C9*1/*3 exhibited a lower losartan intrinsic clearance (Vmax /Km ) than other HLMs including those with CYP2C9*1/*1 and CYP2C9*1/*2. Significant inhibition of 6α-hydroxypaclitaxel formation by EXP-3174 could only be found at levels that were 50 times higher (100 μmol/L) than the maximum concentration generated in the inhibition study using losartan. These results suggest that the metabolic interaction between losartan and paclitaxel is dependent on losartan itself rather than its metabolite and that the CYP2C8 inhibition by losartan is not affected by the CYP2C9 genotype. Further study is needed to define the effect of CYP2C8 genotypes on losartan-paclitaxel interaction. PMID:26551762

  12. Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

    PubMed

    Prasad, G Shyam; Srisailam, K; Sashidhar, R B

    2016-01-01

    Specific inhibitors of Cytochrome P4502C9 enzyme (CYP2C9) viz. clopidogrel, fenofibrate fluvoxamine and sertraline at concentration of 50, 100, 150 and 200 µM were employed to investigate the nature of enzyme involved in bioconversion of meloxicam to its main metabolite 5-OH methyl meloxicam by Cunninghamella blakesleeana. Virtual screening for interaction of specific CYP2C9 inhibitors with human CYP2C9 enzyme was performed by molecular docking using Auto dock vina 4.2 version. The in silico studies were further substantiated by in vitro studies, which indicated fenofibrate to be a potent inhibitor of CYP2C9 enzyme followed by sertraline, clopidogrel and fluvoxamine, respectively. Two-stage fermentation protocol was followed to study metabolism of meloxicam and its inhibition by different CYP2C9 inhibitors. Meloxicam metabolites were identified using HPLC, LC-MS analysis and based on previous reports, as 5-OH methyl meloxicam (M1), 5-carboxy meloxicam (M2) and an unidentified metabolite (M3). All the inhibitors tested in the study showed a clear concentration dependent inhibition of meloxicam metabolism. The results suggest that the enzymes involved in metabolism of meloxicam in C. blakesleeana are akin to mammalian metabolism. Hence, C. blakesleeana can be used as a model organism in studying drug interactions and also in predicting mammalian drug metabolism. PMID:27026863

  13. CYP2C9, CYP2C19, ABCB1 genetic polymorphisms and phenytoin plasma concentrations in Mexican-Mestizo patients with epilepsy.

    PubMed

    Ortega-Vázquez, A; Dorado, P; Fricke-Galindo, I; Jung-Cook, H; Monroy-Jaramillo, N; Martínez-Juárez, I E; Familiar-López, I; Peñas-Lledó, E; LLerena, A; López-López, M

    2016-06-01

    We aimed to explore the possible influence of CYP2C9 (*2, *3 and IVS8-109 A>T), CYP2C19 (*2, *3 and *17) and ABCB1 (1236C>T, 2677G>A/T and 3435C>T) on phenytoin (PHT) plasma concentrations in 64 Mexican Mestizo (MM) patients with epilepsy currently treated with PHT in mono- (n=25) and polytherapy (n=39). Genotype and allele frequencies of these variants were also estimated in 300 MM healthy volunteers. Linear regression models were used to assess associations between the dependent variables (PHT plasma concentration and dose-corrected PHT concentration) with independent variables (CYP2C9, CYP2C19 and ABCB1 genotypes, ABCB1 haplotypes, age, sex, weight, and polytherapy). In multivariate models, CYP2C9 IVS8-109 T was significantly associated with higher PHT plasma concentrations (t(64)=2.27; P=0.03). Moreover, this allele was more frequent in the supratherapeutic group as compared with the subtherapeutic group (0.13 versus 0.03, respectively; P=0.05, Fisher's exact test). Results suggest that CYP2C9 IVS8-109 T allele may decrease CYP2C9 enzymatic activity on PHT. More research is needed to confirm findings. PMID:26122019

  14. CYP2C9 and VKORC1 Genotypes in Puerto Ricans: A Case for Admixture-Matching in Clinical Pharmacogenetic Studies

    PubMed Central

    Villagra, David; Duconge, Jorge; Windemuth, Andreas; Cadilla, Carmen L; Kocherla, Mohan; Gorowski, Krystyna; Bogaard, Kali; Renta, Jessica Y; Cruz, Irelys A; Mirabal, Sara; Seip, Richard L; Ruaño, Gualberto

    2010-01-01

    Backgrounds Admixture is of great relevance to the clinical application of pharmacogenetics and personalized medicine, but unfortunately these studies have been scarce in Puerto Ricans. Besides, allele frequencies for clinically relevant genetic markers in warfarin response (i.e., CYP2C9 and VKORC1) have not yet been fully characterized in this population. Accordingly, this study is aimed at investigating whether a correlation between overall genetic similarity and CYP2C9 and/or VKORC1 genotypes could be established. Methods 98 DNA samples from Puerto Ricans were genotyped for major CYP2C9 and VKORC1 polymorphisms and tested on a physiogenomic (PG)-array to infer population structure and admixture pattern. Results Analysis affirmed that Puerto Ricans are broadly admixed. A genetic distance dendrogram was constructed by clustering those subjects with similar genetic profiles. Individual VKORC1 and CYP2C9 genotypes were visually overlaid atop the three dendrogram sectors. Sector-1, representing Amerindian ancestry, showed higher VKORC1-1639G>A variant frequency than the rest of the population (p=0.051). Although CYP2C9*3 allele frequencies matched the expected HapMap values, admixture may explain deviations from published findings regarding VKORC1-1639G>A and CYP2C9*2 allele frequencies in sector-3. Conclusions Results suggest that the observed inter-individual variations in ancestral contributions have significant implications for the way each Puerto Rican responds to warfarin therapy. Our findings provide valuable evidence on the importance of controlling for admixture in pharmacogenetic studies of Puerto Rican Hispanics. PMID:20488169

  15. Warfarin dose requirement in Turkish patients: the influences of patient characteristics and polymorphisms in CYP2C9, VKORC1 and factor VII

    PubMed Central

    Yildirim, E; Erol, K; Birdane, A

    2014-01-01

    Background: To determine the contribution of cytochrome P4502C9 (CYP2C9), vitamin K epoxide reductase (VKORC1) and factor VII genotypes, age, body mass index (BMI), international normalized ratio (INR) and other individual patient characteristics on warfarin dose requirements in an adult Turkish population. Methods: Blood samples were collected from 101 Turkish patients. Genetic analyses for CYP2C9*2 and *3, VKORC1 -1639 G>A and factor VII -401 G>T polymorphisms were performed. Age, INR, BMI values and other individual patient characteristics were also recorded. Results: The mean daily warfarin dosage was significantly higher in patients with the CYP2C9*1/*1 genotype than in the CYP2C9*2/*2 and CYP2C9*1/*3 groups (p ≤ 0.05). With respect to the VKORC1 -1639 G>A polymorphism, the mean warfarin daily dose requirement was higher in the wild type group compared to the heterozygous group (p≤0.001). The mean daily dose requirement for patients with the GG form of factor VII was significantly higher than that of patients with the TT genotype (p ≤ 0.05). Age, gender, BMI, INR had no statistically significant correlation with warfarin dose (p ≥ 0.05). Conclusions: Polymorphisms in CYP2C9, VKORC1 and factor VII did partially affect daily warfarin dose requirements, while age, gender, BMI and INR do not. However, further case-control studies with a larger study size and different genetic loci are needed to confirm our study. PMID:26052198

  16. Cytochrome P-450 2C9 exerts a vasoconstrictor influence on coronary resistance vessels in swine at rest and during exercise.

    PubMed

    Zhou, Zhichao; Hemradj, Veemal; de Beer, Vincent J; Gao, Fen; Hoekstra, Maaike; Merkus, Daphne; Duncker, Dirk J

    2012-04-15

    A significant endothelium-dependent vasodilation persists after inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the coronary vasculature, which has been linked to the activation of cytochrome P-450 (CYP) epoxygenases expressed in endothelial cells and subsequent generation of vasodilator epoxyeicosatrienoic acids. Here, we investigated the contribution of CYP 2C9 metabolites to regulation of porcine coronary vasomotor tone in vivo and in vitro. Twenty-six swine were chronically instrumented. Inhibition of CYP 2C9 with sulfaphenazole (5 mg/kg iv) alone had no effect on bradykinin-induced endothelium-dependent coronary vasodilation in vivo but slightly attenuated bradykinin-induced vasodilation in the presence of combined NOS/COX blockade with N(ω)-nitro-L-arginine (20 mg/kg iv) and indomethacin (10 mg/kg iv). Sulfaphenazole had minimal effects on coronary resistance vessel tone at rest or during exercise. Surprisingly, in the presence of combined NOS/COX blockade, a significant coronary vasodilator response to sulfaphenzole became apparent, both at rest and during exercise. Subsequently, we investigated in isolated porcine coronary small arteries (∼250 μm) the possible involvement of reactive oxygen species (ROS) in the paradoxical vasoconstrictor influence of CYP 2C9 activity. The vasodilation by bradykinin in vitro in the presence of NOS/COX blockade was markedly potentiated by sulfaphenazole under control conditions but not in the presence of the ROS scavenger N-(2-mercaptoproprionyl)-glycine. In conclusion, CYP 2C9 can produce both vasoconstrictor and vasodilator metabolites. Production of these metabolites is enhanced by combined NOS/COX blockade and is critically dependent on the experimental conditions. Thus production of vasoconstrictors slightly outweighed the production of vasodilators at rest and during exercise. Pharmacological stimulation with bradykinin resulted in vasodilator CYP 2C9 metabolite production when administered in

  17. CYP2C9 Genotype vs. Metabolic Phenotype for Individual Drug Dosing—A Correlation Analysis Using Flurbiprofen as Probe Drug

    PubMed Central

    Vogl, Silvia; Lutz, Roman W.; Schönfelder, Gilbert; Lutz, Werner K.

    2015-01-01

    Currently, genotyping of patients for polymorphic enzymes responsible for metabolic elimination is considered a possibility to adjust drug dose levels. For a patient to profit from this procedure, the interindividual differences in drug metabolism within one genotype should be smaller than those between different genotypes. We studied a large cohort of healthy young adults (283 subjects), correlating their CYP2C9 genotype to a simple phenotyping metric, using flurbiprofen as probe drug. Genotyping was conducted for CYP2C9*1, *2, *3. The urinary metabolic ratio MR (concentration of CYP2C9-dependent metabolite divided by concentration of flurbiprofen) determined two hours after flurbiprofen (8.75 mg) administration served as phenotyping metric. Linear statistical models correlating genotype and phenotype provided highly significant allele-specific MR estimates of 0.596 for the wild type allele CYP2C9*1, 0.405 for CYP2C9*2 (68 % of wild type), and 0.113 for CYP2C9*3 (19 % of wild type). If these estimates were used for flurbiprofen dose adjustment, taking 100 % for genotype *1/*1, an average reduction to 84 %, 60 %, 68 %, 43 %, and 19 % would result for genotype *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3, respectively. Due to the large individual variation within genotypes with coefficients of variation ≥ 20 % and supposing the normal distribution, one in three individuals would be out of the average optimum dose by more than 20 %, one in 20 would be 40 % off. Whether this problem also applies to other CYPs and other drugs has to be investigated case by case. Our data for the given example, however, puts the benefit of individual drug dosing to question, if it is exclusively based on genotype. PMID:25775139

  18. 22 CFR 3a.4 - Procedure for requesting approval.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 22 Foreign Relations 1 2012-04-01 2012-04-01 false Procedure for requesting approval. 3a.4 Section 3a.4 Foreign Relations DEPARTMENT OF STATE GENERAL ACCEPTANCE OF EMPLOYMENT FROM FOREIGN GOVERNMENTS BY MEMBERS OF THE UNIFORMED SERVICES § 3a.4 Procedure for requesting approval. (a) An applicant...

  19. Warfarin Dosing in a Patient with CYP2C9(∗)3(∗)3 and VKORC1-1639 AA Genotypes.

    PubMed

    Johnson, Mark; Richard, Craig; Bogdan, Renee; Kidd, Robert

    2014-01-01

    Genetic factors most correlated with warfarin dose requirements are variations in the genes encoding the enzymes cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKOR). Patients receiving warfarin who possess one or more genetic variations in CYP2C9 and VKORC1 are at increased risk of adverse drug events and require significant dose reductions to achieve a therapeutic international normalized ratio (INR). A 74-year-old white female with atrial fibrillation was initiated on a warfarin dose of 2 mg PO daily, which resulted in multiple elevated INR measurements and three clinically significant hemorrhagic events and four vitamin K antidote treatments over a period of less than two weeks. Genetic analysis later revealed that she had the homozygous variant genotypes of CYP2C9∗3∗3 and VKORC1-1639 AA. Warfarin dosing was subsequently restarted and stabilized at 0.5 mg PO daily with therapeutic INRs. This is the first case report of a white female with these genotypes stabilized on warfarin, and it highlights the value of pharmacogenetic testing prior to the initiation of warfarin therapy to maximize efficacy and minimize the risk of adverse drug events. PMID:24627811

  20. Warfarin Dosing in a Patient with CYP2C9∗3∗3 and VKORC1-1639 AA Genotypes

    PubMed Central

    Bogdan, Renee

    2014-01-01

    Genetic factors most correlated with warfarin dose requirements are variations in the genes encoding the enzymes cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKOR). Patients receiving warfarin who possess one or more genetic variations in CYP2C9 and VKORC1 are at increased risk of adverse drug events and require significant dose reductions to achieve a therapeutic international normalized ratio (INR). A 74-year-old white female with atrial fibrillation was initiated on a warfarin dose of 2 mg PO daily, which resulted in multiple elevated INR measurements and three clinically significant hemorrhagic events and four vitamin K antidote treatments over a period of less than two weeks. Genetic analysis later revealed that she had the homozygous variant genotypes of CYP2C9∗3∗3 and VKORC1-1639 AA. Warfarin dosing was subsequently restarted and stabilized at 0.5 mg PO daily with therapeutic INRs. This is the first case report of a white female with these genotypes stabilized on warfarin, and it highlights the value of pharmacogenetic testing prior to the initiation of warfarin therapy to maximize efficacy and minimize the risk of adverse drug events. PMID:24627811

  1. Oriented Attachment of Cytochrome P450 2C9 to a Self-Assembled Monolayer on a Gold Electrode as a Biosensor Design

    NASA Astrophysics Data System (ADS)

    Schneider, Elizabeth Ann

    Cytochrome P450s (CYPs) are a family of enzymes implicated in the metabolism of drugs in the body. Consequently, P450 reactions are of high interest to the pharmaceutical industry, where lead compounds in drug development are screened as potential substrates of CYPs. The P450 reaction involves electron transfer to an iron heme via NADPH and the electron transfer partner enzyme P450 reductase (CPR). By immobilizing CYPs on an electrode however, NADPH and CPR are potentially no longer needed and the immobilized CYP can act as a biosensor by accepting electrons directly from the electrode. Such a biosensor could be used as an initial screening tool for CYP reactivity of pharmaceuticals in development. In this study, the drug-metabolizing enzyme CYP 2C9 was immobilized to a self-assembled monolayer (SAM) on a gold electrode in three different orientations to investigate the effect that orientation has on the direct electrochemistry of CYP and to evaluate oriented attachment of CYP to an electrode as a biosensor design. Three attachment methods were investigated: random attachment via amine coupling to a carboxy-terminated SAM, oriented attachment via C-terminal His-tag coupling to a Ni-NTA-functionalized SAM, and oriented attachment via maleimide/thiol coupling to a maleimide-functionalized SAM. Three 2C9 mutants (R125C, R132C, and K432C) were developed with a single cysteine mutation at the binding site for CPR on the side of the enzyme closest to the heme; attachment of these mutants to a gold electrode via maleimide/thiol coupling would orient the enzyme such that electron transfer occurs on the electrode in the same orientation that it does in vivo with CPR. Therefore, we expected oriented attachment via maleimide/thiol coupling to produce the most electroactive CYP biosensor. Electrochemical analysis and surface characterization of the SAMs on gold electrodes confirmed that electron transfer occurs through the SAMs, and activity assays of the 2C9 electrodes

  2. Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

    PubMed

    Satoh, T; Fujita, K I; Munakata, H; Itoh, S; Nakamura, K; Kamataki, T; Itoh, S; Yoshizawa, I

    2000-11-15

    To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information. PMID:11067738

  3. Impact of genetic factors (VKORC1, CYP2C9, CYP4F2 and EPHX1) on the anticoagulation response to fluindione

    PubMed Central

    Lacut, Karine; Ayme-Dietrich, Estelle; Gourhant, Lenaick; Poulhazan, Elise; Andro, Marion; Becquemont, Laurent; Mottier, Dominique; Le Gal, Gregoire; Verstuyft, Celine

    2012-01-01

    AIM Genetic variants of the enzyme that metabolizes warfarin, cytochrome P-450 2C9 (CYP2C9) and of a key pharmacologic target of vitamin K antagonists, vitamin K epoxide reductase (VKORC1), contribute to differences in patients' responses to coumarin derivatives. The role of these variants in fluindione response is unknown. Our aim was to assess whether genetic factors contribute to the variability in the response to fluindione. METHODS Four hundred sixty-five patients with a venous thromboembolic event treated by fluindione for at least 3 months with a target international normalized ratio (INR) of 2.0 to 3.0 were studied. VKORC1, CYP2C9, CYP4F2 and EPHX1 genotypes were assessed. INR checks, fluindione doses and bleeding events were collected. RESULTS VKORC1 genotype had a significant impact on early anticoagulation (INR value ≥2 after the first two intakes) (P < 0.0001), on the time required to reach a first INR within the therapeutic range (P < 0.0001) and on the time to obtain a first INR value > 4 (P = 0.0002). The average daily dose of fluindione during the first period of stability was significantly associated with the VKORC1 genotype: 19.8 mg (±5.5) for VKORC1 CC, 14.7 mg (±6.2) for VKORC1 CT and 8.2 mg (±2.5) for VKORC1 TT (P < 0.0001). CYP2C9, CYP4F2 and EPHX1 genotypes did not significantly influence the response to fluindione. CONCLUSIONS VKORC1 genotype strongly affected anticoagulation induced by fluindione whereas CYP2C9, CYP4F2 and EPHX1 genotypes seemed less determining. PMID:21883387

  4. Mechanism-Based Inactivation of Cytochrome P450 2C9 by Tienilic Acid and (±)-Suprofen: A Comparison of Kinetics and Probe Substrate Selection

    PubMed Central

    Hutzler, J. Matthew; Balogh, Larissa M.; Zientek, Michael; Kumar, Vikas; Tracy, Timothy S.

    2009-01-01

    In vitro experiments were conducted to compare kinact, KI and inactivation efficiency (kinact/KI) of cytochrome P450 (P450) 2C9 by tienilic acid and (±)-suprofen using (S)-flurbiprofen, diclofenac, and (S)-warfarin as reporter substrates. Although the inactivation of P450 2C9 by tienilic acid when (S)-flurbiprofen and diclofenac were used as substrates was similar (efficiency of ∼9 ml/min/μmol), the inactivation kinetics were characterized by a sigmoidal profile. (±)-Suprofen inactivation of (S)-flurbiprofen and diclofenac hydroxylation was also described by a sigmoidal profile, although inactivation was markedly less efficient (∼1 ml/min/μmol). In contrast, inactivation of P450 2C9-mediated (S)-warfarin 7-hydroxylation by tienilic acid and (±)-suprofen was best fit to a hyperbolic equation, where inactivation efficiency was moderately higher (10 ml/min/μmol) and ∼3-fold higher (3 ml/min/μmol), respectively, relative to that of the other probe substrates, which argues for careful consideration of reporter substrate when mechanism-based inactivation of P450 2C9 is assessed in vitro. Further investigations into the increased inactivation seen with tienilic acid relative to that with (±)-suprofen revealed that tienilic acid is a higher affinity substrate with a spectral binding affinity constant (Ks) of 2 μM and an in vitro half-life of 5 min compared with a Ks of 21 μM and a 50 min in vitro half-life for (±)-suprofen. Lastly, a close analog of tienilic acid with the carboxylate functionality replaced by an oxirane ring was devoid of inactivation properties, which suggests that an ionic binding interaction with a positively charged residue in the P450 2C9 active site is critical for recognition and mechanism-based inactivation by these close structural analogs. PMID:18838506

  5. Potent inhibition of CYP1A2 by Frutinone A, an active ingredient of the broad spectrum antimicrobial herbal extract from P. fruticosa.

    PubMed

    Thelingwani, Roslyn S; Dhansay, Kariema; Smith, Peter; Chibale, Kelly; Masimirembwa, Collen M

    2012-10-01

    1. Frutinone is an active ingredient extracted from the lipophilic fraction of the Polygala Fruticosa demonstrating various antibacterial and fungal properties. The aim of this study was to characterize its metabolism in an effort to understand metabolism based drug-herb interactions. 2. In vitro metabolic clearance and metabolite identification studies were done using cryopreserved hepatocytes. Reaction phenotyping and inhibition studies were done using human liver microsomes and recombinant cytochrome P450s (CYPs). Frutinone A-CYP1A2 interactions were rationalized using docking simulations. 3. Hepatic clearance was predicted to be low (7.17 mL/min/kg), with reaction phenotyping studies indicating no clearance by the enzymes tested. Frutinone was identified as a potent inhibitor of CYP1A2 with moderate effects on CYP2C19, 2C9, 2D6 and 3A4. CYP1A2 inhibition was reversible and characterised by an IC(50) of 0.56 µM. Inhibition was differential showing mixed (K(i) = 0.48 µM) and competitive (K(i) = 0.31 µM) inhibition with 3-cyano-7-ethoxycoumarin and ethoxyresorufin, respectively. Two binding sites, one for inhibitors and the other for substrates were identified in silico. 4. The potent CYP1A2 inhibition by Frutinone A could be predictive of the potential drug-herb interaction risk in the use of herbal extracts from P. fruticosa. The data suggest future pharmacological research on this chromocoumarin should take metabolic properties into account. PMID:22533317

  6. Expansion of a PBPK model to predict disposition in pregnant women of drugs cleared via multiple CYP enzymes, including CYP2B6, CYP2C9 and CYP2C19

    PubMed Central

    Ke, Alice Ban; Nallani, Srikanth C; Zhao, Ping; Rostami-Hodjegan, Amin; Unadkat, Jashvant D

    2014-01-01

    Aim Conducting PK studies in pregnant women is challenging. Therefore, we asked if a physiologically-based pharmacokinetic (PBPK) model could be used to predict the disposition in pregnant women of drugs cleared by multiple CYP enzymes. Methods We expanded and verified our previously published pregnancy PBPK model by incorporating hepatic CYP2B6 induction (based on in vitro data), CYP2C9 induction (based on phenytoin PK) and CYP2C19 suppression (based on proguanil PK), into the model. This model accounted for gestational age-dependent changes in maternal physiology and hepatic CYP3A, CYP1A2 and CYP2D6 activity. For verification, the pregnancy-related changes in the disposition of methadone (cleared by CYP2B6, 3A and 2C19) and glyburide (cleared by CYP3A, 2C9 and 2C19) were predicted. Results Predicted mean post-partum to second trimester (PP : T2) ratios of methadone AUC, Cmax and Cmin were 1.9, 1.7 and 2.0, vs. observed values 2.0, 2.0 and 2.6, respectively. Predicted mean post-partum to third trimester (PP : T3) ratios of methadone AUC, Cmax and Cmin were 2.1, 2.0 and 2.4, vs. observed values 1.7, 1.7 and 1.8, respectively. Predicted PP : T3 ratios of glyburide AUC, Cmax and Cmin were 2.6, 2.2 and 7.0 vs. observed values 2.1, 2.2 and 3.2, respectively. Conclusions Our PBPK model integrating prior physiological knowledge, in vitro and in vivo data, allowed successful prediction of methadone and glyburide disposition during pregnancy. We propose this expanded PBPK model can be used to evaluate different dosing scenarios, during pregnancy, of drugs cleared by single or multiple CYP enzymes. PMID:23834474

  7. Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase.

    PubMed

    El Adlouni, C; Pinelli, E; Azémar, B; Zaoui, D; Beaune, P; Pfohl-Leszkowicz, A

    2000-01-01

    Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9. PMID:10712746

  8. The role of nitric oxide- and prostacyclin-independent vasodilatation in the human cutaneous microcirculation: effect of cytochrome P450 2C9 inhibition.

    PubMed

    Lenasi, Helena

    2009-07-01

    The component of the flow- or agonist-dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium-dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9-derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO- and prostacyclin (PGI(2))-independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser-Doppler fluxmetry (LDF). The inhibitors of NOS, N(omega)-monomethyl-L-arginine (L-NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh-induced increase in LDF (t-test, P<0.05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L-NMMA- and diclofenac-pretreated site. In addition, sulfaphenazole did not attenuate the ACh-induced vasodilatation in either site. We conclude that a NO- and PGI(2)-independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh-induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9-derived metabolite. PMID:19291087

  9. Frequency distribution of polymorphisms of CYP2C19, CYP2C9, VKORC1 and SLCO1B1 genes in the Yakut population

    PubMed Central

    Vasilyev, Filipp Filippovich; Danilova, Diana Aleksandrovna; Kaimonov, Vladimir Sergeevich; Chertovskih, Yana Valerievna; Maksimova, Nadezda Romanovna

    2016-01-01

    Allele frequencies of single nucleotide polymorphisms (SNPs) are variable among different populations; therefore the study of SNPs in ethnic groups is important for establishing the clinical significance of the screening of these polymorphisms. The main goal of the research is to study the polymorphisms of CYP2C9, CYP2C19, VKORC1, and SLCO1B1 in Yakuts. Genomic DNA from 229 Yakut subjects were analyzed by real-time polymerase chain reaction (PCR) (SLCO1B1 +521T > C, VKORC1 -1639G>A, CYP2C19 +681G>A, +636G>A, CYP2C9 +430С>T, +1075A>C). Genotype frequencies of polymorphisms in the population of the Yakuts were more characteristic of the Asian population. The results have been included in the software application “Lekgen” that we developed for the interpretation of pharmacogenetic testing. The data of our study obtained on frequency carriers of polymorphisms of genes SLCO1B1, CYP2C19, CYP2C9, VKORC1 among the Yakuts may be useful in developing recommendations for a personalized therapy.

  10. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

  11. Interactions between CYP3A4 and Dietary Polyphenols

    PubMed Central

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  12. Interactions between CYP3A4 and Dietary Polyphenols.

    PubMed

    Basheer, Loai; Kerem, Zohar

    2015-01-01

    The human cytochrome P450 enzymes (P450s) catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols. PMID:26180597

  13. Effect of CYP2C9 and VKORC1 Gene Variants on Warfarin Response in Patients with Continuous-Flow Left Ventricular Assist Devices.

    PubMed

    Topkara, Veli K; Knotts, Robert J; Jennings, Douglas L; Garan, A Reshad; Levin, Allison P; Breskin, Alexander; Castagna, Francesco; Cagliostro, Barbara; Yuzefpolskaya, Melana; Takeda, Koji; Takayama, Hiroo; Uriel, Nir; Mancini, Donna M; Eisenberger, Andrew; Naka, Yoshifumi; Colombo, Paolo C; Jorde, Ulrich P

    2016-01-01

    Bleeding and thrombotic complications continue to plague continuous-flow left ventricular assist device (CF-LVAD) therapy in patients with end-stage heart failure. Warfarin genotyping information can be incorporated into decision making for initial dosing as recommended by the Food and Drug Administration; however, clinical utility of this data in the CF-LVAD population has not been well studied. Genotypes testing for CYP2C9 and VCORC1 polymorphisms were determined in 90 CF-LVAD patients. Outcomes studied were the association of CYP2C9 (*1, *2, or *3) and VKORC1 (-1639 G>A) gene variants with time-to-target international normalized ratio (INR), total warfarin dose, maintenance warfarin dose. Continuous-flow left ventricular assist device patients carrying a rare variant in the VKORC1 gene had a significantly lower cumulative warfarin dose until target INR achieved (18.9 vs. 35.0 mg, p = 0.002), days spent until INR target achieved (4.9 vs. 7.0 days, p = 0.021), and discharge warfarin dose (3.2 vs. 5.6 mg, p = 0.001) compared with patients with wild-type genotype. Genotype-guided warfarin dosing may lead to safer anticoagulation and potentially improve outcomes in CF-LVAD patients. PMID:27258224

  14. 7 CFR 1a.2 - Authorization.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Authorization. 1a.2 Section 1a.2 Agriculture Office of the Secretary of Agriculture LAW ENFORCEMENT AUTHORITIES § 1a.2 Authorization. Any official of the Office of Inspector General who is designated by the Inspector General according to §§ 1a.3 and 1a.5...

  15. Lack of Association of Clinical Factors (SAMe-TT2R2) with CYP2C9/VKORC1 Genotype and Anticoagulation Control Quality

    PubMed Central

    Park, Yun Kyung; Lee, Mi ji; Kim, Jae Ha; Kim, Suk Jae; Kim, June Soo; Lee, Soo-Youn

    2015-01-01

    Background and Purpose Advantages of new oral anticoagulations may be greater in atrial fibrillation (AF) patients of poor anticoagulation control with warfarin. The SAMe-TT2R2 scoring system, based on clinical variables, was recently developed to aid in identifying these patients. In this study, we investigated the association of this clinical composite score with genetic factors related warfarin dosing and the quality of anticoagulation control. Methods Clinical and genetic data were collected from 380 consecutive Korean patients with AF (CHA2DS2-VASc score, 3.5±1.8) who were followed for an average of 4 years. We evaluated factors associated with time in therapeutic range (TTR, INR 2-3), including the CYP2C9 and VKORC1 genotypes and the SAMe-TT2R2 score (Sex female, Age <60 years, Medical history [>two co-morbidities], Treatment [interacting drugs, e.g., amiodarone], Tobacco use within 2 years [doubled], and Race non-white [doubled]). Results The average SAMe-TT2R2 score was 3.4±0.9, range 2-7; and 153 patients (40.2%) had SAMe-TT2R2 scores ≥4. Time in specific INR ranges varied depending on the VKORC1 genotype but not with the CYP2C9 genotype or the SAMe-TT2R2 score. TTR was higher in patients with the VKORC1 1173C>T than in VKORC1 TT (61.7±16% vs. 56.7±17.4%, P=0.031). Multivariate testing showed that VKORC1 genotype but not the SAMe-TT2R2 score was significantly associated with labile INRs. There was no correlation between the SAMe-TT2R2 scores and pharmacogenetic data. Conclusions A genetic factor, but none of the common clinical and demographic factors, as combined in the SAMe-TT2R2 score, was associated with the quality of anticoagulation control in Korean patients with AF. PMID:26060806

  16. Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver.

    PubMed

    Hasegawa, Maki; Tahara, Harunobu; Inoue, Ryo; Kakuni, Masakazu; Tateno, Chise; Ushiki, Junko

    2012-03-01

    The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4. PMID:22126990

  17. Drug Modulation of Water–Heme Interactions in Low-Spin P450 Complexes of CYP2C9d and CYP125A1

    PubMed Central

    Conner, Kip P.; Cruce, Alex A.; Krzyaniak, Matthew D.; Schimpf, Alina M.; Frank, Daniel J.; de Montellano, Paul Ortiz; Atkins, William M.; Bowman, Michael K.

    2015-01-01

    Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP–inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenyl-propyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of “classic” type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A 15N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution. PMID:25591012

  18. Study of the relative response factors of various gas chromatograph-flame ionisation detector systems for measurement of C2-C9 hydrocarbons in air.

    PubMed

    Slemr, J; Slemr, F; D'Souza, H; Partridge, R

    2004-12-17

    The assumption of an instrument response that is linear with carbon number is frequently used to quantify atmospheric non-methane hydrocarbons (NMHCs) when using gas chromatography (GC) and detection by flame ionisation detector (FID). In order to assess the validity of this widely used method the results of intercomparison measurements by 14 laboratories across Europe were evaluated. The intercomparison measurements were made on synthetic, gravimetrically-prepared, gas mixtures containing 30 hydrocarbons (C2-C9) in the low ppbv range, using various different GC-FID systems. The response per carbon atom of GC-FID systems to individual NMHCs, relative to that of butane, were found to differ by more than 25% across different systems. The differences were mostly caused by analytical errors within particular GC-FID systems and to a more minor degree by systematic deviations related to the molecular structure. (Correction factors due to the molecular structure would lessen the differences, e.g. by about 5% for olefin compounds.) The differences were larger than 10% even after elimination of obvious outliers. Thus, calibration of GC-FID systems with multicomponent NMHC mixtures is found to be essential whenever the accuracy of NMHC measurements is required to be better than about 10%. If calibration by multicomponent gas mixtures is not possible and effective carbon atom response factors are used to quantify the individual NMHC compounds then the particular analytical system should be carefully characterised and its responses to individual compounds be verified. PMID:15633746

  19. Drug modulation of water-heme interactions in low-spin P450 complexes of CYP2C9d and CYP125A1.

    PubMed

    Conner, Kip P; Cruce, Alex A; Krzyaniak, Matthew D; Schimpf, Alina M; Frank, Daniel J; Ortiz de Montellano, Paul; Atkins, William M; Bowman, Michael K

    2015-02-10

    Azoles and pyridines are commonly incorporated into small molecule inhibitor scaffolds that target cytochromes P450 (CYPs) as a strategy to increase drug binding affinity, impart isoform-dependent selectivity, and improve metabolic stability. Optical absorbance spectra of the CYP-inhibitor complex are widely used to infer whether these inhibitors are ligated directly to the heme iron as catalytically inert, low-spin (type II) complexes. Here, we show that the low-spin complex between a drug-metabolizing CYP2C9 variant and 4-(3-phenylpropyl)-1H-1,2,3-triazole (PPT) retains an axial water ligand despite exhibiting elements of "classic" type II optical behavior. Hydrogens of the axial water ligand are observed by pulsed electron paramagnetic resonance (EPR) spectroscopy for both inhibitor-free and inhibitor-bound species and show that inhibitor binding does not displace the axial water. A (15)N label incorporated into PPT is 0.444 nm from the heme iron, showing that PPT is also in the active site. The reverse type I inhibitor, LP10, of CYP125A1 from Mycobacterium tuberculosis, known from X-ray crystal structures to form a low-spin water-bridged complex, is found by EPR and by visible and near-infrared magnetic circular dichroism spectroscopy to retain the axial water ligand in the complex in solution. PMID:25591012

  20. Development of Flavone Propargyl Ethers as Potent and Selective Inhibitors of Cytochrome P450 Enzymes 1A1 and 1A2

    PubMed Central

    Sridhar, Jayalakshmi; Ellis, Jamie; Dupart, Patrick; Liu, Jiawang; Stevens, Cheryl L.; Foroozesh, Maryam

    2014-01-01

    Naturally occurring flavonoids are known to be metabolized by several cytochrome P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, 3A4, and 3A5. In general flavonoids can act as substrates, inducers, and/or inhibitors of P450 enzymes. The position of the substituents on the flavone backbone has been shown to impact the biological activity against P450 enzymes. To explore the effect of a propargyl ether substitution on flavones and flavanones, 2′-flavone propargyl ether (2′-PF), 3′-flavone propargyl ether (3′-PF), 4′-flavone propargyl ether (4′-PF), 5-flavone propargyl ether (5-PF), 6-flavone propargyl ether (6-PF), 7-flavone propargyl ether (7-PF), 6-flavanone propargyl ether (6-PFN), and 7-flavanone propargyl ether (7-PFN) were synthesized. All of the newly synthesized compounds and the parent hydroxy flavones were tested for both direct inhibition and mechanism-based inhibition of cytochrome P450 enzymes 1A1, 1A2, 2A6, and 2B1. The flavone propargyl ether derivatives were found to be more potent inhibitors of P450s 1A1 and 1A2. None of the flavones and flavanones in our study showed any inhibition of P450 2A6. Only 2′-PF and 6-PFN inhibited P450 2B1. 3′-PF showed direct inhibition of P450 1A1 with the highest observed potency of 0.02 μM, in addition to its ability to cause mechanism-based inhibition with KI and kinactivation values of 0.24 μM and 0.09 min−1 for this enzyme. 7-Hydroxy flavone also exhibited mechanism-based inhibition of P450 1A1 with KI and kinactivation values of 2.43 μM and 0.115 min−1. Docking studies and QSAR studies on P450 enzymes 1A1 and 1A2 were performed which revealed important insights into the nature of binding of these molecules and provided us with good QSAR models that can be used to design new flavone derivatives. PMID:23506553

  1. A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes.

    PubMed

    Mitsui, Tetsuya; Nemoto, Takayuki; Miyake, Taiji; Nagao, Shunsuke; Ogawa, Kotaro; Kato, Motohiro; Ishigai, Masaki; Yamada, Hideyuki

    2014-09-01

    The accurate prediction for the body clearance of a novel drug candidate by humans during the preclinical stage contributes to its successful development. To improve the predictability of human hepatic clearance, we focused on CYP3A4, which is involved in the metabolism of more than 50% of all currently marketed drugs. In this study, we investigated the validity of the in vivo model using transgenic mice carrying the human CYP3A4 gene and lacking their own Cyp3a genes (CYP3A4-Tg mice). The CYP3A4 activity toward its substrates in liver microsomes was similar in CYP3A4-Tg mice and humans. As for the clearance, six CYP3A4 substrates (alprazolam, felodipine, midazolam, nifedipine, nitrendipine, and quinidine) were given intravenously to CYP3A4-Tg mice, and their hepatic intrinsic clearance (CLint,h) was evaluated. A regression analysis of the data obtained indicated that the CLint,h values of six substrates in CYP3A4-Tg mice were highly correlated with those in humans (R(2) = 0.95). This correlation could be improved by correcting the CLint,h values by the relative contribution of artificially expressed CYP3A4 to the overall metabolism in the mice. From these findings, it is reasonable to expect that the CLint,h of a particular drug in humans is predictable by applying the CLint,h obtained in CYP3A4-Tg mice to a regression line prepared in advance. The variance of the CLint,h prediction by this method was evaluated and found to be within a range of 2-fold of the regression value. These results suggest that the CYP3A4-Tg mouse model has the potential to accurately predict the human hepatic clearance of CYP3A4 substrates. PMID:25005602

  2. Gene-gene-environment interactions between drugs, transporters, receptors, and metabolizing enzymes: Statins, SLCO1B1, and CYP3A4 as an example.

    PubMed

    Sadee, Wolfgang

    2013-09-01

    Pharmacogenetic biomarker tests include mostly specific single gene-drug pairs, capable of accounting for a portion of interindividual variability in drug response and toxicity. However, multiple genes are likely to contribute, either acting independently or epistatically, with the CYP2C9-VKORC1-warfarin test panel, an example of a clinically used gene-gene-dug interaction. I discuss here further instances of gene-gene-drug interactions, including a proposed dynamic effect on statin therapy by genetic variants in both a transporter (SLCO1B1) and a metabolizing enzyme (CYP3A4) in liver cells, the main target site where statins block cholesterol synthesis. These examples set a conceptual framework for developing diagnostic panels involving multiple gene-drug combinations. PMID:23436703

  3. Unique CYP3A4 genetic variant in Brazilian tuberculosis patients with/without HIV.

    PubMed

    Jeovanio-Silva, André L; Monteiro, Thaís P; El-Jaick, Kênia B; do Brasil, Pedro E A A; Rolla, Valéria C; de Castro, Liane

    2012-01-01

    CYP3A4 is involved in tuberculosis (TB) and human immunodeficiency virus (HIV) drug metabolism. Transcriptional activation by rifampicin involves the CYP3A4 gene 5'-upstream region. Consequently, variation may interfere with transcription and enzymatic activity and even drug response. However, genetic polymorphisms and distribution of CYP3A4 allelic frequencies in individuals from Rio de Janeiro remain unknown. The aim of this study was to conduct research into sequencing the CYP3A4 5'-upstream region in Brazilian patients with and without HIV. This follow-up study involved 106 individuals undergoing treatment for TB and/or HIV. The CYP3A4 5'-upstream region was analyzed using PCR, sequencing and clinical data. Male patients revealed a higher HIV frequency (p=0.021). The TB forms observed were pulmonary (48.1%), extrapulmonary (22.64%) and disseminated (27.36%). Lymph node form was the most frequent (70.83%) extrapulmonary form of TB. The only single nucleotide polymorphism detected in the population was c.-392A>G. Genotypes observed were CYP3A4*1A/CYP3A4*1A (45.3%), CYP3A4*1A/CYP3A4*1B (40.6%) and CYP3A4*1B/CYP3A4*1B (14.2%), revealing a different distribution with extrapulmonary TB cases (17.6% CYP3A4*1A/CYP3A4*1B and 23.5% CYP3A4*1B/CYP3A4*1B). The CYP3A4*1A allele was found to be associated with tobacco use. The CYP3A4*1B mutant allele occurred in 34% of patients. This study revealed that the CYP3A4 5'-upstream regulatory region was highly conserved with the exception of the -392 position. Genotype association with tobacco suggests that CYP3A4 may participate in tobacco metabolism. Genotype distribution inversion in extrapulmonary TB cases suggests that CYP3A4 may be involved in TB prognosis. PMID:21964586

  4. Scutellarin inhibits cytochrome P450 isoenzyme 1A2 (CYP1A2) in rats.

    PubMed

    Jian, Tun-Yu; He, Jian-Chang; He, Gong-Hao; Feng, En-Fu; Li, Hong-Liang; Bai, Min; Xu, Gui-Li

    2012-08-01

    Scutellarin is the most important flavone glycoside in the herbal drug Erigeron breviscapus (Vant.) Hand.-Mazz. It is used frequently in the clinic to treat ischemic vascular diseases in China. However, the direct relationship between scutellarin and cytochrome P450 (CYP450) is unclear. The present study investigated the in vitro and in vivo effects of scutellarin on cytochrome P450 1A2 (CYP 1A2) metabolism. According to in vitro experiments, scutellarin (10-250 µM) decreased the formation of 4-acetamidophenol in a concentration-dependent manner, with an IC₅₀ value of 108.20 ± 0.657 µM. Furthermore, scutellarin exhibited a weak mixed-type inhibition against the activity of CYP1A2 in rat liver microsomes, with a K(i) value of 95.2 µM. Whereas in whole animal studies, scutellarin treatment for 7 days (at 5, 15, 30 mg/kg, i.p.) decreased the clearance (CL), and increased the T(1/2) (at 15, 30 mg/kg, i.p.), it did not affect the V(d) of phenacetin. Scutellarin treatment (at 5, 15, 30 mg/kg, i.p.) increased the AUC(0-∞) by 14.3%, 67.3% and 159.2%, respectively. Scutellarin at 30 mg/kg also weakly inhibited CYP1A2 activity, in accordance with our in vitro study. Thus, the results indicate that CYP1A2 is inhibited directly, but weakly, by scutellarin in vivo, and provide useful information on the safe and effective use of scutellarin in clinical practice. PMID:22228482

  5. CYP3A4-based drug-drug interaction: CYP3A4 substrates' pharmacokinetic properties and ketoconazole dose regimen effect.

    PubMed

    Boulenc, Xavier; Nicolas, Olivier; Hermabessière, Stéphanie; Zobouyan, Isabelle; Martin, Valérie; Donazzolo, Yves; Ollier, Céline

    2016-02-01

    The aim of the study was to assess the magnitude of the CYP3A4 inhibitory effect of 2 dosing regimens of ketoconazole and the influence of the pharmacokinetic properties of the CYP3A4 substrate on the extent of the substrate exposure increase. For this purpose, a clinical study was conducted and PBPK modeling simulations were performed. A crossover study was conducted in healthy subjects. The study was designed to compare the effects of different regimens of reversible CYP3A4 inhibitors, i.e., ketoconazole 400 mg OD, ketoconazole 200 mg BID, on two CYP3A4 substrates, alprazolam and midazolam, reflecting different pharmacokinetic properties in terms of first-pass effect and elimination. In parallel, time-based simulations were performed using the Simcyp population-based Simulator to address the usefulness of modeling to assess interaction clinical study design with CYP3A4 substrates. Comparison of the OD versus BID regimens for ketoconazole showed an opposite trend for the 2 substrates: BID (200 mg) dosing regimen provided the maximal clearance inhibition for alprazolam, while it was OD (400 mg) dosing regimen for midazolam. However, these effects are moderate despite the well-known pharmacokinetic differences between these substrates, suggesting that these differences are not enough. In the other way round, these investigations show how two CYP3A4 substrates can be different without leading to a major impact of the ketoconazole dosing regimen. The clinical findings are consistent with the Simcyp predictions, in particular the opposite trend observed with midazolam and alprazolam and the ketoconazole dosing regimen. These clinical investigations showed the influence of the CYP3A4 substrates' pharmacokinetic properties and the relevance of ketoconazole dose regimen on the magnitude of the interaction ratios. In addition, PBPK Simcyp simulations demonstrated how they can be used to help clinical study design assessment to capture the maximum effect. PMID:25374256

  6. In vitro inhibition of cytochrome P450 3A4 by Aronia melanocarpa constituents.

    PubMed

    Bräunlich, Marie; Christensen, Hege; Johannesen, Siri; Slimestad, Rune; Wangensteen, Helle; Malterud, Karl E; Barsett, Hilde

    2013-01-01

    Extracts, subfractions, isolated anthocyanins and procyanidins, and two phenolic acids from aronia [Aronia melanocarpa] were investigated for their CYP3A4 inhibitory effects, using midazolam as the probe substrate and recombinant insect cell microsomes expressing CYP3A4 as the enzyme source. Procyanidin B5 was a considerably stronger CYP3A4 inhibitor in vitro than the isomeric procyanidin B2 and comparable to bergamottin, a known CYP3A4 inhibitor from grapefruit juice. The inhibitory activity of proanthocyanidin-containing fractions was correlated to the degree of polymerization. Among the anthocyanins, cyanidin 3-arabinoside showed stronger CYP3A4 inhibition than cyanidin 3-galactoside and cyanidin 3-glucoside. Thus, the ability to inhibit CYP3A4 in vitro seems to be influenced by the sugar unit linked to the anthocyanidin. PMID:23250807

  7. Inhibitory Effects of Vegetable Juices on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    PubMed

    Tsujimoto, Masayuki; Uchida, Tomoe; Kozakai, Hiroyuki; Yamamoto, Saori; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2016-01-01

    It is thought that eating habits induces individual variation in intestinal absorption and metabolism of drugs. The objective of this research was to clarify the influence of vegetables juices on CYP3A4 activity, which is an important enzyme in intestine. Five vegetables juices (VJ-o, Kagome Original(®); VJ-g, Kagome 30 kinds of vegetables and fruits(®); VJ-p, Kagome Purple vegetables(®); VJ-r, Kagome Sweet Tomato(®); and VJ-y, Kagome Fruity Salada(®); KAGOME Co., Ltd., Aichi, Japan) were centrifuged (1630×g, 10 min) and filtered using filter paper and 0.45-µm membrane filters. In this study, recombinant CYP3A4 and LS180 cells were used for the evaluation of CYP3A4 activity. The metabolisms to 6β-hydroxytestosterone by recombinant CYP3A4 were significantly inhibited by VJ-o, VJ-g, and VJ-y in a preincubation time-dependent manner, and CYP3A4 activity in LS180 cells were significantly inhibited by VJ-o and VJ-y. These results show that the difference in ingestion volume of vegetable juices and vegetables might partially induce individual difference in intestinal drug metabolism. PMID:27582329

  8. In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer patients.

    PubMed

    Engdal, Silje; Nilsen, Odd Georg

    2009-07-01

    The herbal remedies Natto K2, Agaricus, mistletoe, noni juice, green tea and garlic, frequently used by cancer patients, were investigated for their in vitro inhibition potential of cytochrome P-450 3A4 (CYP3A4) metabolism. To our knowledge, only garlic and green tea had available data on the possible inhibition of CYP3A4 metabolism. Metabolic studies were performed with human c-DNA baculovirus expressed CYP3A4. Testosterone was used as a substrate and ketoconazole as a positive quantitative inhibition control. The formation of 6-beta-OH-testosterone was quantified by a validated HPLC methodology. Green tea was the most potent inhibitor of CYP3A4 metabolism (IC(50): 73 microg/mL), followed by Agaricus, mistletoe and noni juice (1324, 3594, >10 000 microg/mL, respectively). All IC(50) values were high compared with those determined for crude extracts of other herbal remedies. The IC(50)/IC(25) ratios for the inhibiting herbal remedies ranged from 2.15 to 2.67, indicating similar inhibition profiles of the herbal inhibitors of CYP3A4. Garlic and Natto K2 were classified as non-inhibitors. Although Agaricus, noni juice, mistletoe and green tea inhibited CYP3A4 metabolism in vitro, clinically relevant systemic or intestinal interactions with CYP3A4 were considered unlikely, except for a probable inhibition of intestinal CYP3A4 by the green tea product. PMID:19170155

  9. Pharmacogenetics of the organic anion transporting polypeptide 1A2

    PubMed Central

    Franke, Ryan M; Scherkenbach, Lisa A; Sparreboom, Alex

    2016-01-01

    The solute carrier, human organic anion transporting polypeptide 1A2 (OATP1A2, OATP-A, OATP1 and OATP) is highly expressed in the intestine, kidney, cholangiocytes and the blood–brain barrier. This localization suggests that OATP1A2 may be vitally important in the absorption, distribution and excretion of a broad array of clinically important drugs. Several nonsynonymous polymorphisms have been identified in the gene encoding OATP1A2, SLCO1A2 (SLC21A3), with some of these variants demonstrating functional changes in the transport of OATP1A2 substrates. PMID:19290786

  10. UNDERSTANDING THE MECHANISM OF CYTOCHROME P450 3A4: RECENT ADVANCES AND REMAINING PROBLEMS

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2013-01-01

    Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of the administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research. PMID:23018626

  11. Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

    PubMed Central

    Poulos, Thomas L.

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major and most important drug-metabolizing enzyme in humans that oxidizes and clears over a half of all administered pharmaceuticals. This is possible because CYP3A4 is promiscuous with respect to substrate binding and has the ability to catalyze diverse oxidative chemistries in addition to traditional hydroxylation reactions. Furthermore, CYP3A4 binds and oxidizes a number of substrates in a cooperative manner and can be both induced and inactivated by drugs. In vivo, CYP3A4 inhibition could lead to undesired drug-drug interactions and drug toxicity, a major reason for late-stage clinical failures and withdrawal of marketed pharmaceuticals. Owing to its central role in drug metabolism, many aspects of CYP3A4 catalysis have been extensively studied by various techniques. Here, we give an overview of experimental and theoretical methods currently used for investigation and prediction of CYP3A4-ligand interactions, a defining factor in drug metabolism, with an emphasis on the problems addressed and conclusions derived from the studies. PMID:26002732

  12. Structure-Based Inhibitor Design for Evaluation of a CYP3A4 Pharmacophore Model.

    PubMed

    Kaur, Parminder; Chamberlin, A Richard; Poulos, Thomas L; Sevrioukova, Irina F

    2016-05-12

    Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug-drug interactions, toxicity, and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of coadministered pharmaceuticals that are metabolized by CYP3A4. On the basis of our investigations of analogs of ritonavir, a potent CYP3A4 inactivator and pharmacoenhancer, we have built a pharmacophore model for a CYP3A4-specific inhibitor. This study is the first attempt to test this model using a set of rationally designed compounds. The functional and structural data presented here agree well with the proposed pharmacophore. In particular, we confirmed the importance of a flexible backbone, the H-bond donor/acceptor moiety, and aromaticity of the side group analogous to Phe-2 of ritonavir and demonstrated the leading role of hydrophobic interactions at the sites adjacent to the heme and phenylalanine cluster in the ligand binding process. The X-ray structures of CYP3A4 bound to the rationally designed inhibitors provide deeper insights into the mechanism of the CYP3A4-ligand interaction. Most importantly, two of our compounds (15a and 15b) that are less complex than ritonavir have comparable submicromolar affinity and inhibitory potency for CYP3A4 and, thus, could serve as templates for synthesis of second generation inhibitors for further evaluation and optimization of the pharmacophore model. PMID:26371436

  13. Association of CYP3A4 genotype with treatment-related leukemia

    PubMed Central

    Felix, Carolyn A.; Walker, Amy H.; Lange, Beverly J.; Williams, Terence M.; Winick, Naomi J.; Cheung, Nai-Kong V.; Lovett, Brian D.; Nowell, Peter C.; Blair, Ian A.; Rebbeck, Timothy R.

    1998-01-01

    Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5′ promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher’s Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0.016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone. PMID:9789061

  14. Energetics of Heterotropic Cooperativity between α-Naphthoflavone and Testosterone Binding to CYP3A4

    PubMed Central

    Roberts, Arthur G.; Atkins, William M.

    2007-01-01

    Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of a majority of drugs. Heterotropic cooperativity of drug binding to CYP3A4 was examined with the flavanoid, α-naphthoflavone (ANF) and the steroid, testosterone (TST). UV-vis and EPR spectroscopy of CYP3A4 show that ANF binding to CYP3A4 occurs with apparent negative cooperativity and that there are at least two binding sites: 1) a relatively tight spin-state insensitive binding site (CYP●ANF) and 2) a relatively low affinity spin-state sensitive binding site (CYP●ANF●ANF). Since binding to the spin-state insensitive binding site is considerably tighter for ANF than TST, the spin-state insensitive binding site could be occupied by ANF, while titrating TST at the other site(s). The spin-state insensitive binding site of ANF appears to compete with the spin-state insensitive binding site of TST. The formation of the spin-state insensitive CYP●ANF complex is strongly temperature dependent, when compared to the formation of the CYP●TST complex, suggesting that the formation of the CYP3A4●ANF complex leads to long-range conformational changes within the protein. When the CYP●ANF complex is titrated with TST, the formation of CYP●ANF●TST is favored by 3:1 over the formation of CYP●TST●TST, suggesting that there is an allosteric interaction between ANF and TST. A model of heterotropic cooperativity of CYP3A4 is presented, where the spin-state insensitive binding of ANF occurs at the same peripheral binding site of CYP3A4 as TST. PMID:17459328

  15. CYP3A4 drug interactions: correlation of 10 in vitro probe substrates

    PubMed Central

    Kenworthy, K E; Bloomer, J C; Clarke, S E; Houston, J B

    1999-01-01

    Aims Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug–drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. Methods The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6β-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 μm. Results While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. Conclusions It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions. PMID

  16. Association of CYP3A4 genotype with treatment-related leukemia.

    PubMed

    Felix, C A; Walker, A H; Lange, B J; Williams, T M; Winick, N J; Cheung, N K; Lovett, B D; Nowell, P C; Blair, I A; Rebbeck, T R

    1998-10-27

    Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5' promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher's Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0. 016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone. PMID:9789061

  17. Inhibition of Human Cytochrome P450 3A4 by Cholesterol*

    PubMed Central

    Shinkyo, Raku; Guengerich, F. Peter

    2011-01-01

    Cholesterol has been shown to be hydroxylated at the 4β-position by cytochrome P450 3A4, and the reaction occurs in vivo (Bodin, K., Andersson, U., Rystedt, E., Ellis, E., Norlin, M., Pikuleva, I., Eggertsen, G., Björkhem, I., and Diczfalusy, U. (2002) J. Biol. Chem. 277, 31534–31540). If cholesterol is a substrate of P450 3A4, then it follows that it should also be an inhibitor, particularly in light of the high concentrations found in liver. Heme perturbation spectra indicated a Kd value of 8 μm for the P450 3A4-cholesterol complex. Cholesterol inhibited the P450 3A4-catalyzed oxidations of nifedipine and quinidine, two prototypic substrates, in liver microsomes and a reconstituted enzyme system with Ki ∼ 10 μm in an apparently non-competitive manner. The concentration of cholesterol could be elevated 4–6-fold in cultured human hepatocytes by incubation with cholesterol; the level of P450 3A4 and cell viability were not altered under the conditions used. Nifedipine oxidation was inhibited when the cholesterol level was increased. We conclude that cholesterol is both a substrate and an inhibitor of P450 3A4, and a model is presented to explain the kinetic behavior. We propose that the endogenous cholesterol in hepatocytes should be considered in models of prediction of metabolism of drugs and steroids, even in the absence of changes in the concentrations of free cholesterol. PMID:21471209

  18. Stereospecific Metabolism of Itraconazole by CYP3A4: Dioxolane Ring Scission of Azole Antifungals

    PubMed Central

    Peng, Chi-Chi; Shi, Wei; Lutz, Justin D.; Kunze, Kent L.; Liu, Jun O.; Nelson, Wendel L.

    2012-01-01

    Itraconazole (ITZ) is a mixture of four cis-stereoisomers that inhibit CYP3A4 potently and coordinate CYP3A4 heme via the triazole nitrogen. However, (2R,4S,2′R)-ITZ and (2R,4S,2′S)-ITZ also undergo stereoselective sequential metabolism by CYP3A4 at a site distant from the triazole ring to 3′-OH-ITZ, keto-ITZ, and N-desalkyl-ITZ. This stereoselective metabolism demonstrates specific interactions of ITZ within the CYP3A4 active site. To further investigate this process, the binding and metabolism of the four trans-ITZ stereoisomers by CYP3A4 were characterized. All four trans-ITZ stereoisomers were tight binding inhibitors of CYP3A4-mediated midazolam hydroxylation (IC50 16–26 nM), and each gave a type II spectrum upon binding to CYP3A4. However, instead of formation of 3′-OH-ITZ, they were oxidized at the dioxolane ring, leading to ring scission and formation of two new metabolites of ITZ. These two metabolites were also formed from the four cis-ITZ stereoisomers, although not as efficiently. The catalytic rates of dioxolane ring scission were similar to the dissociation rates of ITZ stereoisomers from CYP3A4, suggesting that the heme iron is reduced while the triazole moiety coordinates to it and no dissociation of ITZ is necessary before catalysis. The triazole containing metabolite [1-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)ethanone] also inhibited CYP3A4 (IC50 >15 μM) and showed type II binding with CYP3A4. The dioxolane ring scission appears to be clinically relevant because this metabolite was detected in urine samples from subjects that had been administered the mixture of cis-ITZ isomers. These data suggest that the dioxolane ring scission is a metabolic pathway for drugs that contain this moiety. PMID:22106171

  19. Cytochrome P450 3A4 and CYP3A5-Catalyzed Bioactivation of Lapatinib.

    PubMed

    Towles, Joanna K; Clark, Rebecca N; Wahlin, Michelle D; Uttamsingh, Vinita; Rettie, Allan E; Jackson, Klarissa D

    2016-10-01

    Metabolic activation of the dual-tyrosine kinase inhibitor lapatinib by cytochromes CYP3A4 and CYP3A5 has been implicated in lapatinib-induced idiosyncratic hepatotoxicity; however, the relative enzyme contributions have not been established. The objective of this study was to examine the roles of CYP3A4 and CYP3A5 in lapatinib bioactivation leading to a reactive, potentially toxic quinoneimine. Reaction phenotyping experiments were performed using individual human recombinant P450 enzymes and P450-selective chemical inhibitors. Lapatinib metabolites and quinoneimine-glutathione (GSH) adducts were analyzed using liquid chromatography-tandem mass spectrometry. A screen of cDNA-expressed P450s confirmed that CYP3A4 and CYP3A5 are the primary enzymes responsible for quinoneimine-GSH adduct formation using lapatinib or O-dealkylated lapatinib as the substrate. The mean kinetic parameters (Km and kcat) of lapatinib O-dealkylation revealed that CYP3A4 was 5.2-fold more efficient than CYP3A5 at lapatinib O-dealkylation (CYP3A4 kcat/Km = 6.8 μM(-1) min(-1) versus CYP3A5 kcat/Km = 1.3 μM(-1) min(-1)). Kinetic analysis of GSH adduct formation indicated that CYP3A4 was also 4-fold more efficient at quinoneimine-GSH adduct formation as measured by kcat (maximum relative GSH adduct levels)/Km (CYP3A4 = 0.0082 vs. CYP3A5 = 0.0021). In human liver microsomal (HLM) incubations, CYP3A4-selective inhibitors SR-9186 and CYP3cide reduced formation of GSH adducts by 78% and 72%, respectively, compared with >90% inhibition by the pan-CYP3A inhibitor ketoconazole. The 16%-22% difference between CYP3A- and CYP3A4-selective inhibition indicates the involvement of remaining CYP3A5 activity in generating reactive metabolites from lapatinib in pooled HLMs. Collectively, these findings support the conclusion that both CYP3A4 and CYP3A5 are quantitatively important contributors to lapatinib bioactivation. PMID:27450182

  20. Metabolic behavior prediction of pazopanib by cytochrome P450 (CYP) 3A4 by molecular docking.

    PubMed

    Liu, Xing-Jie; Lu, Hua; Sun, Ju-Xiang; Wang, Su-Rong; Mo, Yan-Shuai; Yang, Xing-Sheng; Shi, Ben-Kang

    2016-08-01

    Metabolism-mediated drug adverse effects (e.g., drug-drug interaction, bioactivation, etc.) strongly limit the utilization of clinical drugs. The present study aims to predict the metabolic capability of cytochrome P450 (CYP) 3A4 toward pazopanib which is an excellent drug exhibiting therapeutic role toward various cancers especially for ovarian cancer. Pazopanib can be well docked into the activity cavity of CYP3A4, and the interaction structure in pazopanib was methyl group located besides nitrogen in the five-membered ring. The distance between the hydrogen atom in methyl group and active center is 3.64 Å. The interaction amino acid is Glu374. Furthermore, both pazopanib and ketoconazole were docked into the activity cavity of CYP3A4 to compare their binding potential. The distance between ketoconazole and activity center (2.10 Å) is closer than the distance between pazopanib and activity center of CYP3A4, indicating the easy influence of CYP3A4 inhibitor toward the metabolism of pazopanib. All these data were helpful for the clinical application of pazopanib, and R&D of other tinib drug candidates as new anti-tumor drugs. PMID:25737032

  1. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4.

    PubMed

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  2. Effect of Glutathione on Homo- and Heterotropic Cooperativity in Cytochrome P450 3A4

    PubMed Central

    Davydov, Dmitri R.; Davydova, Nadezhda Y.; Tsalkova, Tamara N.; Halpert, James R.

    2008-01-01

    Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1–4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the KS value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo. PMID:18206979

  3. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  4. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  5. High frequency and founder effect of the CYP3A4*20 loss-of-function allele in the Spanish population classifies CYP3A4 as a polymorphic enzyme.

    PubMed

    Apellániz-Ruiz, M; Inglada-Pérez, L; Naranjo, M E G; Sánchez, L; Mancikova, V; Currás-Freixes, M; de Cubas, A A; Comino-Méndez, I; Triki, S; Rebai, A; Rasool, M; Moya, G; Grazina, M; Opocher, G; Cascón, A; Taboada-Echalar, P; Ingelman-Sundberg, M; Carracedo, A; Robledo, M; Llerena, A; Rodríguez-Antona, C

    2015-06-01

    Cytochrome P450 3A4 (CYP3A4) is a key drug-metabolizing enzyme. Loss-of-function variants have been reported as rare events, and the first demonstration of a CYP3A4 protein lacking functional activity is caused by CYP3A4*20 allele. Here we characterized the world distribution and origin of CYP3A4*20 mutation. CYP3A4*20 was determined in more than 4000 individuals representing different populations, and haplotype analysis was performed using CYP3A polymorphisms and microsatellite markers. CYP3A4*20 allele was present in 1.2% of the Spanish population (up to 3.8% in specific regions), and all CYP3A4*20 carriers had a common haplotype. This is compatible with a Spanish founder effect and classifies CYP3A4 as a polymorphic enzyme. This constitutes the first description of a CYP3A4 loss-of-function variant with high frequency in a population. CYP3A4*20 results together with the key role of CYP3A4 in drug metabolism support screening for rare CYP3A4 functional alleles among subjects with adverse drug events in certain populations. PMID:25348618

  6. Anthocyanins and their metabolites are weak inhibitors of cytochrome P450 3A4.

    PubMed

    Dreiseitel, Andrea; Schreier, Peter; Oehme, Anett; Locher, Sanja; Hajak, Goeran; Sand, Philipp G

    2008-12-01

    The cytochrome P450 enzyme cytochrome P450 3A4 (CYP3A4) controls the metabolism of about 60% of all drugs, and its inhibition may dramatically affect drug safety. Modulation of cytochrome P450 activity has been observed by constituents of fruit extracts including several flavonoids. The present investigation addresses CYP3A4 inhibition by anthocyanins, their aglycons, proanthocyanidins, and phenolic metabolites using a chemiluminescent assay. Test compounds inhibited CYP3A4 activity in a concentration-dependent manner featuring IC(50) values from 12.2 up to 7,842 microM. In the order of decreasing effect size, anthocyanidins were followed by anthocyanins, proanthocyanidins, and phenolic acids. When compared to earlier data on furanocoumarins from grapefruit extract, the inhibitory activity of tested anthocyanins, and anthocyanidins was shown to be about 10,000-fold weaker, and was negligible for phenolic acids (>100 000-fold weaker). Future studies are invited to address effects of the above flavonoids on other CYP isoforms for more detailed toxicity profiles. PMID:18727015

  7. Comparison of Paeoniflorin and Albiflorin on Human CYP3A4 and CYP2D6

    PubMed Central

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Akinyi, Olunga Mary

    2015-01-01

    Peony (Paeonia lactiflora Pall-) is a plant medicine and a functional food ingredient with wide application for more than 2000 years. It can be coadministrated with many other drugs, composed of traditional Chinese medicine compound such as shaoyao-gancao decoction. In order to explore the efficacy and safety of peony, effects of paeoniflorin and albiflorin (the principal components of peony) on cytochrome P450 (CYP) 3A4 and CYP2D6 were analyzed in human hepatoma HepG2 cells and evaluated from the level of recombinant CYP enzymes in vitro. The findings indicated that albiflorin possessed stronger regulation on the mRNA expression of CYP3A4 and CYP2D6 than paeoniflorin. For the protein level of CYP3A4, albiflorin showed significant induction or inhibition with the concentration increasing from 10−7 M to 10−5 M, but no remarkable variation was observed in paeoniflorin-treated group. Enzyme activity assay implied that both paeoniflorin and albiflorin could regulate CYP3A4 and CYP2D6 with varying degrees. The results showed that albiflorin should be given more attention because it may play a vital role on the overall efficacy of peony. The whole behavior of both paeoniflorin and albiflorin should be focused on ensuring the rationality and effectiveness of clinical application. PMID:26089940

  8. N-Heterocyclic Carbene Capture by Cytochrome P450 3A4.

    PubMed

    Jennings, Gareth K; Ritchie, Caroline M; Shock, Lisa S; Lyons, Charles E; Hackett, John C

    2016-07-01

    Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ≥7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

  9. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    SciTech Connect

    Petersen, Maria Skaalum Halling, Jonrit; Damkier, Per; Nielsen, Flemming; Grandjean, Philippe; Weihe, Pal; Brosen, Kim

    2007-10-15

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6{beta}-hydroxycortisol/cortisol (6{beta}-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6{beta}-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6{beta}-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6{beta}-OHC/FC ratios and {sigma}PCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only.

  10. Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4.

    PubMed

    Cheng, Chloe Y S; Slominski, Andrzej T; Tuckey, Robert C

    2016-05-01

    20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3. PMID:26970587

  11. Fungal lactone ring opening of 6', 7'-dihydroxybergamottin diminishes cytochrome P450 3A4 inhibitory activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furanocoumarins (FCs) are a class of aromatic compounds in grapefruit that inhibit human intestinal cytochrome P450 3A4 (CYP3A4). Since fungi metabolize polycyclic aromatic hydrocarbons, we hypothesized that certain fungi might also metabolize FCs into forms that may be inactive as CYP3A4 inhibitors...

  12. Elucidation of distinct ligand binding sites for cytochrome P450 3A4.

    PubMed

    Hosea, N A; Miller, G P; Guengerich, F P

    2000-05-23

    Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M(r) 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH(2)), bind with spectral changes indicative of Fe-NH(2) bonding. The binding of the YPFP-NH(2) N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M(r) 521) fits within P450 3A4. YPFP-NH(2) was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K(i) values similar to the spectral binding constant (K(s)) for YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of several typical P450 substrates with K(i) values 10-fold greater than the K(s) for binding YPFP-NH(2) and its K(i) for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH(2). YPFP-NH(2) inhibited the oxidations of midazolam at two different positions (1'- and 4-) with 20-fold different K(i) values. The differences in the K(i) values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH(2) to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH(2)) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and alpha-naphthoflavone occupying distinct sites. Midazolam appears to be able to

  13. Pharmacokinetics of ruboxistaurin are significantly altered by rifampicin-mediated CYP3A4 induction

    PubMed Central

    Yeo, Kwee Poo; Lowe, Stephen L; Lim, Ming Tung; Voelker, James R; Burkey, Jennifer L; Wise, Stephen D

    2006-01-01

    Aims The aim of this study was to evaluate the effect of rifampicin co-administration on the pharmacokinetics of ruboxistaurin and its active metabolite, N-desmethyl ruboxistaurin and, in addition, to compare the changes in pharmacokinetics of ruboxistaurin and N-desmethyl ruboxistaurin with the urinary 6β-hydroxycortisol : cortisol ratio. Ruboxistaurin is a specific protein-kinase-C β inhibitor in clinical development for the treatment of diabetic microvascular complications. Methods This was a two-period, one-sequence study. Sixteen healthy male subjects completed both study periods. In period one, a single 64 mg oral dose of ruboxistaurin was administered. In period two, 600 mg rifampicin was administered daily for 9 days, during which another single 64 mg ruboxistaurin dose was administered on day 7. Blood samples were collected and assayed for ruboxistaurin and N-desmethyl ruboxistaurin. CYP3A4 induction was assessed by ratios of urinary 6β-hydroxycortisol : cortisol (6β-OHC : C) obtained via 24 h and morning-spot sampling techniques. Results Following repeated doses of rifampicin, both the mean Cmax and AUC(0,∞) of ruboxistaurin were significantly reduced by approximately 95% (P ≤ 0.001). For the metabolite, the mean Cmax decreased by 68% (P ≤ 0.001), and AUC(0,∞) decreased by 77% (P ≤ 0.001). The tmax values did not appear affected. The 6β-OHC : C ratios from both 24 h and morning spot methods increased significantly, consistent with CYP3A4 induction. Conclusions The effect of rifampicin co-administration on the exposure of ruboxistaurin is consistent with ruboxistaurin being a substrate of CYP3A4. Therefore, co-administration with known CYP3A4 inducing agents (rifampicin, carbamazepine, phenobarbital, etc.) may decrease the concentrations of ruboxistaurin and N-desmethyl-ruboxistaurin. In this study, 6β OHC : C ratios substantially underestimated the impact of rifampicin on ruboxistaurin. PMID:16433874

  14. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides.

    PubMed

    Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

    2011-11-15

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p<0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37±2.15 vs. 6.24±1.37 tail% DNA, p<0.001). Further, the workers with CYP2D6*3PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p<0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. PMID:21907728

  15. Mixing Apples and Oranges: Analysis of Heterotropic Cooperativity in Cytochrome P450 3A4

    PubMed Central

    Frank, Daniel J.; Denisov, Ilia G.; Sligar, Stephen G.

    2016-01-01

    Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme’s catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, α-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38 µM respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities, and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates. PMID:19560436

  16. Mixing apples and oranges: Analysis of heterotropic cooperativity in cytochrome P450 3A4.

    PubMed

    Frank, Daniel J; Denisov, Ilia G; Sligar, Stephen G

    2009-08-15

    Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme's catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, alpha-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38 microM, respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates. PMID:19560436

  17. PROCEEDINGS: 1991 INTERNATIONAL CONFERENCE ON MUNICIPAL WASTE COMBUSTION - VOLUME 1. SESSIONS P, 0, 1A, 2A, 3A, 4A, 6A, 6B, 9C, AND 10B

    EPA Science Inventory

    The three-volumes document 82 presentations by authors from 15 countries at the Second International Conference on Municipal Waste Combustion (MWC) in Tampa, Florida, April 16-19, 1991. The Conference fostered the exchange of current information on research concerning MWC, ash di...

  18. Parkinson's disease and CYP1A2 activity

    PubMed Central

    Forsyth, J T; Grünewald, R A; Rostami-Hodjegan, A; Lennard, M S; Sagar, H J; Tucker, G T

    2000-01-01

    Aims MPTP, a neurotoxin which induces parkinsonism is partially metabolized by the enzyme CYP1A2. Smoking appears to protect against Parkinson's disease (PD) and cigarette smoke induces CYP1A2 activity. Thus, we investigated the hypothesis that idiopathic PD is associated with lower CYP1A2 activity using caffeine as a probe compound. Methods CYP1A2 activity was assessed using saliva paraxanthine (PX) to caffeine (CA) ratios. Caffeine half-life was also estimated from salivary concentrations of caffeine at 2 and 5 h post dose. 117 treated and 40 untreated patients with PD and 105 healthy control subjects were studied. Results PX/CA ratios were 0.57, 0.93 and 0.77 in treated patients, untreated patients and healthy control subjects, respectively, with no significant differences between study groups (95% CI: treated patients vs controls −0.24, 0.57; untreated patients vs controls −0.75, 0.35). However, patients with PD (treated or untreated) had caffeine half-lives shorter than that in controls (treated patients: 262 min, untreated patients: 244 min, controls: 345 min; 95% CI: controls vs treated patients 23, 143 (P = 0.003); controls vs untreated patients 19, 184 (P = 0.011)). Amongst the patients with PD, caffeine half-life was also inversely related to the age of onset of disease (P = 0.012); gender and concomitant drugs did not influence this significantly. Conclusions Based on PX/CA ratio, there was no evidence of decreased CYP1A2 activity in patients compared with control subjects. The observed decrease in the elimination half-life of caffeine in PD may be caused by increased CYP2E1 activity, an enzyme that also contributes to the metabolism of caffeine. The latter warrants further investigation. PMID:11012552

  19. Affine Kac-Moody symmetric spaces related with A1^{(1)}, A2^{(1)},} A2^{(2)}

    NASA Astrophysics Data System (ADS)

    Nayak, Saudamini; Pati, K. C.

    2014-08-01

    Symmetric spaces associated with Lie algebras and Lie groups which are Riemannian manifolds have recently got a lot of attention in various branches of Physics for their role in classical/quantum integrable systems, transport phenomena, etc. Their infinite dimensional counter parts have recently been discovered which are affine Kac-Moody symmetric spaces. In this paper we have (algebraically) explicitly computed the affine Kac-Moody symmetric spaces associated with affine Kac-Moody algebras A1^{(1)}, A2^{(1)}, A2^{(2)}. We hope these types of spaces will play similar roles as that of symmetric spaces in many physical systems.

  20. The effect of the CYP1A2 *1F mutation on CYP1A2 inducibility in pregnant women

    PubMed Central

    Nordmark, Anna; Lundgren, Stefan; Ask, Birgitta; Granath, Fredrik; Rane, Anders

    2002-01-01

    Aims To investigate the influence of the CYP1A2*1F mutation on CYP1A2 activity in smoking and nonsmoking pregnant women. Methods Pregnant women (n = 904) who served as control subjects in a case-control study of early fetal loss were investigated. They were phenotyped for CYP1A2 using dietary caffeine and the urinary ratio AFMU + 1X + 1 U/1,7 U. An assay for CYP1A2*1F using 5′-nuclease assay (Taqman) was developed to genotype the population. Results The frequencies of *1 A and *1F alleles among Swedish women were 0.29 and 0.71, respectively. There was no statistically significant difference in CYP1A2 activity between the genotypes, although a trend towards enhanced activity was observed in *1F/*1F (log MRc 0.77) and *1F/*1 A (log MRc 0.82) genotypes compared with the *1 A/*1 A genotype (log MRc 0.71) (anovaP = 0.07). The mean difference between the *1 A homozygotes and the heterozygotes was 0.11 [95% confidence interval of the difference: (−0.21, −0.01)] and that between the *1 A and *1F homozygotes was 0.05 [95% confidence interval of the difference: (−0.13, 0.03)]. No significant effect (P = 0.22) of the *1F on CYP1A2 activity was observed in smokers, tested using an interaction term (smoking * genotype) in the anova model (*1F/*1F log MRc 0.79, *1F/*1 A log MRc 0.86, and *1 A/*1 A log MRc 0.73). In smokers, there was no difference in ratio between homozygotes for the *1 A and *1F alleles [mean difference −0.06; 95% confidence interval of the difference: −0.22, 0.11] or between *1 A/*1 A and *1 A/*1F genotypes [mean difference −0.13; 95% confidence interval of the difference: −0.29, 0.04]. Conclusions The effect of the CYP1A2*1F mutation on CYP1A2 activity in smoking pregnant women could not be confirmed. PMID:12445029

  1. Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

    PubMed

    Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda; Halpert, James R; Hubbell, Wayne L

    2016-04-12

    We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes. PMID:27074675

  2. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

    SciTech Connect

    Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

    2011-11-15

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 {+-} 2.15 vs. 6.24 {+-} 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: Black-Right-Pointing-Pointer Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. Black-Right-Pointing-Pointer Workers exposed to some OPs demonstrated increased DNA damage. Black-Right-Pointing-Pointer CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. Black-Right-Pointing-Pointer Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.

  3. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    SciTech Connect

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  4. Induction of human CYP3A4 by huperzine A, ligustrazine and oridonin through pregnane X receptor-mediated pathways.

    PubMed

    Zhang, Yi-Wen; Bao, Mei-Hua; Wang, Guo; Qu, Qiang; Zhou, Hong-Hao

    2014-07-01

    The pregnane X receptor (PXR) is a key regulator of CYP3A4, which is involved in catalyzing the metabolic conversion of a number of endogenous substrates. In this study, we screened 22 compounds isolated from traditional Chinese herbal medicines using luciferase reporter gene assays for inspecting their capabilities in inducing PXR-mediated transactivation of CYP3A4 expression. In addition, the mRNA and protein expressions of CYP3A4 and PXR as well as the enzymatic activites of CYP3A4 were analyzed by real-time PCR, Western blot analysis and UPLC-MS/MS-based metabolite assay in LS174T cells. Huperzine A, ligustrazine and oridonin were identified to be the inducers of CYP3A4. These compounds induced the CYP3A4 reporter luciferase activity, and up-regulated CYP3A4 mRNA and protein levels significantly. Besides, huperzine A, ligustrazine and oridonin significantly up-regulated enzymatic activities of CYP3A4. However, the three compounds showed no effects on PXR mRNA and protein expression. To our knowledge, it is the first identification of these three compounds as PXR activators to induce CYP3A4. These results indicate that huperzine A, ligustrazine and oridonin induced CYP3A4 expression and activation via PXR dependent pathways, and might contribute to drug-drug interactions. PMID:25073399

  5. Redox Potential Control by Drug Binding to Cytochrome P450 3A4

    PubMed Central

    Das, Aditi; Grinkova, Yelena V.; Sligar, Stephen G.

    2008-01-01

    The cytochrome P450s are ubiquitous heme proteins that utilize two reducing equivalents to cleave a ferrous iron - dioxygen complex to produce a single water molecule with the insertion of one oxygen atom into a bound substrate. For the case of soluble cytochrome P450 CYP101, it has been shown that there is a linear free energy relationship between heme redox potential and the spin state of the ferric protein. However, the universality of this relationship has been challenged in the case of mammalian enzymes. Most cytochrome P450s are integral membrane proteins, and detailed redox potential measurements have proved difficult due protein aggregation or the necessary presence of detergent. In this communication we utilize a soluble nanometer scale membrane bilayer disc (Nanodisc) to stabilize monomeric human cytochrome P450 CYP3A4. The Nanodisc system allows facile redox potential measurements to be made on substrate-free CYP3A4 as well as with several drug molecules bound at the active site. We show that substrate binding can dramatically effect the redox potential of the heme protein through modulation of the ferric spin state. A linear free energy relationship is observed, analogous to that noted for the soluble P450s, indicating a common mechanism for this linkage and providing a means for control of electron input in response to the presence of a metabolizable substrate, this potentially limiting the unwanted production of reduced oxygen species. PMID:17948999

  6. Dipeptide Prodrug Approach to Evade Efflux Pumps and CYP3A4 Metabolism of Lopinavir

    PubMed Central

    Patel, Mitesh; Sheng, Ye; Mandava, Nanda K.; Pal, Dhananjay; Mitra, Ashim K.

    2014-01-01

    Oral absorption of lopinavir (LPV) is limited due to P-glycoprotein (P-gp) and multidrug resistance-associated protein2 (MRP2) mediated efflux by intestinal epithelial cells. Moreover, LPV is extensively metabolized by CYP3A4 enzymes. In the present study, dipeptide prodrug approach was employed to circumvent efflux pumps (P-gp and MRP2) and CYP3A4 mediated metabolism of LPV. Valine-isoleucine-LPV (Val-Ile-LPV) was synthesized and identified by LCMS and NMR techniques. The extent of LPV and Val-Ile-LPV interactions with P-gp and MRP2 was studied by uptake and transport studies across MDCK-MDR1 and MDCK-MRP2 cells. To determine the metabolic stability, time and concentration dependent degradation study was performed in liver microsomes. Val-Ile-LPV exhibited significantly higher aqueous solubility relative to LPV. This prodrug generated higher stability under acidic pH. Val-Ile-LPV demonstrated significantly lower affinity towards P-gp and MRP2 relative to LPV. Transepithelial transport of Val-Ile-LPV was significantly higher in the absorptive direction (apical to basolateral) relative to LPV. Importantly, Val-Ile-LPV was recognized as an excellent substrate by peptide transporter. Moreover, Val-Ile-LPV displayed significantly higher metabolic stability relative to LPV. Results obtained from this study suggested that dipeptide prodrug approach is a viable option to elevate systemic levels of LPV following oral administration PMID:25261710

  7. Trainable structure-activity relationship model for virtual screening of CYP3A4 inhibition.

    PubMed

    Didziapetris, Remigijus; Dapkunas, Justas; Sazonovas, Andrius; Japertas, Pranas

    2010-11-01

    A new structure-activity relationship model predicting the probability for a compound to inhibit human cytochrome P450 3A4 has been developed using data for >800 compounds from various literature sources and tested on PubChem screening data. Novel GALAS (Global, Adjusted Locally According to Similarity) modeling methodology has been used, which is a combination of baseline global QSAR model and local similarity based corrections. GALAS modeling method allows forecasting the reliability of prediction thus defining the model applicability domain. For compounds within this domain the statistical results of the final model approach the data consistency between experimental data from literature and PubChem datasets with the overall accuracy of 89%. However, the original model is applicable only for less than a half of PubChem database. Since the similarity correction procedure of GALAS modeling method allows straightforward model training, the possibility to expand the applicability domain has been investigated. Experimental data from PubChem dataset served as an example of in-house high-throughput screening data. The model successfully adapted itself to both data classified using the same and different IC₅₀ threshold compared with the training set. In addition, adjustment of the CYP3A4 inhibition model to compounds with a novel chemical scaffold has been demonstrated. The reported GALAS model is proposed as a useful tool for virtual screening of compounds for possible drug-drug interactions even prior to the actual synthesis. PMID:20814717

  8. Pitavastatin Concentrations Are Not Increased by CYP3A4 Inhibitor Itraconazole in Healthy Subjects.

    PubMed

    Nakagawa, Shunji; Gosho, Masahiko; Inazu, Yuji; Hounslow, Neil

    2013-04-01

    Itraconazole is a synthetic triazole antifungal agent which is known to be a potent inhibitor of cytochrome P450 (CYP) 3A4, and may cause drug-drug interactions with the many drugs metabolized by this route, including some statins. In this study, the influence of concomitant administration of a single oral dose of pitavastatin with itraconazole at steady state was investigated to determine the potential for pharmacokinetic interaction and any effects on safety. Eighteen subjects were enrolled into the study. The AUC and Cmax of pitavastatin alone were 138 ng h/mL and 63.8 ng/mL, and pitavastatin with itraconazole were 106 ng h/mL and 49.5 ng/mL, respectively. Comparison of the 90% confidence interval of the geometric mean ratio of AUC0-t and Cmax against a standard reference of 0.80-1.25 demonstrated that the lower limit was breached for both pitavastatin and its lactone metabolite (0.71-0.84 and 0.69-0.88 for AUC0-t and Cmax , respectively, for pitavastatin, 0.86-0.97 and 0.76-0.86 for AUC0-t and Cmax , respectively, for pitavastatin lactone). The safety and tolerability of pitavastatin was not affected by co-administration with itraconazole. This study suggests that pitavastatin is not a CYP3A4 substrate in humans. PMID:27121674

  9. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    PubMed Central

    Liu, Wenqin; Shi, Jian; Zhu, Lijun; Dong, Lingna; Luo, Feifei; Zhao, Min; Wang, Ying; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2015-01-01

    Oxymatrine (OMT) is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT), and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs) and human intestinal microsomes (HIMs) and the cytochrome P450 (CYP) isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT. PMID:26586934

  10. MDR- and CYP3A4-mediated drug-herbal interactions.

    PubMed

    Pal, Dhananjay; Mitra, Ashim K

    2006-03-27

    According to recent epidemiological reports, almost 40% of American population use complimentary and alternative medicine (CAM) during their lifetime. Patients detected with HIV or cancer often consume herbal products especially St. John's wort (SJW) for antidepressants in combination with prescription medicines. Such self-administered herbal products along with prescribed medicines raise concerns of therapeutic activity due to possible drug-herbal interactions. P-glycoprotein (P-gp) and cytochrome P450 3A4 (CYP3A4) together constitute a highly efficient barrier for many orally absorbed drugs. Available literature, clinical reports and in vitro studies from our laboratory indicate that many drugs and herbal active constituents are substrates for both P-gp and CYP3A4. Results from clinical studies and case reports indicate that self-administered SJW reduce steady state plasma concentrations of amitriptyline, cyclosporine, digoxin, fexofenadine, amprenavir, indonavir, lopinavir, ritonavir, saquinavir, benzodiazepines, theophyline, irinotecan, midazolan and warfarin. This herbal agent has been also reported to cause bleeding and unwanted pregnancies when concomitantly administered with oral contraceptives. Most of these medicinal agents and SJW are substrates for P-gp and/or CYP3A4. In vitro studies from our laboratory suggest that short-term exposure with pure herbal agents such as hypericin, kaempferol and quercetin or extract of SJW resulted in higher uptake or influx of ritonavir and erythromycin. Hypericin, kaempferol and quercetin also caused a remarkable inhibition of cortisol metabolism with the percent intact cortisol values of 64.58%, 89.6% and 90.1%, respectively, during short-term in vitro experiments. Conversely, long-term exposure of herbal agents (hyperforin, kaempferol and quercetin) showed enhanced expression of CYP3A4 mRNA in Caco-2 cells. In another study, we observed that long-term exposure of hypericin, kaempferol, quercetin and silibinin resulted

  11. Trichostatin A, a histone deacetylase inhibitor stimulate CYP3A4 proximal promoter activity in Hepa-I cells.

    PubMed

    Ahn, Mee Ryung; Kim, Dae-Kee; Sheen, Yhun Yhong

    2004-04-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant CYPs in human liver, comprising approximately 30% of the total liver CYPs contents and is involved in the metabolism of more than 60% of currently used therapeutic drugs. However, the molecular mechanisms underlying regulation of CYP3A4 gene expression have not been understood. Thus, this study has been carried out to gain the insight of the molecular mechanism of CYP3A4 gene expression, investigating if the histone deacetylation is involved in the regulation of CYP3A4 gene expression by proximal promoter. Also SXR was investigated to see if they were involved in the regulation of CYP3A4 proximal promoter activity. Hepa-I cells were transfected with a plasmid containing approximately 1 kb of the human CYP3A4 proximal promoter region (863 to +64 bp) cloned in front of a reporter gene, luciferase, in the presence or absence of SXR. Transfected cells were treated with CYP3A4 inducers such as rifampicin, PCN and RU 486, in order to examine the regulation of CYP3A4 gene expression in the presence or absence of trichostatin A (TSA). In Hepa-I cells, CYP3A4 inducers increased modestly the luciferase activity when TSA was co-treated, but this increment was not enhanced by SXR cotransfection. Taken together, these results indicated that the inhibition of histone deacetylation was required to SXR-mediated increase in CYP3A4 proximal promoter region when rifampicin, or PCN was treated. Further a trans-activation by SXR may demand other species-specific transcription factors. PMID:15180307

  12. Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene expression by activating human PXR

    PubMed Central

    Pondugula, Satyanarayana R.; Flannery, Patrick C.; Abbott, Kodye L.; Coleman, Elaine S.; Mani, Sridhar; Samuel, Temesgen; Xie, Wen

    2015-01-01

    Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important role in mediating adverse drug interactions. Given the common use of natural products as part of adjunct human health behavior, there is a growing concern about natural products for their potential to induce undesired drug interactions through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied whether 3,3′-diindolylmethane (DIM), a natural health supplement, could induce hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal cells. DIM, at its physiologically relevant concentrations, not only induced hPXR transactivation of CYP3A4 promoter activity but also induced gene expression of CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate rhodamine 123, suggesting that DIM induces the functional expression of MDR1. Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4 and MDR1 in an hPXR-dependent manner. Together, these results support our conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in conjunction with other medications metabolized and transported via CYP3A4 and MDR1, respectively. PMID:25542144

  13. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  14. Tritium analyses of COBRA-1A2 beryllium pebbles

    SciTech Connect

    Baldwin, D.L.

    1998-03-01

    Selected tritium measurements have been completed for the COBRA-1A2 experiment C03 and D03 beryllium pebbles. The completed results, shown in Tables 1, 2, and 3, include the tritium assay results for the 1-mm and 3-mm C03 pebbles, and the 1-mm D03 pebbles, stepped anneal test results for both types of 1-mm pebbles, and the residual analyses for the stepped-anneal specimens. All results have been reported with date-of-count and are not corrected for decay. Stepped-anneal tritium release response is provided in addenda.

  15. The role of CYP3A4 in the biotransformation of bile acids and therapeutic implication for cholestasis

    PubMed Central

    Zhao, Kong-Nan; Chen, Chen

    2014-01-01

    CYP3A4 is a major cytochrome P450. It catalyses a broad range of substrates including xenobiotics such as clinically used drugs and endogenous compounds bile acids. Its function to detoxify bile acids could be used for treating cholestasis, which is a condition characterised by accumulation of bile acids. Although bile acids have important physiological functions, they are very toxic when their concentrations are excessively high. The accumulated bile acids in cholestasis can cause liver and other tissue injuries. Thus, control of the concentrations of bile acids is critical for treatment of cholestasis. CYP3A4 is responsively upregulated in cholestasis mediated by the nuclear receptors farnesol X receptor (FXR) and pregnane X receptor (PXR) as a defence mechanism. However, the regulation of CYP3A4 is complicated by estrogen, which is increased in cholestasis and down regulates CYP3A4 expression. The activity of CYP3A4 is also inhibited by accumulated bile acids due to their property of detergent effect. In some cholestasis cases, genetic polymorphisms of the CYP3A4 and PXR genes may interfere with the adaptive response. Further stimulation of CYP3A4 activity in cholestasis could be an effective approach for treatment of the disease. In this review, we summarise recent progress about the roles of CYP3A4 in the metabolism of bile acids, its regulation and possible implication in the treatment of cholestasis. PMID:25332983

  16. Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice.

    PubMed

    Cheng, Jie; Fang, Zhong-Ze; Kim, Jung-Hwan; Krausz, Kristopher W; Tanaka, Naoki; Chiang, John Y L; Gonzalez, Frank J

    2014-03-01

    Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr(ΔIEpC)) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr(ΔIEpC) mice to produce the Vdr(ΔIEpC)/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr(ΔIEpC), and Vdr(ΔIEpC)/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr(ΔIEpC) mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr(ΔIEpC)/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity. PMID:24343899

  17. Magic-Angle Spinning Solid-State NMR Spectroscopy of Nanodisc– Embedded Human CYP3A4

    PubMed Central

    Kijac, Aleksandra; Li, Ying; Sligar, Stephen G.; Rienstra, Chad M.

    2008-01-01

    Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because x-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Δ3−12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields ∼2.5 mg of the enzymatically active, uniformly 13C, 15N-enriched CYP3A4 from a liter of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are ∼0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free x-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality. PMID:17985934

  18. High Content Imaging and Analysis Enable Quantitative In Situ Assessment of CYP3A4 Using Cryopreserved Differentiated HepaRG Cells

    PubMed Central

    Ranade, Aarati R.; Wilson, Melinda S.; McClanahan, Amy M.; Ball, Andrew J.

    2014-01-01

    High-throughput imaging-based hepatotoxicity studies capable of analyzing individual cells in situ hold enormous promise for drug safety testing but are frequently limited by a lack of sufficient metabolically competent human cells. This study examined cryopreserved HepaRG cells, a human liver cell line which differentiates into both hepatocytes and biliary epithelial cells, to determine if these cells may represent a suitable metabolically competent cellular model for novel High Content Analysis (HCA) applications. Characterization studies showed that these cells retain many features characteristic of primary human hepatocytes and display significant CYP3A4 and CYP1A2 induction, unlike the HepG2 cell line commonly utilized for HCA studies. Furthermore, this study demonstrates that CYP3A4 induction can be quantified via a simple image analysis-based method, using HepaRG cells as a model system. Additionally, data demonstrate that the hepatocyte and biliary epithelial subpopulations characteristic of HepaRG cultures can be separated during analysis simply on the basis of nuclear size measurements. Proof of concept studies with fluorescent cell function reagents indicated that further multiparametric image-based assessment is achievable with HepaRG. In summary, image-based screening of metabolically competent human hepatocyte models cells such as HepaRG offers novel approaches for hepatotoxicity assessment and improved drug screening tools. PMID:25276124

  19. Interaction of Human Cytochrome P450 3A4 with Hydrophobicity Probe Nile Red Shows Heterogeneous, Strong Binding

    NASA Astrophysics Data System (ADS)

    Hansen, Jennifer; Guengerich, F.; Martin, Martha; Marsch, Glenn

    2009-03-01

    Human cytochrome P450 3A4 (CYP 3A4) binds an unusually wide variety of substrates, and metabolizes about 50% of all drugs. Steady-state fluorescence spectra were acquired for complexes of CYP 3A4 and the fluorescence probe Nile Red. Difference fluorescence spectra and Hill plots were generated, and Hill coefficients were determined. The fluorescence from multiple Nile Red bound states was observed, with all bound states having higher emission energies than the fluorescence from free Nile Red. Nile Red was titrated into 150nM CYP 3A4, and fluorescence difference spectra showed the quenching of CYP 3A4 tryptophan fluorescence by Nile Red. CYP 3A4 was also added to Nile Red, and changes in the Nile Red fluorescence spectra were monitored. The dissociation constant showed tight binding, with Kd = 44nM. Good fits to the Hill plots were obtained with n = 1, suggesting non-cooperative binding. This study revealed strong, heterogeneous, non-cooperative binding of Nile Red to CYP 3A4.

  20. The effect of interferon-{alpha} on the expression of cytochrome P450 3A4 in human hepatoma cells

    SciTech Connect

    Flaman, Anathea S.; Gravel, Caroline; Hashem, Anwar M.; Tocchi, Monika; Li Xuguang

    2011-06-01

    Interferon {alpha} (IFN{alpha}) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFN{alpha} treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFN{alpha} on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFN{alpha} inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFN{alpha} did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFN{alpha} does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFN{alpha} down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFN{alpha} suppresses CYP3A4 expression, caution is warranted when IFN{alpha} is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

  1. Mechanism-Based Inactivation of Human Cytochrome P450 3A4 by Two Piperazine-Containing Compounds

    PubMed Central

    Bolles, Amanda K.; Fujiwara, Rina; Briggs, Erran D.; Nomeir, Amin A.

    2014-01-01

    Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4. PMID:25273356

  2. A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Mansell, Sara E; Leon-Ponte, Matilde; Yu, Janice; Pin, Christopher L; Adams, Paul C; Kim, Richard B; Tirona, Rommel G

    2016-10-01

    Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD. PMID:27482056

  3. Study Liver Cytochrome P450 3A4 Inhibition and Hepatotoxicity Using DMSO-Differentiated HuH-7 Cells.

    PubMed

    Liu, Yitong

    2016-01-01

    Metabolically competent, inexpensive, and robust in vitro cell models are needed for studying liver drug-metabolizing enzymes and hepatotoxicity. Human hepatoma HuH-7 cells develop into a differentiated in vitro model resembling primary human hepatocytes after a 2-week dimethyl sulfoxide (DMSO) treatment. DMSO-treated HuH-7 cells express elevated cytochrome P450 3A4 (CYP3A4) enzyme gene expression and activity compared to untreated HuH-7 cells. This cell model could be used to study CYP3A4 inhibition by reversible and time-dependent inhibitors, including drugs, food-related substances, and environmental chemicals. The DMSO-treated HuH-7 model is also a suitable tool for investigating hepatotoxicity. This chapter describes a detailed methodology for developing DMSO-treated HuH-7 cells, which are subsequently used for CYP3A4 inhibition and hepatotoxicity studies. PMID:27518624

  4. SPR and electrochemical analyses of interactions between CYP3A4 or 3A5 and cytochrome b5

    NASA Astrophysics Data System (ADS)

    Gnedenko, O. V.; Yablokov, E. O.; Usanov, S. A.; Mukha, D. V.; Sergeev, G. V.; Bulko, T. V.; Kuzikov, A. V.; Moskaleva, N. E.; Shumyantseva, V. V.; Ivanov, A. S.; Archakov, A. I.

    2014-02-01

    The combination of SPR biosensor with electrochemical analysis was used for the study of protein-protein interaction between cytochromes CYP3A4 or 3А5 and cytochromes b5: the microsomal, mitochondrial forms of this protein, and 2 ≪chimeric≫ proteins. Kinetic constants of CYP3A4 and CYP3А5 complex formation with cytochromes b5 were determined by the SPR biosensor. Essential distinction between CYP3A4 and CYP3A5 was observed upon their interactions with mitochondrial cytochrome b5. The electrochemical analysis of CYP3A4, CYP3A5, and cytochromes b5 immobilized on screen printed graphite electrodes modified with membranous matrix revealed that these proteins have very close reduction potentials -0.435 to -0.350 V (vs. Ag/AgCl).

  5. Arsenite and its metabolites, MMA{sup III} and DMA{sup III}, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

    SciTech Connect

    Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.; Ramirez, P.; Vega, L.; Elizondo, G.

    2009-09-01

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA{sup III}) and dimethylarsinous acid (DMA{sup III}) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA{sup III} induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA{sup III} increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA{sup III} induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

  6. Arsenite and its metabolites, MMA(III) and DMA(III), modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice.

    PubMed

    Medina-Díaz, I M; Estrada-Muñiz, E; Reyes-Hernández, O D; Ramírez, P; Vega, L; Elizondo, G

    2009-09-01

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA(III) induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA(III) increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA(III) induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice. PMID:19084030

  7. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

    PubMed Central

    Ande, Anusha; Wang, Lei; Vaidya, Naveen K.; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  8. Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

    PubMed Central

    Liu, Yitong; Flynn, Thomas J.; Xia, Menghang; Wiesenfeld, Paddy L.; Ferguson, Martine S.

    2016-01-01

    A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity. PMID:26377104

  9. Mechanism-based inhibition of recombinant human cytochrome P450 3A4 by tomato juice extract.

    PubMed

    Sunaga, Katsuyoshi; Ohkawa, Kenichi; Nakamura, Kenichi; Ohkubo, Atsuko; Harada, Sonoko; Tsuda, Tadashi

    2012-01-01

    This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6β-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6β-hydroxylation activity by 19.3-26.2% with 0-min preincubation. Residual activity was strongly reduced by 69.9-83.5% at 20-min preincubation, a reduction similar to that of grapefruit juice extract, known to contain constituents of mechanism-based inhibitors. One juice extract (tomato juice C) showed irreversible dose- and preincubation time-dependent and partial nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inhibition of CYP3A4 activity. Furthermore, we examined whether the CYP3A4 inhibitory effect of tomato juice was substrate dependent by examining midazolam 1'-hydroxylation activity and nifedipine oxidation activity, in addition to testosterone 6β-hydroxylation activity. Tomato juice showed a potent inhibitory effect on nifedipine oxidation activity, which was comparable to that on testosterone 6β-hydroxylation activity; however, it showed a weak inhibitory effect on midazolam 1'-hydroxylation activity. We conclude that tomato juice contains one or more mechanism-based and competitive inhibitor(s) of CYP3A4. Additionally, significant CYP3A4 inhibitory activity did not result from lycopene, a major compound in tomato. Although the active compound was uncertain, a strong CYP3A4 inhibitory activity was observed in other solanaceous plants, i.e., potato, eggplant, sweet pepper, and capsicum. Therefore, responsible compounds in tomato are likely commonly shared among solanaceous vegetables. PMID:22382318

  10. Pharmacogenomics of Cytochrome P450 3A4: Recent Progress Toward the “Missing Heritability” Problem

    PubMed Central

    Klein, Kathrin; Zanger, Ulrich M.

    2013-01-01

    CYP3A4 is the most important drug metabolizing enzyme in adult humans because of its prominent expression in liver and gut and because of its broad substrate specificity, which includes drugs from most therapeutic categories and many endogenous substances. Expression and function of CYP3A4 vary extensively both intra- and interindividually thus contributing to unpredictable drug response and toxicity. A multitude of environmental, genetic, and physiological factors are known to influence CYP3A4 expression and activity. Among the best predictable sources of variation are drug–drug interactions, which are either caused by pregnane X-receptor (PXR), constitutive androstane receptor (CAR) mediated gene induction, or by inhibition through coadministered drugs or other chemicals, including also plant and food ingredients. Among physiological and pathophysiological factors are hormonal status, age, and gender, the latter of which was shown to result in higher levels in females compared to males, as well as inflammatory processes that downregulate CYP3A4 transcription. Despite the influence of these non-genetic factors, the genetic influence on CYP3A4 activity was estimated in previous twin studies and using information on repeated drug administration to account for 66% up to 88% of the interindividual variation. Although many single nucleotide polymorphisms (SNPs) within the CYP3A locus have been identified, genetic association studies have so far failed to explain a major part of the phenotypic variability. The term “missing heritability” has been used to denominate the gap between expected and known genetic contribution, e.g., for complex diseases, and is also used here in analogy. In this review we summarize CYP3A4 pharmacogenetics/genomics from the early inheritance estimations up to the most recent genetic and clinical studies, including new findings about SNPs in CYP3A4 (*22) and other genes (P450 oxidoreductase (POR), peroxisome proliferator

  11. Molecular docking of chemotherapeutic agents to CYP3A4 in non-small cell lung cancer.

    PubMed

    Subhani, Syed; Jamil, Kaiser

    2015-07-01

    CYP3A4, a "heme" containing isoform, abundantly found in the liver, gastro-intestinal tract, lungs and renal cells, also known as drug metabolising enzyme (DME) may be responsible for the disease progression in cancers such as lung cancer. Hence, we have targeted this protein for improving drug selection and in preventing adverse reactions. The aim of this study was to examine chemotherapeutic drug binding to CYP3A4 and the interactions therein. We have used Schrödinger suite 2014, to perform molecular docking of human CYP3A4, by Induced Fit Docking using gemcitabine, cisplatin, carboplatin, docetaxel and paclitaxel drugs. We evaluated drug-binding affinities using Prime/MMGBSA and using these scores we compared the affinities of combination therapies against CYP3A4. Analysis of the docking results showed gemcitabine>carboplatin>cisplatin as the order of binding affinities, with gemcitabine having the best docking score. Interestingly, docetaxel and paclitaxel did not bind to CYP3A4*1B. The combination drug-binding affinity analysis showed gemcitabine+carboplatin to have the best docking score and hence, efficacy. Our investigation has identified the residue Arg 105 to be more frequently involved in drug binding to CYP3A4. Our results suggest that gemcitabine or combination of gemcitabine+carboplatin could serve as an excellent therapy against CYP3A4 in NSCLC patients. Thus, our study depicts binding of chemotherapeutic drugs to CYP3A4 and has identified the residues that may be targeted for therapy in NSCLC patients. PMID:26211584

  12. Relationship between genotypes Sult1a2 and Cyp2d6 and tamoxifen metabolism in breast cancer patients.

    PubMed

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen. PMID:23922954

  13. Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

    PubMed Central

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen. PMID:23922954

  14. Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

    PubMed

    Budzinski, Jason W; Trudeau, Vance L; Drouin, Cathy E; Panahi, Mitra; Arnason, J Thor; Foster, Brian C

    2007-09-01

    In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions. PMID:18066144

  15. Furanocoumarin derivatives in Kampo extract medicines inhibit cytochrome P450 3A4 and P-glycoprotein.

    PubMed

    Iwanaga, Kazunori; Hayashi, Manami; Hamahata, Yukimi; Miyazaki, Makoto; Shibano, Makio; Taniguchi, Masahiko; Baba, Kimiye; Kakemi, Masawo

    2010-08-01

    Furanocoumarins in grapefruit are known to show inhibitory effects against P-glycoprotein (P-gp) and CYP3A4 in intestinal epithelial cells; however, furanocoumarin derivatives are widely contained in the plants of Rutaceae and Umbelliferae families, which are used as components of Kampo extract medicines. In this study, we investigated the inhibitory effects of 12 furanocoumarins extracted from plants in the Umbelliferae family against P-gp and CYP3A4 activity. Furthermore, we studied their inhibitory effect on P-gp when furanocoumarins are used as Kampo extract medicine rather than as an isolated single compound. From screening of the CYP3A4 inhibitory effect, notopterol and rivulobirin A, the only dimer types of furanocoumarin, were found to be potent inhibitors of CYP3A4. On the other hand, byakangelicol and rivulobirin A showed strong P-gp inhibition from the screening of P-gp inhibitor evaluated by quinidine permeation through the Caco-2 monolayer; however, the chemical structural relationship of furanocoumarins between P-gp and CYP3A4 inhibitory effects could not be obtained. We also investigated the effect of these furanocoumarins on the transport of digoxin through the Caco-2 monolayer. The inhibitory effect of rivulobirin A was more potent than that of byakangelicol. Application of either Senkyu-cha-cho-san or Sokei-kakketsu-to, which are composed of herbal remedies in the Umbelliferae group, significantly decreased the efflux ratio of digoxin. In conclusion, it was found that some furanocoumarins extracted from the plants in the Umbelliferae family strongly inhibited P-gp and CYP3A4. Kampo extract medicines containing herbal remedies belonging to the Umbelliferae family may cause a drug-drug interaction with P-gp or a CYP3A4 substrate drug. PMID:20463004

  16. Co-medication of statins and CYP3A4 inhibitors before and after introduction of new reimbursement policy

    PubMed Central

    Devold, Helene M; Molden, Espen; Skurtveit, Svetlana; Furu, Kari

    2009-01-01

    AIMS To assess the prevalence of co-medication of statins and CYP3A4 inhibitors before and after introduction of a new Norwegian reimbursement policy, which states that all patients should be prescribed simvastatin as first-line lipid-lowering therapy. METHODS Data from patients receiving simvastatin, lovastatin, pravastatin, fluvastatin or atorvastatin in 2004 and 2006, including co-medication of potent CYP3A4 inhibitors, were retrieved from the Norwegian Prescription Database covering the total population of Norway. Key measurements were prevalence of continuous statin use (two or more prescriptions on one statin) and proportions of different statin types among all patients and those co-medicated with CYP3A4 inhibitors. RESULTS In 2004, 5.9% (n = 272 342) of the Norwegian population received two or more prescriptions on one statin compared with 7.0% (n = 324 267) in 2006. The relative number of simvastatin users increased from 39.7% (n = 112 122) in 2004 to 63.1% (n = 226 672) in 2006. A parallel increase was observed within the subpopulation co-medicated with statins and CYP3A4 inhibitors, i.e. from 42.9% (n = 7706) in 2004 to 63.6% (n = 13 367) in 2006. For all other statins the number of overall users decreased to a similar extent to those co-medicated with CYP3A4 inhibitors. CONCLUSIONS In both 2004 and 2006, the choice of statin type did not depend on whether the patient used a CYP3A4 inhibitor or not. Considering the pronounced interaction potential of simvastatin with CYP3A4 inhibitors, a negative influence of the new policy on overall statin safety seems likely. PMID:19220274

  17. Prediction of inter-individual variability on the pharmacokinetics of CYP1A2 substrates in non-smoking healthy volunteers.

    PubMed

    Haraya, Kenta; Kato, Motohiro; Chiba, Koji; Sugiyama, Yuichi

    2016-08-01

    The activity of CYP1A2, a major drug-metabolizing enzyme, is known to be affected by various environmental factors. Our study aimed to predict inter-individual variability of AUC/Dose of CYP1A2 substrates in non-smoking healthy volunteers using the Monte Carlo simulation. Inter-individual variability in hepatic intrinsic clearance of CYP1A2 substrates (CLint,h,1A2) was estimated using dispersion model based on the inter-individual variability (N = 96) of the AUC of caffeine, a major CYP1A2 substrate. The estimated coefficient of variation (CV) of CLint,h,1A2 was 55%, similar to previously reported CLint,h,2D6 (60%) but larger than CLint,h,3A4 (33%). Then, this estimated CV was validated by predicting the CVs of AUC/Dose of tizanidine and phenacetin, which are mainly metabolized by CYP1A2 and have negligible renal clearance. As a result, reported CVs were successfully predicted within 2.5-97.5 percentile range of predicted values. Moreover, CVs for AUC/Dose of the CYP1A2 substrates theophylline and lidocaine, which are affected by other CYPs and renal clearance, were also successfully predicted. The inter-individual variability of AUC/Dose of CYP1A2 substrates was successfully predicted using 55% CV for CLint,h,1A2, and the results, along with those reported by our group for other CYPs, support the prediction of inter-individual variability of pharmacokinetics in the clinical setting. PMID:27318879

  18. Inactivation of Human Cytochrome P450 3A4 and 3A5 by Dronedarone and N-Desbutyl Dronedarone.

    PubMed

    Hong, Yanjun; Chia, Yvonne Mei Fen; Yeo, Ray Hng; Venkatesan, Gopalakrishnan; Koh, Siew Kwan; Chai, Christina Li Lin; Zhou, Lei; Kojodjojo, Pipin; Chan, Eric Chun Yong

    2016-01-01

    Dronedarone is an antiarrhythmic agent approved in 2009 for the treatment of atrial fibrillation. An in-house preliminary study demonstrated that dronedarone inhibits cytochrome P450 (CYP) 3A4 and 3A5 in a time-dependent manner. This study aimed to investigate the inactivation of CYP450 by dronedarone. We demonstrated for the first time that both dronedarone and its main metabolite N-desbutyl dronedarone (NDBD) inactivate CYP3A4 and CYP3A5 in a time-, concentration-, and NADPH-dependent manner. For the inactivation of CYP3A4, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 0.87 µM and 0.039 minute(-1), respectively, for dronedarone, and 6.24 µM and 0.099 minute(-1), respectively, for NDBD. For CYP3A5 inactivation, the inactivator concentration at the half-maximum rate of inactivation and inactivation rate constant at an infinite inactivator concentration are 2.19 µM and 0.0056 minute(-1) for dronedarone and 5.45 µM and 0.056 minute(-1) for NDBD. The partition ratios for the inactivation of CYP3A4 and CYP3A5 by dronedarone are 51.1 and 32.2, and the partition ratios for the inactivation of CYP3A4 and CYP3A5 by NDBD are 35.3 and 36.6. Testosterone protected both CYP3A4 and CYP3A5 from inactivation by dronedarone and NDBD. Although the presence of Soret peak confirmed the formation of a quasi-irreversible metabolite-intermediate complex between dronedarone/NDBD and CYP3A4/CYP3A5, partial recovery of enzyme activity by potassium ferricyanide illuminated an alternative irreversible mechanism-based inactivation (MBI). MBI of CYP3A4 and CYP3A5 was further supported by the discovery of glutathione adducts derived from the quinone oxime intermediates of dronedarone and NDBD. In conclusion, dronedarone and NDBD inactivate CYP3A4 and CYP3A5 via unique dual mechanisms of MBI and formation of the metabolite-intermediate complex. Our novel findings contribute new knowledge for

  19. Aloe vera juice: IC₅₀ and dual mechanistic inhibition of CYP3A4 and CYP2D6.

    PubMed

    Djuv, Ane; Nilsen, Odd Georg

    2012-03-01

    The aim of this study was to evaluate the inhibitory potency (IC₅₀ values) of ethanol extracts of two commercially available aloe vera juice (AVJ) products, on CYP3A4 and CYP2D6 activities in vitro and to determine if such inhibitions could be mechanism-based. Recombinant human CYP3A4 and CYP2D6 enzymes were used and the activities were expressed by the metabolism of testosterone and dextromethorphan with ketoconazole and quinidine as positive inhibitor controls, respectively. The formed metabolites were quantified by validated HPLC techniques. Time- and NADPH- dependent inhibition assays were performed to evaluate a possible mechanism-based inhibition. One of the AVJ extracts showed about twice the inhibitory potency towards both CYP enzymes over the other with IC₅₀ values of 8.35 ± 0.72 and 12.5 ± 2.1 mg/mL for CYP3A4 and CYP2D6, respectively. The AVJ was found to exert both CYP mediated and non-CYP mediated inhibition of both CYP3A4 and CYP2D6. This dual mechanistic inhibition, however, seems to be governed by different mechanisms for CYP3A4 and CYP2D6. Estimated IC₅₀ inhibition values indicate no major interference of AVJ with drug metabolism in man, but the dual mechanistic inhibition of both enzymes might be of clinical significance. PMID:21842479

  20. Modulation of CYP2D6 and CYP3A4 metabolic activities by Ferula asafetida resin

    PubMed Central

    Al-Jenoobi, Fahad I.; Al-Thukair, Areej A.; Alam, Mohd Aftab; Abbas, Fawkeya A.; Al-Mohizea, Abdullah M.; Alkharfy, Khalid M.; Al-Suwayeh, Saleh A.

    2014-01-01

    Present study investigated the potential effects of Ferula asafetida resin on metabolic activities of human drug metabolizing enzymes: CYP2D6 and CYP3A4. Dextromethorphan (DEX) was used as a marker to assess metabolic activities of these enzymes, based on its CYP2D6 and CYP3A4 mediated metabolism to dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. In vitro study was conducted by incubating DEX with human liver microsomes and NADPH in the presence or absence of Asafetida alcoholic extract. For clinical study, healthy human volunteers received a single dose of DEX alone (phase-I) and repeated the same dose after a washout period and four-day Asafetida treatment (phase-II). Asafetida showed a concentration dependent inhibition on DOR formation (in vitro) and a 33% increase in DEX/DOR urinary metabolic ratio in clinical study. For CYP3A4, formation of 3-MM in microsomes was increased at low Asafetida concentrations (10, 25 and 50 μg/ml) but slightly inhibited at the concentration of 100 μg/ml. On the other hand, in vivo observations revealed that Asafetida significantly increased DEX/3-MM urinary metabolic ratio. The findings of this study suggest that Asafetida may have a significant effect on CYP3A4 metabolic activity. Therefore, using Ferula asafetida with CYP3A4 drug substrates should be cautioned especially those with narrow therapeutic index such as cyclosporine, tacrolimus and carbamazepine. PMID:25561870

  1. Mechanism-based inhibition of CYP3A4 and CYP2D6 by Indonesian medicinal plants.

    PubMed

    Subehan; Usia, Tepy; Iwata, Hiroshi; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2006-05-24

    Thirty samples of Indonesian medicinal plants were tested for their mechanism-based inhibition on cytochrome P450 3A4 (CYP3A4) and CYP2D6 via erythromycin N-demethylation and dextromethorphan O-demethylation activities in human liver microsomes. From screening with 0 and 20min preincubation at 0.5mg/ml of methanol extracts, five plants (Cinnamomum burmani bark, Foeniculum vulgare seed, Strychnos ligustrina wood, Tinospora crispa stem, and Zingiber cassumunar rhizome) showed more than 30% increase of CYP3A4 inhibition, while three (Alpinia galanga rhizome, Melaleuca leucadendron leaf, and Piper nigrum fruit) showed more than 30% increase of CYP2D6 inhibition. In these eight plants, Foeniculum vulgare seed, Cinnamomum burmani bark, and Strychnos ligustrina wood showed time-dependent inhibition on CYP3A4 and Piper nigrum fruit and Melaleuca leucadendron leaf on CYP2D6. Among these, four plants other than Melaleuca leucadendron revealed NADPH-dependent inhibition. Thus, Foeniculum vulgare, Cinnamomum burmani, and Strychnos ligustrina should contain mechanism-based inhibitors on CYP3A4 and Piper nigrum contain that on CYP2D6. PMID:16414224

  2. An in vitro evaluation of human cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures.

    PubMed

    Budzinski, J W; Foster, B C; Vandenhoek, S; Arnason, J T

    2000-07-01

    Serial dilutions of 21 commercial ethanolic herbal extracts and tinctures, and 13 related pure plant compounds have been analyzed for their in vitro cytochrome P450 3A4 (CYP3A4) inhibitory capability via a fluorometric microtitre plate assay. Roughly 75% of the commercial products and 50% of the pure compounds showed significant inhibition of CYP3A4 metabolite formation. For each herbal product and pure compound exhibiting dose-dependency, the inhibition values were used to generate median inhibitory concentration (IC50) curves using linear regression. Among the commercial extracts, Hydrastis canadensis (goldenseal), Hypericum perforatum (St. John's wort), and Uncaria tomentosa (cat's claw) had the lowest IC50 values at < 1% full strength, followed by Echinacea angustifolia roots, Trifolium pratense (wild cherry), Matricaria chamomilla (chamomile), and Glycyrrhiza glabra (licorice), which had IC50 values ranging from 1%-2% of full strength. Dillapiol, hypericin, and naringenin had the lowest IC50 values among the pure plant compounds at < 0.5 mM; dillapiol was the most potent inhibitor at 23.3 times the concentration of the positive CYP3A4 inhibitor ketoconazole. Utilizing high-throughput screening methodologies for assessing CYP3A4 inhibition by natural products has important implications for predicting the likelihood of potential herbal-drug interactions, as well as determining candidates for further in-depth analyses. PMID:10969720

  3. The role of CYP 3A4 and 1A1 in amiodarone-induced hepatocellular toxicity.

    PubMed

    Wu, Qiangen; Ning, Baitang; Xuan, Jiekun; Ren, Zhen; Guo, Lei; Bryant, Matthew S

    2016-06-24

    Amiodarone is a widely used potent antiarrhythmic for the treatment of cardiac disease; however, its use is often discontinued due to numerous adverse effects, including hepatotoxicity. To investigate the role of drug metabolism in this liver toxicity, amiodarone and its major metabolite desethylamiodarone were incubated with HepG2 cells overexpressing a series of cytochrome P450 (CYP) isoforms. Significantly higher cytotoxicity of amiodarone was observed in HepG2 cells overexpressing CYP3A4 or CYP1A1, compared with that observed in empty vector transduced control cells. Further, higher levels of the more potent hepatotoxic metabolite desethylamiodarone were detected in CYP3A4 or CYP1A1 expressed cells. The CYP3A4 inhibitor ketoconazole and the CYP1A1 inhibitor α-naphthoflavone drastically inhibited the metabolism of amiodarone to desethylamiodarone. Along with the inhibition of CYP1A1 or CYP3A4, the cytotoxicity of amiodarone was significantly reduced. These data indicate that the metabolism of amiodarone to desethylamiodarone by CYP1A1 or CYP3A4 plays an important role in the hepatocellular toxicity of amiodarone. PMID:27113703

  4. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    SciTech Connect

    Istrate, Monica A.; Nussler, Andreas K.; Eichelbaum, Michel; Burk, Oliver

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  5. Camptothecin Attenuates Cytochrome P450 3A4 Induction by Blocking the Activation of Human Pregnane X ReceptorS⃞

    PubMed Central

    Chen, Yakun; Tang, Yong; Robbins, Gregory T.

    2010-01-01

    Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs. PMID:20504912

  6. Characterizing the Membrane-Bound State of Cytochrome P450 3A4: Structure, Depth of Insertion, and Orientation

    PubMed Central

    2013-01-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant membrane-associated isoform of the P450 family in humans and is responsible for biotransformation of more than 50% of drugs metabolized in the body. Despite the large number of crystallographic structures available for CYP3A4, no structural information for its membrane-bound state at an atomic level is available. In order to characterize binding, depth of insertion, membrane orientation, and lipid interactions of CYP3A4, we have employed a combined experimental and simulation approach in this study. Taking advantage of a novel membrane representation, highly mobile membrane mimetic (HMMM), with enhanced lipid mobility and dynamics, we have been able to capture spontaneous binding and insertion of the globular domain of the enzyme into the membrane in multiple independent, unbiased simulations. Despite different initial orientations and positions of the protein in solution, all the simulations converged into the same membrane-bound configuration with regard to both the depth of membrane insertion and the orientation of the enzyme on the surface of the membrane. In tandem, linear dichroism measurements performed on CYP3A4 bound to Nanodisc membranes were used to characterize the orientation of the enzyme in its membrane-bound form experimentally. The heme tilt angles measured experimentally are in close agreement with those calculated for the membrane-bound structures resulted from the simulations, thereby verifying the validity of the developed model. Membrane binding of the globular domain in CYP3A4, which appears to be independent of the presence of the transmembrane helix of the full-length enzyme, significantly reshapes the protein at the membrane interface, causing conformational changes relevant to access tunnels leading to the active site of the enzyme. PMID:23697766

  7. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    PubMed

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-01

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated. PMID:26580496

  8. Effects of Commonly Used Excipients on the Expression of CYP3A4 in Colon and Liver Cells

    PubMed Central

    Tompkins, Leslie; Lynch, Caitlin; Haidar, Sam; Polli, James; Wang, Hongbing

    2013-01-01

    Purpose The objective of this investigation was to assess whether common pharmaceutical excipients regulate the expression of drug-metabolizing enzymes in human colon and liver cells. Methods Nineteen commonly used excipients were evaluated using a panel of experiments including cell-based human PXR activation assays, real-time RT-PCR assays for CYP3A4 mRNA expression, and immunoblot analysis of CYP3A4 protein expression in immortalized human liver cells (HepG2 and Fa2N4), human primary hepatocytes, and the intestinal LS174T cell models. Results No excipient activated human PXR or practically induced CYP3A4. However, three excipients (polysorbate 80, pregelatinized starch, and hydroxypropyl methylcellulose) tended to decrease mRNA and protein expression across experimental models. Conclusion This study represents the first investigation of the potential role of excipients in the expression of drug-metabolizing enzymes. Findings imply that some excipients may hold potential for excipient-drug interactions by repression of CYP3A4 expression. PMID:20503067

  9. Priapism Induced by Boceprevir-CYP3A4 Inhibition and α-Adrenergic Blockade: Case Report

    PubMed Central

    Hammond, Kyle P.; Nielsen, Craig; Linnebur, Sunny A.; Langness, Jacob A.; Ray, Graham; Maroni, Paul; Kiser, Jennifer J.

    2014-01-01

    A 44-year-old white man presented to the emergency department with a 3-day history of priapism requiring a surgically performed distal penile shunt. A drug–drug interaction is the suspected cause whereby CYP3A4 inhibition by boceprevir led to increased exposures of doxazosin, tamsulosin, and/or quetiapine, resulting in additional α-adrenergic blockade. PMID:24092799

  10. Herb-drug interaction of 50 Chinese herbal medicines on CYP3A4 activity in vitro and in vivo.

    PubMed

    Pao, Li-Heng; Hu, Oliver Yoa-Pu; Fan, Hsien-Yuan; Lin, Chang-Ching; Liu, Liang-Chun; Huang, Pei-Wei

    2012-01-01

    The purpose of this study is to evaluate the effects of Chinese herbal medicines on the enzymatic activity of CYP3A4 and the possible metabolism-based herb-drug interactions in human liver microsomes and in rats. Fifty single-herbal preparations were screened for the activity of CYP3A4 using human liver microsomes for an in vitro probe reaction study. The enzymatic activity of CYP3A4 was estimated by determing the 6β-hydroxytestosterone metabolized from testosterone performed on a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Huang Qin (Scutellaria baicalensis Geprgi), Mu Dan Pi (Paeonia suffruticosa Andr.), Ji Shiee Terng (Spatholobus suberectus Dunn.) and Huang Qi (Astragalus membranaceus [Fisch] Bge) have been demonstrated to have remarkable inhibiting effects on the metabolism of CYP3A4, whereas Xi Yi Hua (Magnolia biondii Pamp.) exhibited a moderate inhibition. These five single herbs were further investigated in an animal study using midazolam. Mu Dan Pi, Ji Shiee Terng and Huang Qi were observed to have greatly increased in the C(max) and AUC of midazolam. This study provides evidence of possible herb-drug interactions involved with certain single herbs. PMID:22298448

  11. Identification and characterization of reactive metabolites in myristicin-mediated mechanism-based inhibition of CYP1A2.

    PubMed

    Yang, Ai-Hong; He, Xin; Chen, Jun-Xiu; He, Li-Na; Jin, Chun-Huan; Wang, Li-Li; Zhang, Fang-Liang; An, Li-Jun

    2015-07-25

    Myristicin belongs to the methylenedioxyphenyl or allyl-benzene family of compounds, which are found widely in plants of the Umbelliferae family, such as parsley and carrot. Myristicin is also the major active component in the essential oils of mace and nutmeg. However, this compound can cause adverse reactions, particularly when taken inappropriately or in overdoses. One important source of toxicity of natural products arises from their metabolic biotransformations into reactive metabolites. Myristicin contains a methylenedioxyphenyl substructure, and this specific structural feature may allow compounds to cause a mechanism-based inhibition of cytochrome P450 enzymes and produce reactive metabolites. Therefore, the aim of this work was to identify whether the role of myristicin in CYP enzyme inhibition is mechanism-based inhibition and to gain further information regarding the structure of the resulting reactive metabolites. CYP cocktail assays showed that myristicin most significantly inhibits CYP1A2 among five CYP enzymes (CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) from human liver microsomes. The 3.21-fold IC50 shift value of CYP1A2 indicates that myristicin may be a mechanism-based inhibitor of CYP1A2. Next, reduced glutathione was shown to block the inhibition of CYP1A2, indicating that myristicin utilized a mechanism-based inhibition. Phase I metabolism assays identified two metabolites, 5-allyl-1-methoxy-2,3-dihydroxybenzene (M1) and 1'-hydroxymyristicin or 2',3'-epoxy-myristicin (M2). Reduced glutathione capturing assays captured the glutathione-M1 adduct, and the reactive metabolites were identified using UPLC-MS(2) as a quinone and its tautomer. Thus, it was concluded that myristicin is a mechanism-based inhibitor of CYP1A2, and the reactive metabolites are quinone tautomers. Additionally, the cleavage process of the glutathione-M1 adduct was analyzed in further detail. This study provides additional information on the metabolic mechanism of myristicin

  12. Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus

    PubMed Central

    Dostalek, Miroslav; Court, Michael H; Yan, Bingfang; Akhlaghi, Fatemeh

    2011-01-01

    BACKGROUND AND PURPOSE Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. EXPERIMENTAL APPROACH Human liver microsomal fractions (HLMs) were prepared from diabetic (n = 12) and demographically matched nondiabetic (n = 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. KEY RESULTS Midazolam 1′- or 4-hydroxylation and testosterone 6β-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. CONCLUSIONS AND IMPLICATIONS These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted. PMID:21323901

  13. Safety of Herbal Medicinal Products: Echinacea and Selected Alkylamides Do Not Induce CYP3A4 mRNA Expression.

    PubMed

    Modarai, Maryam; Silva, Elisabete; Suter, Andy; Heinrich, Michael; Kortenkamp, Andreas

    2011-01-01

    A major safety concern with the use of herbal medicinal products (HMP) is their interactions with conventional medicines, which are often mediated via the cytochrome P450 (CYP) system. Echinacea is a widely used over-the-counter HMP, with proven immunomodulatory properties. Its increasing use makes research into its safety an urgent concern. Previously, we showed that Echinacea extracts and its alkylamides (thought to be important for Echinacea's immunomodulatory activity) mildly inhibit the enzymatic activity of the main drug metabolising CYP isoforms, but to this date, there is insufficient work on its ability to alter CYP expression levels. We now report for the first time the effect of a commercial Echinacea extract (Echinaforce) and four Echinacea alkylamides on the transcription of the major drug metabolizing enzyme CYP3A4. HepG2 cells were exposed for 96 h to clinically relevant concentrations of Echinaforce (22, 11.6 and 1.16 μg mL(-1)) or the alkylamides (1.62 and 44 nM). CYP3A4 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Neither Echinaforce nor the alkylamides produced any significant changes in the steady-state CYP3A4 mRNA levels, under these conditions. In contrast, treatment with 50 μM rifampicin resulted in a 3.8-fold up-regulation over the vehicle control. We conclude that Echinaforce is unlikely to affect CYP3A4 transcriptional levels, even at concentrations which can inhibit the enzymatic activity of CYP3A4. Overall, our data provides further evidence for the lack of interactions between Echinacea and conventional drugs. PMID:19906827

  14. Amlodipine metabolism in human liver microsomes and roles of CYP3A4/5 in the dihydropyridine dehydrogenation.

    PubMed

    Zhu, Yanlin; Wang, Fen; Li, Quan; Zhu, Mingshe; Du, Alicia; Tang, Wei; Chen, Weiqing

    2014-02-01

    Amlodipine is a commonly prescribed calcium channel blocker for the treatment of hypertension and ischemic heart disease. The drug is slowly cleared in humans primarily via dehydrogenation of its dihydropyridine moiety to a pyridine derivative (M9). Results from clinical drug-drug interaction studies suggest that CYP3A4/5 mediate metabolism of amlodipine. However, attempts to identify a role of CYP3A5 in amlodipine metabolism in humans based on its pharmacokinetic differences between CYP3A5 expressers and nonexpressers failed. Objectives of this study were to determine the metabolite profile of amlodipine (a racemic mixture and S-isomer) in human liver microsomes (HLM), and to identify the cytochrome P450 (P450) enzyme(s) involved in the M9 formation. Liquid chromatography/mass spectrometry analysis showed that amlodipine was mainly converted to M9 in HLM incubation. M9 underwent further O-demethylation, O-dealkylation, and oxidative deamination to various pyridine derivatives. This observation is consistent with amlodipine metabolism in humans. Incubations of amlodipine with HLM in the presence of selective P450 inhibitors showed that both ketoconazole (an inhibitor of CYP3A4/5) and CYP3cide (an inhibitor of CYP3A4) completely blocked the M9 formation, whereas chemical inhibitors of other P450 enzymes had little effect. Furthermore, metabolism of amlodipine in expressed human P450 enzymes showed that only CYP3A4 had significant activity in amlodipine dehydrogenation. Metabolite profiles and P450 reaction phenotyping data of a racemic mixture and S-isomer of amlodipine were very similar. The results from this study suggest that CYP3A4, rather than CYP3A5, plays a key role in metabolic clearance of amlodipine in humans. PMID:24301608

  15. CYP1A2, GSTM1, and GSTT1 polymorphisms and diet effects on CYP1A2 activity in a crossover feeding trial*

    PubMed Central

    Peterson, Sabrina; Schwarz, Yvonne; Li, Shuying S.; Li, Lin; King, Irena B.; Chen, Chu; Eaton, David L.; Potter, John D.; Lampe, Johanna W.

    2009-01-01

    Cytochrome P-450 1A2 (CYP1A2) is a biotransformation enzyme that activates several procarcinogens. CYP1A2 is induced by cruciferous and inhibited by apiaceous vegetable intake. Using a randomized, cross-over feeding trial in humans, we investigated dose effects of cruciferous vegetables and effects of any interaction between cruciferous and apiaceous vegetables on CYP1A2 activity. We also investigated whether response varied by CYP1A2*1F, GSTM1, and GSTT1 genotypes (glutathione S-transferases that metabolize crucifer constituents) and whether CYP1A2 activity rebounds after apiaceous vegetables are removed from the diet. Participants (N = 73), recruited based on genotypes, consumed four diets for two weeks each: low-phytochemical diet (basal), basal plus single dose of cruciferous (1C), basal plus double dose of cruciferous (2C), and basal plus single dose of cruciferous and apiaceous vegetables (1C+A). CYP1A2 activity was determined by urine caffeine tests administered at baseline and the end of each feeding period. Compared with basal diet, the 1C diet increased CYP1A2 activity (P < 0.0001) and the 2C diet resulted in further increases (P < 0.0001) with men experiencing greater dose-response than women. The 1C+A diet decreased CYP1A2 activity compared to the 1C and 2C diets (P < 0.0001 for both). Although there was no overall effect of CYP1A2*1F or GSTM1-null/GSTT1-null genotypes or genotype-by-diet interactions, there were significant diet response differences within each genotype. Additionally, CYP1A2 activity recovered modestly one day after the removal of apiaceous vegetables. These results suggest complex interactions among dietary patterns, genetic variation, and modulation of biotransformation that may not be apparent in observational studies. PMID:19843669

  16. 2,3,7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)-MEDIATED OXIDATIVE STRESS IN FEMALE CYP1A-2 KNOCKOUT (CYP1A2-/-) MICE

    EPA Science Inventory

    2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD)-Mediated Oxidative Stress in Female CYP1A2 Knockout (CYP1A2-/-) Mice

    Deborah Burgin1, Janet Diliberto2, Linda Birnbaum2
    1UNC Toxicology; 2USEPA/ORD/NHEERL, RTP, NC

    Most of the effects due to TCDD exposure are mediated via...

  17. Sertraline-induced potentiation of the CYP3A4-dependent neurotoxicity of carbamazepine: An in vitro study

    PubMed Central

    Ghosh, Chaitali; Hossain, Mohammad; Spriggs, Addison; Ghosh, Arnab; Grant, Gerald A.; Marchi, Nicola; Perucca, Emilio; Janigro, Damir

    2015-01-01

    SUMMARY Objective Drug toxicity is a hurdle to drug development and to clinical translation of basic research. Antiepileptic drugs such as carbamazepine (CBZ) and selective serotonin reuptake inhibitors such as sertraline (SRT) are commonly co-prescribed to patients with epilepsy and comorbid depression. Because SRT may interfere with cytochrome P450 (CYP) enzyme activity and CYPs have been implicated in the conversion of CBZ to reactive cytotoxic metabolites, we investigated in vitro models to determine whether SRT affects the neurotoxic potential of CBZ and the mechanisms involved. Methods Human fetal brain-derived dopaminergic neurons, human brain microvascular endothelial cells (HBMECs), and embryonic kidney (HEK) cells were used to evaluate cytotoxicity of CBZ and SRT individually and in combination. Nitrite and glutathione (GSH) levels were measured with drug exposure. To validate the role of CYP3A4 in causing neurotoxicity, drug metabolism was compared to cell death in HEK CYP3A4 overex-pressed and cells pretreated with the CYP3A4 inhibitor ketoconazole. Results In all cellular systems tested, exposure to CBZ (127 μM) or SRT (5 μM) alone caused negligible cytotoxicity. By contrast CBZ, tested at a much lower concentration (17 μM) in combination with SRT (5 μM), produced prominent cytotoxicity within 15 min exposure. In neurons and HBMECs, cytotoxicity was associated with increased nitrite levels, suggesting involvement of free radicals as a pathogenetic mechanism. Pretreatment of HBMECs with reduced GSH or with the GSH precursor N-acetyl-L-cysteine prevented cytotoxic response. In HEK cells, the cytotoxic response to the CBZ + SRT combination correlated with the rate of CBZ biotransformation and production of 2-hydroxy CBZ, further suggesting a causative role of reactive metabolites. In the same system, cytotoxicity was potentiated by overexpression of CYP3A4, and prevented by CYP3A4 inhibitor. Significance These results demonstrate an unexpected

  18. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

    PubMed

    Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. PMID:20361990

  19. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    SciTech Connect

    Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 {mu}M. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 {mu}M. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 {mu}M, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 {mu}M and 10 {mu}M, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

  20. Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Hammond, Geoffrey L; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity. Results In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche. Conclusion These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer. PMID:15217502

  1. Hill Parameters and Heterogeneity of alpha-Naphthoflavone Binding to Human Cytochrome P450 3A4 by Fluorescence Spectroscopic Analysis

    NASA Astrophysics Data System (ADS)

    Carlson, Benjamin; Marsch, Glenn; Martin, Martha; Guengerich, F. Peter

    2009-03-01

    Human cytochrome P450 3A4 (CYP 3A4) is an alpha-helical membrane-bound protein that metabolizes approximately 50% of all drugs. The interaction between CYP450 3A4 and alpha-naphthoflavone (ANF) was characterized using fluorescence methods. ANF quenched fluorescence from tryptophan residues in CYP 3A4, and CYP 3A4 quenched bound ANF. The ANF emission energy was unchanged upon binding to CYP 3A4, implying that enzyme-bound 3A4 is completely quenched. Fluorescence difference spectra were fit to the Hill equation by varying the parameters Kd and n. For quenching of tryptophan fluorescence by ANF, no significant sigmoidal behavior was observed with n=1, and the spectral dissociation constant revealed a strong ANF-CYP 3A4 interaction with Kd=27nM. Modest cooperativity and very tight binding was observed in the quenching of ANF by CYP 3A4, with n=1.4 and Kd= 4.9nM. Fluorescence polarization anisotropy decreased at low ANF/CYP 3A4 molar ratios; then increased at higher ratios. Compared to substrate-free CYP 3A4, adding substrate at low molar ratios increases the CYP 3A4 rotation, suggesting the molecular volume decreases.

  2. Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes.

    PubMed

    Girennavar, Basavaraj; Poulose, Shibu M; Jayaprakasha, Guddadarangavvanahally K; Bhat, Narayan G; Patil, Bhimanagouda S

    2006-04-15

    Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively. PMID:16338240

  3. Tamoxifen-associated hot flash severity is inversely correlated with endoxifen concentration and CYP3A4*22.

    PubMed

    Baxter, Simon D; Teft, Wendy A; Choi, Yun-Hee; Winquist, Eric; Kim, Richard B

    2014-06-01

    Tamoxifen use is often limited in some patients due to adverse effects including severe hot flash symptoms. Tamoxifen undergoes hepatic bioactivation by CYP2D6 and CYP3A4 to form the active metabolite endoxifen. It remains unclear whether the extent of attained endoxifen level or genetic polymorphisms in drug metabolizing enzymes is associated with the frequency and severity of hot flashes. We conducted a prospective study using self-reported surveys to assess tamoxifen side effects experienced during the week prior to clinic visits of 132 female breast cancer patients on tamoxifen therapy, and hot flash severity scores were tabulated. At the time of clinic visit, blood samples were obtained to determine tamoxifen and its metabolite levels and to determine CYP2D6 and CYP3A4 genotypes. The majority of participants (77 %) experienced hot flashes, with 11 % experiencing severe or very severe symptoms. We observed an inverse correlation between endoxifen concentration and hot flash severity score following adjustment for age, BMI, and menopausal status in patients with non-zero scores (p < 0.001). Interestingly, CYP2D6 genotype was not significantly associated with hot flash scores in patients on no known inhibitory medications. However, CYP3A4*22 carriers were less likely to have hot flashes with an odds ratio of 8.87 (p < 0.01) even when compared to a cohort with similar endoxifen levels. Our data demonstrate that patients with higher endoxifen levels tended to predict lower hot flash severity scores. Importantly, this is the first study to show CYP3A4*22 genotype as an independent predictor of hot flash severity during tamoxifen therapy. PMID:24744093

  4. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors.

    PubMed

    Yu, Hongyi; Moore, Michael L; Erhard, Karl; Hardwicke, Mary Ann; Lin, Hong; Luengo, Juan I; McSurdy-Freed, Jeanelle; Plant, Ramona; Qu, Junya; Raha, Kaushik; Rominger, Cynthia M; Schaber, Michael D; Spengler, Michael D; Rivero, Ralph A

    2013-02-14

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  5. [3a,4]-Dihydropyrazolo[1,5a]pyrimidines: Novel, Potent, and Selective Phosphatidylinositol-3-kinase β Inhibitors

    PubMed Central

    2013-01-01

    A series of novel [3a,4]dihydropyrazolo[1,5a]pyrimidines were identified, which were highly potent and selective inhibitors of PI3Kβ. The template afforded the opportunity to develop novel SAR for both the hinge-binding (R3) and back-pocket (R4) substitutents. While cellular potency was relatively modest due to high protein binding, the series displayed low clearance in rat, mouse, and monkey. PMID:24900655

  6. Linkage and association of haplotypes at the APOA1/C3/A4/A5 genecluster to familial combined hyperlipidemia

    SciTech Connect

    Eichenbaum-Voline, Sophie; Olivier, Michael; Jones, Emma L.; Naoumova, Rossitza P.; Jones, Bethan; Gau, Brian; Seed, Mary; Betteridge,D. John; Galton, David J.; Rubin, Edward M.; Scott, James; Shoulders,Carol C.; Pennacchio, Len A.

    2002-09-15

    Combined hyperlipidemia (CHL) is a common disorder of lipidmetabolism that leads to an increased risk of cardiovascular disease. Thelipid profile of CHL is characterised by high levels of atherogeniclipoproteins and low levels of high-density-lipoprotein-cholesterol.Apolipoprotein (APO) A5 is a newly discovered gene involved in lipidmetabolism located within 30kbp of the APOA1/C3/A4 gene cluster. Previousstudies have indicated that sequence variants in this cluster areassociated with increased plasma lipid levels. To establish whethervariation at the APOA5 gene contributes to the transmission of CHL, weperformed linkage and linkage disequilibrium (LD) tests on a large cohortof families (n=128) with familial CHL (FCHL). The linkage data producedevidence for linkage of the APOA1/C3/A4/A5 genomic interval to FCHL (NPL= 1.7, P = 0.042). The LD studies substantiated these data. Twoindependent rare alleles, APOA5c.56G and APOC3c.386G of this gene clusterwere over-transmitted in FCHL (P = 0.004 and 0.007, respectively), andthis was associated with a reduced transmission of the most commonAPOA1/C3/A4/A5 haplotype (frequency 0.4425) to affected subjects (P =0.013). The APOA5c.56G allele was associated with increased plasmatriglyceride levels in FCHL probands, whereas the second, andindependent, APOC3c.386G allele was associated with increased plasmatriglyceride levels in FCHL pedigree founders. Thus, this allele (or anallele in LD) may mark a quantitative trait associated with FCHL, as wellas representing a disease susceptibility locus for the condition. Thisstudy establishes that sequence variation in the APOA1/C3/A4/A5 genecluster contributes to the transmission of FCHL in a substantialproportion of affected families, and that these sequence variants mayalso contribute to the lipid abnormalities of the metabolic syndrome,which is present in up to 40 percent of persons with cardiovasculardisease.

  7. Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

    PubMed

    Šemeláková, M; Jendželovský, R; Fedoročko, P

    2016-07-01

    Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4. PMID:27261575

  8. Structural Optimization of Ghrelin Receptor Inverse Agonists to Improve Lipophilicity and Avoid Mechanism-Based CYP3A4 Inactivation.

    PubMed

    Takahashi, Bitoku; Funami, Hideaki; Shibata, Makoto; Maruoka, Hiroshi; Koyama, Makoto; Kanki, Satomi; Muto, Tsuyoshi

    2015-01-01

    Structural optimization of 2-aminonicotinamide derivatives as ghrelin receptor inverse agonists is reported. So as to avoid mechanism-based inactivation (MBI) of CYP3A4, 1,3-benzodioxol ring of the lead compound was modified. Improvement of the main activity and lipophilicity was achieved simultaneously, leading to compound 18a, which showed high lipophilic ligand efficiency (LLE) and low MBI activity. PMID:26423040

  9. PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

    PubMed Central

    Zheng, Jian; Chan, Ting; Cheung, Florence Shin Gee; Zhu, Ling; Murray, Michael; Zhou, Fanfan

    2014-01-01

    The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability. PMID:24728453

  10. Cytochrome P450 1A2 (CYP1A2) activity, mammographic density, and oxidative stress: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Rao, Venketeshwer; Agarwal, Sanjiv; Martin, Lisa; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Mammographically dense breast tissue is a strong predictor of breast cancer risk, and is influenced by both mitogens and mutagens. One enzyme that is able to affect both the mitogenic and mutagenic characteristics of estrogens is cytochrome P450 1A2 (CYP1A2), which is principally responsible for the metabolism of 17β-estradiol. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity, malondialdehyde (MDA) levels, and mammographic density. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Levels of serum and urinary MDA, and MDA–deoxyguanosine adducts in DNA were measured. Mammograms were digitized and measured using a computer-assisted method. Results CYP1A2 activity in postmenopausal women, but not in premenopausal women, was positively associated with mammographic density, suggesting that increased CYP1A2 activity after the menopause is a risk factor for breast cancer. In premenopausal women, but not in postmenopausal women, CYP1A2 activity was positively associated with serum and urinary MDA levels; there was also some evidence that CYP1A2 activity was more positively associated with percentage breast density when MDA levels were high, and more negatively associated with percentage breast density when MDA levels were low. Conclusion These findings provide further evidence that variation in the activity level of enzymes involved in estrogen metabolism is related to levels of mammographic density and potentially to breast cancer risk. PMID:15217501

  11. Feed-forward regulation of bile acid detoxification by CYP3A4: studies in humanized transgenic mice.

    PubMed

    Stedman, Catherine; Robertson, Graham; Coulter, Sally; Liddle, Christopher

    2004-03-19

    Bile acids are potentially toxic end products of cholesterol metabolism and their concentrations must be tightly regulated. Homeostasis is maintained by both feed-forward regulation and feedback regulation. We used humanized transgenic mice incorporating 13 kb of the 5' regulatory flanking sequence of CYP3A4 linked to a lacZ reporter gene to explore the in vivo relationship between bile acids and physiological adaptive CYP3A gene regulation in acute cholestasis after bile duct ligation (BDL). Male transgenic mice were subjected to BDL or sham surgery prior to sacrifice on days 3, 6, and 10, and others were injected with intraperitoneal lithocholic acid (LCA) or vehicle alone. BDL resulted in marked hepatic activation of the CYP3A4/lacZ transgene in pericentral hepatocytes, with an 80-fold increase in transgene activation by day 10. Individual bile acids were quantified by liquid chromatography/mass spectrometry. Serum 6beta-hydroxylated bile acids were increased following BDL, confirming the physiological relevance of endogenous Cyp3a induction to bile acid detoxification. Although concentrations of conjugated primary bile acids increased after BDL, there was no increase in LCA, a putative PXR ligand, indicating that this cannot be the only endogenous bile acid mediating this protective response. Moreover, in LCA-treated animals, 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining showed hepatic activation of the CYP3A4 transgene only on the liver capsular surface, and minimal parenchymal induction, despite significant liver injury. This study demonstrates that CYP3A up-regulation is a significant in vivo adaptive response to cholestasis. However, this up-regulation is not dependent on increases in circulating LCA and the role of other bile acids as regulatory molecules requires further exploration. PMID:14681232

  12. Analysis of Mechanism-Based Inhibition of CYP 3A4 by a Series of Fluoroquinolone Antibacterial Agents.

    PubMed

    Watanabe, Akiko; Takakusa, Hideo; Kimura, Takako; Inoue, Shin-Ichi; Kusuhara, Hiroyuki; Ando, Osamu

    2016-10-01

    A series of fluoroquinolone compounds (compounds 1-9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage. PMID:27469000

  13. Mechanism of Drug-Drug Interactions Mediated by Human Cytochrome P450 CYP3A4 Monomer

    PubMed Central

    Denisov, Ilia G.; Grinkova, Yelena V.; Baylon, Javier L.; Tajkhorshid, Emad; Sligar, Stephen G.

    2016-01-01

    Using Nanodiscs, we quantitate the heterotropic interaction between two different drugs mediated by monomeric CYP3A4 incorporated into a native-like membrane environment. The mechanism of this interaction is deciphered by global analysis of multiple turnover experiments performed under identical conditions using the pure substrates progesterone (PGS) and carbamazepine (CBZ) and their mixtures. Activation of CBZ epoxidation and simultaneous inhibition of PGS hydroxylation are measured and quantitated through differences in their respective affinities towards both a remote allosteric site and the productive catalytic site near the heme iron. Preferred binding of PGS at the allosteric site and higher preference of CBZ binding at the productive site give rise to a non-trivial drug-drug interaction. Molecular dynamics simulations indicate functionally important conformational changes caused by PGS binding at the allosteric site and by two CBZ molecules positioned inside the substrate binding pocket. Structural changes involving Phe-213, Phe-219, and Phe-241 are suggested to be responsible for the observed synergetic effects and positive allosteric interactions between these two substrates. Such a mechanism is likely of general relevance to the mutual heterotropic effects caused by biologically active compounds which exhibit different patterns of interaction with the distinct allosteric and productive sites of CYP3A4, as well as other xenobiotic metabolizing cytochromes P450 that are also involved in drug-drug interactions. Importantly, this work demonstrates that a monomeric CYP3A4 can display the full spectrum of activation and cooperative effects that are observed in hepatic membranes. PMID:25777547

  14. Radical scavenging and cytochrome P450 3A4 inhibitory activity of bergaptol and geranylcoumarin from grapefruit.

    PubMed

    Girennavar, Basavaraj; Jayaprakasha, G K; Jadegoud, Y; Nagana Gowda, G A; Patil, Bhimanagouda S

    2007-06-01

    Grapefruit juice has been shown to increase the oral bioavailability of several clinically important drugs by inhibiting first pass metabolism. Several compounds in grapefruit juice have shown different biological activities. Unique among them are furocoumarins with potent inhibitory activity against cytochrome P450 enzymes. In the present study, two bioactive compounds were isolated from grapefruit juice and grapefruit peel oil. The purity of the isolated compounds has been analyzed by HPLC. Structures of the compounds were elucidated by extensive NMR and mass spectral studies and identified as bergaptol and geranylcoumarin. The isolated compounds were tested for their radical scavenging activity using 2,2'-azobis (3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) methods at different concentrations. Bergaptol showed very good radical scavenging activity at all the tested concentrations. Furthermore, these compounds were evaluated for their inhibitory activity against CYP3A4 enzyme. Bergaptol and geranylcoumarin were found to be potent inhibitors of debenzylation activity of CYP3A4 enzyme with an IC(50) value of 24.92 and 42.93 microM, respectively. PMID:17400460

  15. Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles

    SciTech Connect

    Rodriguez-Antona, Cristina . E-mail: cristina.rodriguez-antona@cnio.es; Sayi, Jane G.; Gustafsson, Lars L.; Bertilsson, Leif; Ingelman-Sundberg, Magnus

    2005-12-09

    The human cytochrome P450 3A (CYP3A) enzymes, which metabolize 50% of currently used therapeutic drugs, exhibit great interindividual differences in activity that have a major impact on drug treatment outcome, but hitherto no genetic background importantly contributing to this variation has been identified. In this study we show that CYP3A4 mRNA and hnRNA contents with a few exceptions vary in parallel in human liver, suggesting that mechanisms affecting CYP3A4 transcription, such as promoter polymorphisms, are relevant for interindividual differences in CYP3A4 expression. Tanzanian (n = 143) healthy volunteers were phenotyped using quinine as a CYP3A probe and the results were used for association studies with CYP3A4 genotypes. Carriers of CYP3A4*1B had a significantly lower activity than those with CYP3A4*1 whereas no differences were seen for five other SNPs investigated. Nuclear proteins from the B16A2 hepatoma cells were found to bind with less affinity to the CYP3A4*1B element around -392 bp as compared to CYP3A4*1. The data indicate the existence of a genetic CYP3A4 polymorphism with functional importance for interindividual differences in enzyme expression.

  16. Fluoxetine reduces CES1, CES2, and CYP3A4 expression through decreasing PXR and increasing DEC1 in HepG2 cells.

    PubMed

    Shang, Wei; Liu, Jie; Chen, Ruini; Ning, Rui; Xiong, Jing; Liu, Wei; Mao, Zhao; Hu, Gang; Yang, Jian

    2016-05-01

    1. This study investigated the mechanisms of the decreases of carboxylesterases (CES) and cytochrome P4503A4 (CYP3A4) and the enzymatic activities induced by fluoxetine (FLX) in HepG2 cells. We found that FLX decreased the carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) expression and the hydrolytic activity. 2. FLX decreased the pregnane X receptor (PXR) expression which regulated the target genes such as CYP3A4, whereas increased the differentiated embryonic chondrocyte-expressed gene 1 (DEC1) expression. 3. FLX repressed the PXR at transcriptional level. 4. Overexpression of PXR alone increased the expression of CES1, CES2, and CYP3A4 and attenuated the decreases of CES1, CES2, and CYP3A4 induced by FLX. On the contrary, knockdown of PXR alone decreased the expression of CES1, CES2, and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX. 5. Knockdown of DEC1 alone increased the expression of PXR and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX. 6. Taken together, the decreases of CES and CYP3A4 expression and enzymatic activities induced by FLX are through decreasing PXR and increasing DEC1 in HepG2 cells. PMID:26340669

  17. Fentanyl Enhances Hepatotoxicity of Paclitaxel via Inhibition of CYP3A4 and ABCB1 Transport Activity in Mice

    PubMed Central

    Pan, Jia-Hao; Bi, Bing-Tian; Feng, Kun-Yao; Huang, Wan; Zeng, Wei-An

    2015-01-01

    Fentanyl, a potent opioid analgesic that is used to treat cancer pain, is commonly administered with paclitaxel in advanced tumors. However, the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanism of action is not well studied. The purpose of this study was to investigate the effect of fentanyl on the hepatotoxicity of paclitaxel and its potential mechanisms of action. Pharmacokinetic parameters of paclitaxel were tested using reversed phase high-performance liquid chromatography (RP-HPLC). Aspartate transaminase (AST), alanine aminotransferase (ALT), and mouse liver histopathology were examined. Moreover, the cytotoxicity of anti-carcinogens was examined using 1-(4, 5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), and the intracellular accumulation of doxorubicin and rhodamine 123 was detected by flow cytometry. Furthermore, the expression of ABCB1 and the activity of ABCB1 ATPase and CYP3A4 were also examined. In this study, the co-administration of fentanyl and paclitaxel prolonged the half-life (t1/2) of paclitaxel from 1.455 hours to 2.344 hours and decreased the clearance (CL) from 10.997 ml/h to 7.014 ml/h in mice. Fentanyl significantly increased the levels of ALT in mice to 88.2 U/L, which is more than 2-fold higher than the level detected in the control group, and it increased the histological damage in mouse livers. Furthermore, fentanyl enhanced the cytotoxicity of anti-carcinogens that are ABCB1 substrates and increased the accumulation of doxorubicin and rhodamine 123. Additionally, fentanyl stimulated ABCB1 ATPase activity and inhibited CYP3A4 activity in the liver microsomes of mice. Our study indicates that the obvious hepatotoxicity during this co-administration was due to the inhibition of CYP3A4 activity and ABCB1 transport activity. These findings suggested that the accumulation-induced hepatotoxicity of paclitaxel when it is combined with fentanyl should be avoided. PMID:26633878

  18. CYP1A2 polymorphism in Chinese patients with acute liver injury induced by Polygonum multiflorum.

    PubMed

    Ma, K F; Zhang, X G; Jia, H Y

    2014-01-01

    The objective of this study was to evaluate the genotype and allelic frequencies of CYP1A2 in Chinese patients with acute liver injury induced by Polygonum multiflorum. We examined the clinical mechanism of acute liver injury induced by P. multiflorum. According to the diagnostic criteria for drug-induced liver injury (DILI), 43 cases of P. multiflorum-induced liver injury admitted to the First Affiliated Hospital, Zhejiang University were identified between January 2008 and December 2012. An additional 43 control subjects were also chosen. Several alleles, including 1C, 1F, 2, 7, 9, and 11 of CYP1A2 were amplified from genomic DNA and sequenced. We used the chi-square test to determine whether CYP1A2 allele polymorphisms are associated with acute liver injury induced by P. multiflorum. The frequency of the CYP1A2 1C allele was 46.5% in P. multiflorum-induced DILI patients, which was significantly different from the frequency of 27.9% observed in healthy subjects. The frequency of the CYP1A2 1F allele was 63.9% in P. multiflorum-induced DILI patients, compared to 57.0% in healthy controls; the difference was not significant. The allelic frequencies of CYP1A2 2, CYP1A2 7, CYP1A2 9, and CYP1A2 11 were too low to be detected. The frequency of the CYP1A2 1C mutation in Chinese patients with P. multiflorum-induced acute liver injury differed from that in healthy Chinese people, indicating that CYP1A2 1C is probably related to metabolism of P. multiflorum, which is followed by acute liver injury. PMID:25117321

  19. Minor furanocoumarins and coumarins in grapefruit peel oil as inhibitors of human cytochrome P450 3A4.

    PubMed

    César, Thaïs B; Manthey, John A; Myung, Kyung

    2009-09-01

    A new cyclic acetal (1) of marmin (6',7'-dihydroxy-7-geranyloxycoumarin), two new cyclic acetals (5, 6) of 6',7'-dihydroxybergamottin, and the known compounds marmin (2), 7-geranyloxycoumarin (3), bergamottin (4), and 6',7'-dihydroxybergamottin (7) were isolated from grapefruit peel oil. All compounds were tested for inhibitory activity against intestinal cytochrome P450 3A4, an enzyme involved in the "grapefruit/drug" interactions in humans. Coumarins (1-3) exhibited negligible inhibitory activity, while the furanocoumarins (4-7) showed potent in vitro inhibitory activity with IC(50) values of 2.42, 0.13, 0.27, and 1.58 microM, respectively. PMID:19689106

  20. Comparison of inhibitory duration of grapefruit juice on organic anion-transporting polypeptide and cytochrome P450 3A4.

    PubMed

    Tanaka, Shimako; Uchida, Shinya; Miyakawa, Sachiko; Inui, Naoki; Takeuchi, Kazuhiko; Watanabe, Hiroshi; Namiki, Noriyuki

    2013-01-01

    Recently, a new type of interaction has been reported in which fruit juices diminish oral drug bioavailability through inhibition of organic anion-transporting polypeptide (OATP). In this study, we aimed to clarify the duration of OATP inhibition by grapefruit juice (GFJ), and to compare it with the duration of GFJ-induced inhibition of cytochrome P450 (CYP) 3A4 activity. Seven healthy volunteers were enrolled in this open-label, single-sequence study. They were orally administered celiprolol (100 mg) and midazolam (15 µg/kg) with water on the control day. Three days later, they ingested GFJ (200 mL) 3 times a day for 3 d. On day 1, the same drugs were administered with GFJ. On days 3 and 7, the same drugs were administered with water. Pharmacokinetics of both drugs were evaluated on each trial day. The peak plasma concentration (Cmax) and the area under the plasma concentration-time curve from 0 to 8 h (AUC0-8) of celiprolol significantly decreased on day 1, and the mean ratios of these values and the corresponding control-day values were 0.18 and 0.25, respectively. The Cmax and AUC0-8 returned to the control levels on days 3 and 7. In contrast, AUC0-8 of midazolam were higher on days 1 and 3 than on the control day (mean ratio, 2.12 and 1.47, respectively). The AUC0-8 returned to the control level on day 7. In conclusion, results of this study indicated that the OATP inhibition caused by GFJ dissipated faster than GFJ-mediated alterations in CYP3A4 activity, which were sustained for at least 48 h. PMID:24292052

  1. Simultaneous evaluation of human CYP3A4 and ABCB1 induction by reporter assay in LS174T cells, stably expressing their reporter genes.

    PubMed

    Inami, Keita; Sasaki, Takamitsu; Kumagai, Takeshi; Nagata, Kiyoshi

    2015-04-01

    The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp). Predicting whether candidate compounds induce CYP3A4 and P-gp is a crucial stage in the drug development process, as drug-drug interactions may result in the induction of intestinal CYP3A4 and P-gp. However, the assay systems needed to evaluate both CYP3A4 and P-gp induction in the intestine are yet to be established. To address this urgent requirement, LS174T cells were used to create two stable cell lines expressing the CYP3A4 or ATP-binding cassette subfamily B member 1 (ABCB1, encoding P-gp) reporter genes. First, these stable cells were tested by treatment with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (9-cis RA) that induce CYP3A4 and P-gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess the induction of CYP3A4 and ABCB1, both stable cells were co-cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH)2 D3, ATRA, and 9-cis RA. These activity levels were maintained after passaging more than 20 times and following multiple freeze-thaw cycles. These results demonstrate that our established cell lines can be used to evaluate simultaneously CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates. PMID:25410880

  2. INHIBITION OF HUMAN AND RAT CYP1A2 BY TCDD AND DIOXIN-LIKE CHEMICALS

    EPA Science Inventory

    Dioxins have been shown to bind and induce rodent CYP1A2, producing a dose-dependent hepatic sequestration in vivo. The induction of CYP1A2 activity has been used as a noninvasive biomarker for human exposure to dioxins; while there is a consistent relationship between exposure ...

  3. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 29 Labor 7 2014-07-01 2014-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH... communication (See also § 1917.1(a)(2)(vi))....

  4. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 7 2011-07-01 2011-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH... communication (See also § 1917.1(a)(2)(vi))....

  5. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 29 Labor 7 2013-07-01 2013-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH... communication (See also § 1917.1(a)(2)(vi))....

  6. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 29 Labor 7 2012-07-01 2012-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH... communication (See also § 1917.1(a)(2)(vi))....

  7. Genetic effects of ATP1A2 in familial hemiplegic migraine type II and animal models

    PubMed Central

    2013-01-01

    Na+/K+-ATPase alpha 2 (Atp1a2) is an integral plasma membrane protein belonging to the P-type ATPase family that is responsible for maintaining the sodium (Na+) and potassium (K+) gradients across cellular membranes with hydrolysis of ATP. Atp1a2 contains two subunits, alpha and beta, with each having various isoforms and differential tissue distribution. In humans, mutations in ATP1A2 are associated with a rare form of hereditary migraines with aura known as familial hemiplegic migraine type II. Genetic studies in mice have revealed other neurological effects of Atp1a2 in mice including anxiety, fear, and learning and motor function disorders. This paper reviews the recent findings in the literature concerning Atp1a2. PMID:23561701

  8. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    PubMed

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR. PMID:26987505

  9. Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

    PubMed

    Wang, Hai-Yan; Zhang, Ji; Zhang, Yue-Jing; Zhang, Bo; Liu, Chong-Xi; He, Hai-Rong; Wang, Xiang-Jing; Xiang, Wen-Sheng

    2014-12-01

    Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime. PMID:25081559

  10. Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice[S

    PubMed Central

    Cheng, Jie; Fang, Zhong-Ze; Kim, Jung-Hwan; Krausz, Kristopher W.; Tanaka, Naoki; Chiang, John Y. L.; Gonzalez, Frank J.

    2014-01-01

    Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (VdrΔIEpC) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into VdrΔIEpC mice to produce the VdrΔIEpC/3A4 line. LCA was administered to control, transgenic-CYP3A4, VdrΔIEpC, and VdrΔIEpC/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the VdrΔIEpC mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the VdrΔIEpC/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity. PMID:24343899

  11. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma

    PubMed Central

    Ren, Jianwai; Chen, George G.; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C. S.; Wang, Y.; Ho, Rocky L. K.; Yang, Shengli; Lu, Gang; Lee, C. G.; Lai, Paul B. S.

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  12. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma.

    PubMed

    Ren, Jianwai; Chen, George G; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C S; Wang, Y; Ho, Rocky L K; Yang, Shengli; Lu, Gang; Lee, C G; Lai, Paul B S

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  13. Multiple substrate-binding sites are retained in cytochrome P450 3A4 mutants with decreased cooperativity

    PubMed Central

    Fernando, Harshica; Rumfeldt, Jessica A. O.; Davydova, Nadezhda Y.; Halpert, James R.; Davydov, Dmitri R.

    2010-01-01

    1. The basis of decreased cooperativity in substrate binding in the cytochrome P450 3A4 mutants F213W, F304W and L211F/D214E was studied with fluorescence resonance energy transfer (FRET) and absorbance spectroscopy. 2. Whereas in the wild type enzyme the absorbance changes reflecting the interactions with 1-pyrenebutanol exhibit a Hill coefficient (nH) around 1.7 (S50 = 11.7 μM), the mutants showed no cooperativity (nH ≤ 1.1) with unchanged S50 values. 3. Contrary to the premise that the mutants lack one of the two binding sites, the mutants exhibited at least two substrate binding events. The high affinity interaction is characterized by a dissociation constant (KD) ≤ 1.0 μM, whereas the KD of the second binding has the same magnitude as the S50. 4. Theoretical analysis of a two-step binding model suggests that nH values may vary from 1.1 to 2.2 depending on the amplitude of the spin shift caused by the first binding event. 5. Alteration of cooperativity in the mutants is caused by a partial displacement of the “spin-shifting” step. Whereas in the wild type the spin shift occurs in the ternary complex only, the mutants exhibit some spin shift upon binding of the first substrate molecule. PMID:21143007

  14. Content of CYP3A4 inhibitors, naringin, naringenin and bergapten in grapefruit and grapefruit juice products.

    PubMed

    Ho, P C; Saville, D J; Coville, P F; Wanwimolruk, S

    2000-04-01

    The flavonoids, naringin and naringenin and the furanocoumarin, bergapten (5-methoxypsoralen), were detected in some fresh grapefruit and commercial grapefruit juices but were not detected in other fruit juices tested (orange; orange with apple base; dark grape; orange and mango with apple base; orange, peach, passion fruit juice). The contents of these three grapefruit constituents in commercial juice and fresh grapefruit varied from brand to brand and also from lot to lot. Juice was prepared from the fresh fruit via different methods (by hand, squeezer or blender). The naringin content, after hand-squeeze, ranged from 115 to 384 mg/l. With hand-squeeze juice production, bergapten was not detected (less than 0.5 mg/l) in two varieties of grapefruit, and naringenin was usually not in detectable levels (less than 2 mg/l) in three varieties. All three constituents were present in New Zealand grapefruit preparations (including juice by hand-squeeze) and different lots showed variation in content (1.5-, 2.3- and 4.7-fold for naringin, naringenin and bergapten, respectively). Differences in the concentrations of these three constituents, which have potential for drug interaction, may contribute to the variability in pharmacokinetics of CYP3A4 drugs and some contradictory results of drug interaction studies with grapefruit juice. PMID:10812937

  15. Prednisone has no effect on the pharmacokinetics of CYP3A4 metabolized drugs - midazolam and odanacatib.

    PubMed

    Marcantonio, Eugene E; Ballard, Jeanine; Gibson, Christopher R; Kassahun, Kelem; Palamanda, Jairam; Tang, Cuyue; Evers, Raymond; Liu, Chengcheng; Zajic, Stefan; Mahon, Chantal; Mostoller, Kate; Hreniuk, David; Mehta, Anish; Morris, Denise; Wagner, John A; Stoch, S Aubrey

    2014-11-01

    We evaluated the effect of prednisone on midazolam and odanacatib pharmacokinetics. In this open-label, 2-period crossover study in healthy male subjects, midazolam 2 mg was administered (Day -1) followed by odanacatib 50 mg (Day 1) during Part 1. In Period 2, prednisone 10 mg once daily (qd) was administered on Days 1-28; odanacatib was co-administered on Day 14 and midazolam 2 mg was co-administered on Days 1 and 28. Subjects were administered midazolam 2 mg on Days 42 and 56. Safety and tolerability were assessed throughout the study. A physiologically-based pharmacokinetic (PBPK) model was also built. There were 15 subjects enrolled; mean age was 31 years. The odanacatib AUC(0- ∞) GMR (90% CI) [odanacatib + prednisone (Day 14, Period 2)/odanacatib alone (Day 1, Period 1] was 1.06 (0.96, 1.17). AUC(0-∞) GMR (90%CI) [midazolam + prednisone (Day 28, Period 2)/midazolam alone (Day -1, Period 1] was 1.08 (0.93,1.26). There were no serious AEs or AEs leading to discontinuation. PBPK modeling showed that prednisone does not cause significant effects on the exposure of sensitive CYP3A4 substrates in vivo at therapeutic doses. Co-administration of prednisone 10 mg qd had no effect on pharmacokinetics of either odanacatib 10 mg or midazolam 2 mg. PMID:24895078

  16. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells.

    PubMed

    Negoro, Ryosuke; Takayama, Kazuo; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. PMID:26966071

  17. Modeling of Rifampicin-Induced CYP3A4 Activation Dynamics for the Prediction of Clinical Drug-Drug Interactions from In Vitro Data

    PubMed Central

    Yamashita, Fumiyoshi; Sasa, Yukako; Yoshida, Shuya; Hisaka, Akihiro; Asai, Yoshiyuki; Kitano, Hiroaki; Hashida, Mitsuru; Suzuki, Hiroshi

    2013-01-01

    Induction of cytochrome P450 3A4 (CYP3A4) expression is often implicated in clinically relevant drug-drug interactions (DDI), as metabolism catalyzed by this enzyme is the dominant route of elimination for many drugs. Although several DDI models have been proposed, none have comprehensively considered the effects of enzyme transcription/translation dynamics on induction-based DDI. Rifampicin is a well-known CYP3A4 inducer, and is commonly used as a positive control for evaluating the CYP3A4 induction potential of test compounds. Herein, we report the compilation of in vitro induction data for CYP3A4 by rifampicin in human hepatocytes, and the transcription/translation model developed for this enzyme using an extended least squares method that can account for inherent inter-individual variability. We also developed physiologically based pharmacokinetic (PBPK) models for the CYP3A4 inducer and CYP3A4 substrates. Finally, we demonstrated that rifampicin-induced DDI can be predicted with reasonable accuracy, and that a static model can be used to simulate DDI once the blood concentration of the inducer reaches a steady state following repeated dosing. This dynamic PBPK-based DDI model was implemented on a new multi-hierarchical physiology simulation platform named PhysioDesigner. PMID:24086247

  18. Prediction of variability in CYP3A4 induction using a combined 1H NMR metabonomics and targeted UPLC-MS approach.

    PubMed

    Rahmioglu, Nilufer; Le Gall, Gwénaëlle; Heaton, James; Kay, Kristine L; Smith, Norman W; Colquhoun, Ian J; Ahmadi, Kourosh R; Kemsley, E Kate

    2011-06-01

    The activity of Cytochrome P450 3A4 (CYP3A4) enzyme is associated with many adverse or poor therapeutic responses to drugs. We used (1)H NMR-based metabonomics to identify a metabolic signature associated with variation in induced CYP3A4 activity. A total of 301 female twins, aged 45--84, participated in this study. Each volunteer was administered a potent inducer of CYP3A4 (St. John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate (300 mg). Pre- and postintervention fasting urine samples were used to obtain metabolite profiles, using (1)H NMR spectroscopy, and were analyzed using UPLC--MS to obtain a marker for CYP3A4 induction, via the ratio of 3-hydroxyquinine to quinine (3OH-Q:Q). Multiple linear regression was used to build a predictive model for 3OH-Q:Q values based on the preintervention metabolite profiles. A combination of seven metabolites and seven covariates showed a strong (r = 0.62) relationship with log(3OH-Q:Q). This regression model demonstrated significant (p < 0.00001) predictive ability when applied to an independent validation set. Our results highlight the promise of metabonomics for predicting CYP3A4-mediated drug response. PMID:21491888

  19. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    PubMed

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease. PMID:26301745

  20. Expression of CYP3A4 and CYP3A7 in Human Foetal Tissues and its Correlation with Nuclear Receptors.

    PubMed

    Betts, Stina; Björkhem-Bergman, Linda; Rane, Anders; Ekström, Lena

    2015-10-01

    Previous reports have suggested that the nuclear receptors vitamin D receptor (VDR), peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are involved in the regulation of the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 expression in adults. The aim of this study was to investigate the gene expression of CYP3A4 and the foetal CYP3A7 in human foetal tissues and their relation to gene expression and genetic variations in the nuclear receptors VDR, PPARα, PXR and CAR. We determined the relative expression of CYP3A4 and CYP3A7 and these nuclear receptors in foetal livers, intestines and adrenals, using quantitative PCR. In addition, the expression of these enzymes was also analysed in adult liver. There was a high interindividual variability in CYP3A4 and CYP3A7, 49 times and 326 times, respectively. Both CYP3A4 and CYP3A7 had the highest expression in the liver. There were significant correlations (p < 0.001) between the nuclear receptors studied and the expression of CYP3A4 and CYP3A7 in foetal liver, as well as the expression of CYP3A4 in foetal intestine. Polymorphisms in the VDR gene, rs1544410 and rs1523130 (TaqI), in the PXR gene, rs1523130, and in the PPARα gene, rs4253728, were not correlated with CYP3A4 or CYP3A7 expression. However, C-homozygous individuals of the TaqI VDR polymorphism had 60% lower VDR gene expression (p < 0.05), than individuals carrying one or two T alleles. In conclusion, differences in the expression of nuclear receptors might determine the variability in CYP3A4 and CYP3A7 expression observed in foetal liver. PMID:25689036

  1. Selection of alternative CYP3A4 probe substrates for clinical drug interaction studies using in vitro data and in vivo simulation.

    PubMed

    Foti, Robert S; Rock, Dan A; Wienkers, Larry C; Wahlstrom, Jan L

    2010-06-01

    Understanding the potential for cytochrome P450-mediated drug-drug interactions (DDIs) is a critical step in the drug discovery process. DDIs of CYP3A4 are of particular importance because of the number of marketed drugs that are cleared by this enzyme. In response to studies that suggested the presence of several binding regions within the CYP3A4 active site, multiple probe substrates are often used for in vitro CYP3A4 DDI studies, including midazolam (the clinical standard), felodipine/nifedipine, and testosterone. However, the design of clinical CYP3A4 DDI studies may be confounded for cases such as 1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458), with which testosterone is predicted to exhibit a clinically relevant DDI whereas midazolam and felodipine/nifedipine are not. To develop an appropriate path forward for such clinical DDI studies, the inhibition potency of 20 known inhibitors of CYP3A4 were measured in vitro using 8 clinically relevant CYP3A4 probe substrates and testosterone. Hierarchical clustering suggested four probe substrate clusters: testosterone; felodipine; midazolam, buspirone, quinidine, and sildenafil; and simvastatin, budesonide, and fluticasone. The in vivo sensitivities of six clinically relevant CYP3A4 probe substrates (buspirone, cyclosporine, nifedipine, quinidine, sildenafil, and simvastatin) were determined in relation to midazolam from literature DDI data. Buspirone, sildenafil, and simvastatin exhibited similar or greater sensitivity than midazolam to CYP3A4 inhibition in vivo. Finally, Simcyp was used to predict the in vivo magnitude of CYP3A4 DDIs caused by AMG 458 using midazolam, sildenafil, simvastatin, and testosterone as probe substrates. PMID:20203109

  2. Comparison of CYP1A2 and NAT2 Phenotypes between Black and White Smokers

    PubMed Central

    Muscat, Joshua E.; Pittman, Brian; Kleinman, Wayne; Lazarus, Philip; Stellman, Steven D.; Richie, John P.

    2008-01-01

    The lower incidence rate of transitional cell carcinoma of the urinary bladder in blacks than in whites may be due to racial differences in the catalytic activity of enzymes that metabolize carcinogenic arylamines in tobacco smoke. To examine this, we compared cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 activities (NAT2) in black and white smokers using urinary caffeine metabolites as a probe for enzyme activity in a community-based study of 165 black and 183 white cigarette smokers. The paraxanthine (1,7-dimethylxanthine, 17X)/caffeine (trimethylxanthine, 137X) ratio or [17X + 1,7-dimethyluric acid (17U)]/137X ratio was used as an indicator of CYP1A2 activity. The 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine (1X) ratio indicated NAT2 activity. The odds ratio for the slow NAT2 phenotype associated with black race was 0.4; 95% confidence intervals 0.2–0.7. The putative combined low risk phenotype (slow CYP1A2/rapid NAT2) was more common in blacks than in whites (25% vs. 15%, P<0.02). There were no significant racial differences in slow and rapid CYP1A2 phenotypes, and in the combined slow NAT2/rapid CYP1A2 phenotype. Age, education, cigarette smoking amount, body mass index, GSTM1 and GSTM3 genotypes were unrelated to CYP1A2 and NAT2 activity. Intake of cruciferous vegetables (primarily broccoli), red meat, carrots, grapefruit and onions predicted CYP1A2 activity either for all subjects or in race-specific analyses. Carrot and grapefruit consumption was related to NAT2 activity. Collectively, these results indicated that phenotypic differences in NAT2 alone or in combination with CYP1A2 might help explain the higher incidence rates of transitional cell bladder cancer in whites. PMID:18703023

  3. Comparison of CYP1A2 and NAT2 phenotypes between black and white smokers.

    PubMed

    Muscat, Joshua E; Pittman, Brian; Kleinman, Wayne; Lazarus, Philip; Stellman, Steven D; Richie, John P

    2008-10-01

    The lower incidence rate of transitional cell carcinoma of the urinary bladder in blacks than in whites may be due to racial differences in the catalytic activity of enzymes that metabolize carcinogenic arylamines in tobacco smoke. To examine this, we compared cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 activities (NAT2) in black and white smokers using urinary caffeine metabolites as a probe for enzyme activity in a community-based study of 165 black and 183 white cigarette smokers. The paraxanthine (1,7-dimethylxanthine, 17X)/caffeine (trimethylxanthine, 137X) ratio or [17X+1,7-dimethyluric acid (17U)]/137X ratio was used as an indicator of CYP1A2 activity. The 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine (1X) ratio indicated NAT2 activity. The odds ratio for the slow NAT2 phenotype associated with black race was 0.4; 95% confidence intervals 0.2-0.7. The putative combined low risk phenotype (slow CYP1A2/rapid NAT2) was more common in blacks than in whites (25% vs. 15%, P<0.02). There were no significant racial differences in slow and rapid CYP1A2 phenotypes, and in the combined slow NAT2/rapid CYP1A2 phenotype. Age, education, cigarette smoking amount, body mass index, GSTM1 and GSTM3 genotypes were unrelated to CYP1A2 and NAT2 activity. Intake of cruciferous vegetables (primarily broccoli), red meat, carrots, grapefruit and onions predicted CYP1A2 activity either for all subjects or in race-specific analyses. Carrot and grapefruit consumption was related to NAT2 activity. Collectively, these results indicated that phenotypic differences in NAT2 alone or in combination with CYP1A2 might help explain the higher incidence rates of transitional cell bladder cancer in whites. PMID:18703023

  4. Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2

    SciTech Connect

    Gorman, Nadia; Trask, Heidi S.; Robinson, Susan W.; Sinclair, Jacqueline F.; Smith, Andrew G.; Sinclair, Peter R. . E-mail: psinc@dartmouth.edu

    2007-06-01

    Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the high affinity form of the receptor (AHRb{sub 1}), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(-/-) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(-/-) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(-/-) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential.

  5. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    SciTech Connect

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V.

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

  6. 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. PMID:26429523

  7. Identification of cytochrome P450 3A4 modification site with reactive metabolite using linear ion trap-Fourier transform mass spectrometry.

    PubMed

    Yukinaga, Hideo; Takami, Tomonori; Shioyama, Sho-Hei; Tozuka, Zenzaburo; Masumoto, Hiroshi; Okazaki, Osamu; Sudo, Ken-Ichi

    2007-10-01

    Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4. PMID:17867646

  8. Establishment of In Silico Prediction Models for CYP3A4 and CYP2B6 Induction in Human Hepatocytes by Multiple Regression Analysis Using Azole Compounds.

    PubMed

    Nagai, Mika; Konno, Yoshihiro; Satsukawa, Masahiro; Yamashita, Shinji; Yoshinari, Kouichi

    2016-08-01

    Drug-drug interactions (DDIs) via cytochrome P450 (P450) induction are one clinical problem leading to increased risk of adverse effects and the need for dosage adjustments and additional therapeutic monitoring. In silico models for predicting P450 induction are useful for avoiding DDI risk. In this study, we have established regression models for CYP3A4 and CYP2B6 induction in human hepatocytes using several physicochemical parameters for a set of azole compounds with different P450 induction as characteristics as model compounds. To obtain a well-correlated regression model, the compounds for CYP3A4 or CYP2B6 induction were independently selected from the tested azole compounds using principal component analysis with fold-induction data. Both of the multiple linear regression models obtained for CYP3A4 and CYP2B6 induction are represented by different sets of physicochemical parameters. The adjusted coefficients of determination for these models were of 0.8 and 0.9, respectively. The fold-induction of the validation compounds, another set of 12 azole-containing compounds, were predicted within twofold limits for both CYP3A4 and CYP2B6. The concordance for the prediction of CYP3A4 induction was 87% with another validation set, 23 marketed drugs. However, the prediction of CYP2B6 induction tended to be overestimated for these marketed drugs. The regression models show that lipophilicity mostly contributes to CYP3A4 induction, whereas not only the lipophilicity but also the molecular polarity is important for CYP2B6 induction. Our regression models, especially that for CYP3A4 induction, might provide useful methods to avoid potent CYP3A4 or CYP2B6 inducers during the lead optimization stage without performing induction assays in human hepatocytes. PMID:27208383

  9. GW4064, an agonist of farnesoid X receptor, represses CYP3A4 expression in human hepatocytes by inducing small heterodimer partner expression.

    PubMed

    Zhang, Shu; Pan, Xian; Jeong, Hyunyoung

    2015-05-01

    Farnesoid X receptor (FXR) functions as a regulator of bile acid and lipid homeostasis and is recognized as a promising therapeutic target for metabolic diseases. The biologic function of FXR is mediated in part by a small heterodimer partner (SHP); ligand-activated FXR enhances SHP expression, and SHP in turn represses the activity of multiple transcription factors. This study aimed to investigate the effect of FXR activation on expression of the major drug-metabolizing enzyme CYP3A4. The effects of 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064), a synthetic agonist of FXR, on the expression and activity of CYP3A4 were examined in primary human hepatocytes by using quantitative real-time polymerase chain reaction and S9 phenotyping. In human hepatocytes, treatment of GW4064 (1 μM) for 48 hours resulted in a 75% decrease in CYP3A4 mRNA expression and a 25% decrease in CYP3A4 activity, accompanied by ∼3-fold increase in SHP mRNA expression. In HepG2 cells, SHP repressed transactivation of CYP3A4 promoter by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and glucocorticoid receptor. Interestingly, GW4064 did not repress expression of CYP2B6, another target gene of PXR and CAR; GW4064 enhanced CYP2B6 promoter activity. In conclusion, GW4064 represses CYP3A4 expression in human hepatocytes, potentially through upregulation of SHP expression and subsequent repression of CYP3A4 promoter activity. Clinically significant drug-drug interaction involving FXR agonists and CYP3A4 substrates may occur. PMID:25725071

  10. ONTOGENIC EXPRESSION OF HUMAN CARBOXYLESTERASE-2 AND CYTOCHROME P450 3A4 IN LIVER AND DUODENUM: POSTNATAL SURGE AND ORGAN-DEPENDENT REGULATION1

    PubMed Central

    Chen, Yi-Tzai; Trzoss, Lynnie; Yang, Dongfang; Yan, Bingfang

    2015-01-01

    Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner. PMID:25724353

  11. Size and surface modification of amorphous silica particles determine their effects on the activity of human CYP3A4 in vitro

    NASA Astrophysics Data System (ADS)

    Imai, Shunji; Yoshioka, Yasuo; Morishita, Yuki; Yoshida, Tokuyuki; Uji, Miyuki; Nagano, Kazuya; Mukai, Yohei; Kamada, Haruhiko; Tsunoda, Shin-ichi; Higashisaka, Kazuma; Tsutsumi, Yasuo

    2014-12-01

    Because of their useful chemical and physical properties, nanomaterials are widely used around the world - for example, as additives in food and medicines - and such uses are expected to become more prevalent in the future. Therefore, collecting information about the effects of nanomaterials on metabolic enzymes is important. Here, we examined the effects of amorphous silica particles with various sizes and surface modifications on cytochrome P450 3A4 (CYP3A4) activity by means of two different in vitro assays. Silica nanoparticles with diameters of 30 and 70 nm (nSP30 and nSP70, respectively) tended to inhibit CYP3A4 activity in human liver microsomes (HLMs), but the inhibitory activity of both types of nanoparticles was decreased by carboxyl modification. In contrast, amine-modified nSP70 activated CYP3A4 activity. In HepG2 cells, nSP30 inhibited CYP3A4 activity more strongly than the larger silica particles did. Taken together, these results suggest that the size and surface characteristics of the silica particles determined their effects on CYP3A4 activity and that it may be possible to develop silica particles that do not have undesirable effects on metabolic enzymes by altering their size and surface characteristics.

  12. Racial Differences in CYP3A4 Genotype and Survival Among Men Treated on Radiation Therapy Oncology Group (RTOG) 9202: A Phase III Randomized Trial

    SciTech Connect

    Roach, Mack Silvio, Michelle de; Rebbick, Timothy; Grignon, David; Rotman, Marvin; Wolkov, Harvey; Fisher, Barbara; Hanks, Gerald; Shipley, William U.; Pollack, Alan; Sandler, Howard; Watkins-Bruner, Deborah Ph.D.

    2007-09-01

    Purpose: Inherited genotypes may explain the inferior outcomes of African American (AA) men with prostate cancer. To understand how variation in CYP3A4 correlated with outcomes, a retrospective examination of the CYP3A4*1B genotype was performed on men treated with Radiation Therapy Oncology Group (RTOG) 92-02. Methods and Materials: From 1,514 cases, we evaluated 56 (28.4%) of 197 AA and 54 (4.3%) of 1,274 European American (EA) patients. All patients received goserelin and flutamide for 2 months before and during RT (STAD-RT) {+-} 24 months of goserelin (long-term androgen deprivation plus radiation [LTAD-RT]). Events studied included overall survival and biochemical progression using American Society for Therapeutic Radiology and Oncology consensus guidelines. Results: There were no differences in outcome in patients in with or without CYP3A4 data. There was an association between race and CYP3A4 polymorphisms with 75% of EAs having the Wild Type compared to only 25% of AA men (p <0.0001). There was no association between CYP3A4 classification or race and survival or progression. Conclusions: The samples analyzed support previously reported observations about the distribution of CYP3A4*1B genotype by race, but race was not associated with poorer outcome. However, patient numbers were limited, and selection bias cannot be completely ruled out.

  13. Induction of cytochrome P-450 1A2 by oxidized tryptophan in Hepa lclc7 cells.

    PubMed

    Sindhu, R K; Mitsuhashi, M; Kikkawa, Y

    2000-03-01

    Recent studies from this laboratory have demonstrated that L-tryptophan, after oxidation either by UV-irradiation or ozone, induces aryl hydrocarbon receptor (AhR) activation and binding of the liganded AhR complex to its specific DNA recognition site, thereby initiating transcription of the cytochrome P-450 1a1 (Cyp1a1) gene with concomitant increase of CYP1A1 protein and 7-ethoxyresorufin O-deethylase activity in wild-type mouse hepatoma cells, Hepa lclc7 (Hepa-1), in culture. Temporary inhibition of protein synthesis by cycloheximide resulted in superinduction of oxidized tryptophan-inducible CYP1A1 mRNA, protein, and 7-ethoxyresorufin O-deethylase activity in Hepa-1 cells. In the present communication, the results obtained by immunoblot analyses with monoclonal CYP1A1/1A2 antibody (NIH 1-7-1) demonstrate that both UV- or ozone-oxidized tryptophan also induce CYP1A2 protein in Hepa-1 cells. CYP1A2 mRNA, detected by reverse transcription-polymerase chain reaction, was markedly induced in the UV- or ozone-oxidized tryptophan-treated cells. Temporary inhibition of protein synthesis by cycloheximide further induced oxidized tryptophan-inducible CYP1A2 mRNA as well as the protein in Hepa-1 cells. This is the first report demonstrating the induction of CYP1A2 mRNA and protein in Hepa-1 cells. PMID:10688617

  14. Allele dependent silencing of COL1A2 using small interfering RNAs

    PubMed Central

    Lindahl, Katarina; Rubin, Carl-Johan; Kindmark, Andreas; Ljunggren, Östen

    2008-01-01

    Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI. PMID:19015742

  15. Part I---Evaluating Effects of Oligomer Formation on Cytochrome P450 2C9 Electron Transfer and Drug Metabolism, Part II---Utilizing Molecular Modeling Techniques to Study the Src-Interacting Proteins Actin Filament Associated Protein of 110 kDa (AFAP-110) and Cortactin

    NASA Astrophysics Data System (ADS)

    Jett, John Edward, Jr.

    The dissertation has been divided into two parts to accurately reflect the two distinct areas of interest pursued during my matriculation in the School of Pharmacy at West Virginia University. In Part I, I discuss research probing the nature of electron transfer in the Cytochrome P450 family of proteins, a group of proteins well-known for their role in drug metabolism. In Part II, I focus on in silico and in vitro work developed in concert to probe protein structure and protein-protein interactions involved in actin filament reorganization and cellular motility. Part I. Cytochrome P450s (P450s) are an important class of enzymes known to metabolize a variety of endogenous and xenobiotic compounds. P450s are most commonly found in liver and intestinal endothelial cells and are responsible for the metabolism of approximately 75% of pharmaceutical drugs on the market. CYP2C9---one of the six major P450 isoforms---is responsible for ˜20% of drug metabolism. Elucidation of the factors that affect in vitro drug metabolism is crucial to the accurate prediction of in vivo drug metabolism kinetics. Currently, the two major techniques for studying in vitro drug metabolism are solution-based. However, it is known that the results of solution-based studies can vary from in vivo drug metabolism. One reason suggested to account for this variation is the state of P450 oligomer formation in solution compared to the in vivo environment, where P450s are membrane-bound. To understand the details of how oligomer formation affects in vitro drug metabolism, it is imperative that techniques be developed which will allow for the unequivocal control of oligomer formation without altering other experimental parameters. Our long term goal of this research is to develop methods to more accurately predict in vivo drug metabolism from in vitro data. This section of the dissertation will discuss the development of a platform consisting of a doped silicon surface containing a large array of gold

  16. Data package addendum for COBRA-1A2 life extension to 400 EFPD

    SciTech Connect

    Hecht, S.L.; Ermi, A.M.

    1994-08-29

    The COBRA-1A experiment was originally designed for irradiations up to 350 effective full power days (EFPD) in EBR-II. Three of the seven B7A test capsules were discharged after 88.6 EFPD (COBRA-1A1; EBR-II designation X516), while the remaining four capsules continued to be irradiated to a goal exposure of 300 EFPD (COBRA-1A2; EBR-II designation X516A). However, it was recently decided that COBRA-1A2 was to remain in the reactor during Run 170, giving and nominal end-of-life (EOL) exposure of 375 EFPD. Since the revised test exposure exceeds the design basis given in supporting analyses, amended analyses are provided herein, giving the technical bases for the extended irradiation. This report describes the safety analysis for the extension of the COBRA-1A2 test (X516A) to 400 effective full power days in FBR-II.

  17. CYP3A4-transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam.

    PubMed

    Cummins, Carolyn L; Jacobsen, Wolfgang; Christians, Uwe; Benet, Leslie Z

    2004-01-01

    CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier. PMID:14569063

  18. PCB Exposure and in Vivo CYP1A2 Activity among Native Americans

    PubMed Central

    Fitzgerald, Edward F.; Hwang, Syni-An; Lambert, George; Gomez, Marta; Tarbell, Alice

    2005-01-01

    Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects. PMID:15743714

  19. A predominate role of CYP1A2 for the metabolism of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid, in human liver microsomes.

    PubMed

    Turpeinen, Miia; Hofmann, Ute; Klein, Kathrin; Mürdter, Thomas; Schwab, Matthias; Zanger, Ulrich M

    2009-05-01

    Nabumetone, a widely used nonsteroidal anti-inflammatory drug, requires biotransformation into 6-methoxy-2-naphthylacetic acid (6-MNA), a close structural analog to naproxen, to achieve its analgesic and anti-inflammatory effects. Despite its wide use, the enzymes involved in metabolism have not been identified. In the present study, several in vitro approaches were used to identify the cytochrome P450 (P450) enzyme(s) responsible for 6-MNA formation. In human liver microsomes (HLMs) 6-MNA formation displayed monophasic Michaelis-Menten kinetics with apparent K(m) and V(max) values (mean +/- S.D.) of 75.1 +/- 15.3 microM and 1304 +/- 226 pmol/min/mg protein, respectively, and formation rate of 6-MNA varied approximately 5.5-fold (179-983 pmol/min/mg protein). 6-MNA activity correlated strongly with both CYP1A2-mediated phenacetin O-deethylation activity and CYP1A2 protein content (r = 0.85 and 0.74, respectively; p < 0.0001 for both). Additional correlations were found with model activities of CYP2C19 and CYP3A4. Of 11 cDNA-expressed recombinant P450s used, recombinant CYP1A2 was the major form catalyzing the 6-MNA formation with an apparent K(m) of 45 microM and V(max) of 8.7 pmol/min/pmol P450. Minor fractions were catalyzed by recombinant P450s CYP1A1, CYP2B6, CYP2C19, CYP2D6, and CYP2E1. Experiments with P450-selective chemical inhibitors and monoclonal anti-P450 antibodies showed that furafylline, a mechanism-based inhibitor CYP1A2, and anti-CYP1A2 antibody markedly inhibited 6-MNA formation, whereas inhibitors for other P450s did not show significant inhibitory effects. Taken together, these studies indicate that the formation of the active metabolite of nabumetone, 6-MNA, is predominantly catalyzed by CYP1A2 in HLMs with only minor contribution of other P450s. PMID:19204080

  20. Identification of the Residue in Human CYP3A4 That Is Covalently Modified by Bergamottin and the Reactive Intermediate That Contributes to the Grapefruit Juice Effect

    PubMed Central

    Lin, Hsia-lien; Kenaan, Cesar

    2012-01-01

    Previous studies have demonstrated that bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of CYP3A4 and contributes, in part, to the grapefruit juice-drug interaction. Although the covalent binding of [14C]BG to the CYP3A4 apoprotein has been demonstrated by SDS-polyacrylamide gel electrophoresis, the identity of the modified amino acid residue and the reactive intermediate species of BG responsible for the inactivation have not been reported. In the present study, we show that inactivation of CYP3A4 by BG results in formation of a modified apoprotein-3A4 and a GSH conjugate, both exhibiting mass increases of 388 Da, which corresponds to the mass of 6′,7′-dihydroxybergamottin (DHBG), a metabolite of BG, plus one oxygen atom. To identify the adducted residue, BG-inactivated 3A4 was digested with trypsin, and the digests were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS). A mass shift of 388 Da was used for the SEQUEST database search, which revealed a mass increase of 388 Da for the peptide with the sequence 272LQLMIDSQNSK282, and MS/MS analysis of the adducted peptide demonstrated that Gln273 is the residue modified. Mutagenesis studies showed that the Gln273 to Val mutant was resistant to inactivation by BG and DHBG and did not generate two of the major metabolites of BG formed by 3A4 wild type. In conclusion, we have determined that the reactive intermediate, oxygenated DHBG, covalently binds to Gln273 and thereby contributes to the mechanism-based inactivation of CYP3A4 by BG. PMID:22344702

  1. The Effect of microRNAs in the Regulation of Human CYP3A4: a Systematic Study using a Mathematical Model

    NASA Astrophysics Data System (ADS)

    Wei, Zhiyun; Jiang, Songshan; Zhang, Yiting; Wang, Xiaofei; Peng, Xueling; Meng, Chunjie; Liu, Yichen; Wang, Honglian; Guo, Luo; Qin, Shengying; He, Lin; Shao, Fengmin; Zhang, Lirong; Xing, Qinghe

    2014-03-01

    CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.

  2. Immunohistochemical Markers of CYP3A4 and CYP3A7: A New Tool Towards Personalized Pharmacotherapy of Hepatocellular Carcinoma

    PubMed Central

    Fanni, D.; Manchia, M.; Lai, F.; Gerosa, C.; Ambu, R.; Faa, G.

    2016-01-01

    Hepatocellular carcinoma (HCC) represents a major global health problem, since more than 90% of primary liver cancers worldwide are HCC. Most cases of HCC are secondary to viral hepatitis infection (hepatitis B or C), alcoholism and cirrhosis. Sorafenib, an oral tyrosine kinase inhibitor that suppresses tumor proliferation and angiogenesis, emerged as the first effective systemic treatment for HCC after 30 years of research, and is currently the standard-of-care for patients with advanced HCC. Sorafenib is metabolized by cytochrome P450 (CYP450), particularly from the 3A4 isoform, producing two main metabolites: the N-oxide and the N-hydroxymethyl metabolite. We studied 11 HCC sample showing the presence of CYP3A4 and CYP3A7 in most of the samples analysed. Specifically, the immunoreactivity of CYP3A4 was stronger and more widespread than that of CYP3A7. The CYP3A4 immunoreactivity was observed in surrounding hepatocytes in 8 out of 11 cases; while the CYP3A7 immunostaining was found in normal liver cells, in 7 out of 11 cases. These results suggest the existence of a marked inter-individual variability regarding the presence of the isoforms of CYP3A. In addition, since sorafenib is metabolized by CYP3A4, but not by CYP3A7, an overexpression of CYP3A4 may lead to an increase in the degradation of the drug and then to clinical ineffectiveness. These results might implicate the necessity of an individualized approach in the treatment of HCC as positivity to CYP3A4 in HCC liver samples might predict a scarce response to sorafenib. PMID:27349315

  3. The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin

    PubMed Central

    Liu, Ju-E; Ren, Bin; Tang, Lan; Tang, Qian-Jie; Liu, Xiao-Ying; Li, Xin; Bai, Xue; Zhong, Wan-Ping; Meng, Jin-Xiu; Lin, Hao-Ming; Wu, Hong; Chen, Ji-Yan; Zhong, Shi-Long

    2016-01-01

    To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R2 = 0.12, P = 0.002) and CYP3A5 mRNA (partial R2 = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3′-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality. PMID:27211076

  4. The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin.

    PubMed

    Liu, Ju-E; Ren, Bin; Tang, Lan; Tang, Qian-Jie; Liu, Xiao-Ying; Li, Xin; Bai, Xue; Zhong, Wan-Ping; Meng, Jin-Xiu; Lin, Hao-Ming; Wu, Hong; Chen, Ji-Yan; Zhong, Shi-Long

    2016-01-01

    To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R(2) = 0.12, P = 0.002) and CYP3A5 mRNA (partial R(2) = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3'-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality. PMID:27211076

  5. EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2

    EPA Science Inventory

    EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC.
    Sponsor: H Barton

    Bromodichlorometh...

  6. Thermal ramp tritium release in COBRA-1A2 C03 beryllium pebbles

    SciTech Connect

    Baldwin, D.L.

    1998-03-01

    Tritium release kinetics, using the method of thermal ramp heating at three linear ramp rates, were measured on the COBRA-1A2 C03 1-mm beryllium pebbles. This report includes a brief discussion of the test, and the test data in graph format.

  7. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) MARINE TERMINALS Marine Terminal Operations § 1917.28...

  8. COMPARING ENVIRONMENTALLY RELEVANT PCBS TO TCDD IN CYP1A2 NULL AND WILDTYPE MICE

    EPA Science Inventory


    The role of CYP1A2 on the interactions of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin), dioxin-like (DL) and non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) was compared in multiple responses of different laboratory-defined mixtures, based on mass ratios found in...

  9. Three-dimensional quantitative structure–activity relationship and docking studies in a series of anthocyanin derivatives as cytochrome P450 3A4 inhibitors

    PubMed Central

    Shityakov, Sergey; Puskás, István; Roewer, Norbert; Förster, Carola; Broscheit, Jens

    2014-01-01

    The cytochrome P450 (CYP)3A4 enzyme affects the metabolism of most drug-like substances, and its inhibition may influence drug safety. Modulation of CYP3A4 by flavonoids, such as anthocyanins, has been shown to inhibit the mutagenic activity of mammalian cells. Considering the previous investigations addressing CYP3A4 inhibition by these substances, we studied the three-dimensional quantitative structure–activity relationship (3D-QSAR) in a series of anthocyanin derivatives as CYP3A4 inhibitors. For the training dataset (n=12), comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) yielded crossvalidated and non-crossvalidated models with a q2 of 0.795 (0.687) and r2 of 0.962 (0.948), respectively. The models were also validated by an external test set of four compounds with r2 of 0.821 (CoMFA) and r2 of 0.812 (CoMSIA). The binding affinity modes associated with experimentally derived IC50 (half maximal inhibitory concentration) values were confirmed by molecular docking into the CYP3A4 active site with r2 of 0.66. The results obtained from this study are useful for a better understanding of the effects of anthocyanin derivatives on inhibition of carcinogen activation and cellular DNA damage. PMID:24741320

  10. Systematic and quantitative assessment of the effect of chronic kidney disease on CYP2D6 and CYP3A4/5.

    PubMed

    Yoshida, K; Sun, B; Zhang, L; Zhao, P; Abernethy, D R; Nolin, T D; Rostami-Hodjegan, A; Zineh, I; Huang, S-M

    2016-07-01

    Recent reviews suggest that chronic kidney disease (CKD) can affect the pharmacokinetics of nonrenally eliminated drugs, but the impact of CKD on individual elimination pathways has not been systematically evaluated. In this study we developed a comprehensive dataset of the effect of CKD on the pharmacokinetics of CYP2D6- and CYP3A4/5-metabolized drugs. Drugs for evaluation were selected based on clinical drug-drug interaction (CYP3A4/5 and CYP2D6) and pharmacogenetic (CYP2D6) studies. Information from dedicated CKD studies was available for 13 and 18 of the CYP2D6 and CYP3A4/5 model drugs, respectively. Analysis of these data suggested that CYP2D6-mediated clearance is generally decreased in parallel with the severity of CKD. There was no apparent relationship between the severity of CKD and CYP3A4/5-mediated clearance. The observed elimination-route dependency in CKD effects between CYP2D6 and CYP3A4/5 may inform the need to conduct clinical CKD studies with nonrenally eliminated drugs for optimal use of drugs in patients with CKD. PMID:26800425

  11. Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives.

    PubMed

    Rauschenbach, R; Gieschen, H; Husemann, M; Salomon, B; Hildebrand, M

    1995-10-01

    Expression of human cytochrome (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6beta-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6beta-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated. PMID:8666035

  12. Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes.

    PubMed

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-05-01

    Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki-64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki-36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC50 values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co-administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb-drug interactions in humans. PMID:25684704

  13. Ophiopogon japonicus strains from different cultivation regions exhibit markedly different properties on cytotoxicity, pregnane X receptor activation and cytochrome P450 3A4 induction

    PubMed Central

    GE, LE-LE; KAN, LIAN-DI; ZHUGE, ZHENG-BING; MA, KE; CHEN, SHU-QING

    2015-01-01

    Maidong, known as Ophiopogon japonicus, is one of the two basic ingredients of Shenmai injection, which is a widely used herbal preparation in traditional Chinese medicine (TCM) for the treatment of atherosclerotic coronary heart disease and viral myocarditis. Previously, the ethanol extract of Maidong activated the pregnane X receptor (PXR) signaling pathway and induced the cytochrome P450 3A4 (CYP3A4) reporter gene and raised the concern of herb-drug interactions (HDIs) when Maidong was used in combination with prescribed drugs metabolized by CYP3A4. Therefore, the present study further investigated and compared the differences of the ethanol and aqueous extracts (ee- and ae-, respectively) of two Maidong strains, known as Zhe Maidong (ZM) and Chuan Maidong (CM). Cytotoxicity, PXR activation and CYP3A4 induction by the 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-diphenytetrazoliumromide assay, reporter gene assay and reverse transcription-quantitative polymerase chain reaction analysis were examined. The observations showed that ee-ZM demonstrated a significantly higher cytotoxicity, a relatively weaker PXR activation capability and a markedly stronger CYP3A4-inducing capacity than ee-CM. Compared to ae-CM, ae-ZM exhibited only a slight or no difference on cytotoxicity and CYP3A4 induction, while a significant lower level of PXR activation was apparent. Collectively, Maidong from different producing areas possess different properties upon cytotoxicity and the drug-metabolizing enzyme inducing effect, and attention should be paid to the selection of Maidong strains from different planting regions into TCM preparations for reducing potential adverse reactions and HDIs. PMID:26137250

  14. Production of {sup 4}He and tritium from Be in the COBRA-1A2 irradiation

    SciTech Connect

    Greenwood, L.R.

    1998-03-01

    The production of {sup 4}He and tritium has been calculated for beryllium irradiated in the COBRA-1A2 experiment in the Experimental Breeder Reactor II. Reaction rates were based on adjusted neutron spectra determined from reactor dosimetry measurements at three different elevations in the region of the beryllium capsules. Equations are given so that gas production can be calculated for any specific capsule elevation.

  15. A Function for the hnRNP A1/A2 Proteins in Transcription Elongation

    PubMed Central

    Lemieux, Bruno; Blanchette, Marco; Monette, Anne; Mouland, Andrew J.; Wellinger, Raymund J.; Chabot, Benoit

    2015-01-01

    The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes. PMID:26011126

  16. Novel action of FOXL2 as mediator of Col1a2 gene autoregulation.

    PubMed

    Marongiu, Mara; Deiana, Manila; Marcia, Loredana; Sbardellati, Andrea; Asunis, Isadora; Meloni, Alessandra; Angius, Andrea; Cusano, Roberto; Loi, Angela; Crobu, Francesca; Fotia, Giorgio; Cucca, Francesco; Schlessinger, David; Crisponi, Laura

    2016-08-01

    FOXL2 belongs to the evolutionarily conserved forkhead box (FOX) superfamily and is a master transcription factor in a spectrum of developmental pathways, including ovarian and eyelid development and bone, cartilage and uterine maturation. To analyse its action, we searched for proteins that interact with FOXL2. We found that FOXL2 interacts with specific C-terminal propeptides of several fibrillary collagens. Because these propeptides can participate in feedback regulation of collagen biosynthesis, we inferred that FOXL2 could thereby affect the transcription of the cognate collagen genes. Focusing on COL1A2, we found that FOXL2 indeed affects collagen synthesis, by binding to a DNA response element located about 65Kb upstream of this gene. According to our hypothesis we found that in Foxl2(-/-) mouse ovaries, Col1a2 was elevated from birth to adulthood. The extracellular matrix (ECM) compartmentalizes the ovary during folliculogenesis, (with type I, type III and type IV collagens as primary components), and ECM composition changes during the reproductive lifespan. In Foxl2(-/-) mouse ovaries, in addition to up-regulation of Col1a2, Col3a1, Col4a1 and fibronectin were also upregulated, while laminin expression was reduced. Thus, by regulating levels of extracellular matrix components, FOXL2 may contribute to both ovarian histogenesis and the fibrosis attendant on depletion of the follicle reserve during reproductive aging and menopause. PMID:27212026

  17. Novel de novo EEF1A2 missense mutations causing epilepsy and intellectual disability

    PubMed Central

    Lam, Wayne W.K.; Millichap, John J.; Soares, Dinesh C.; Chin, Richard; McLellan, Ailsa; FitzPatrick, David R.; Elmslie, Frances; Lees, Melissa M.; Schaefer, G. Bradley

    2016-01-01

    Background Exome sequencing has led to the discovery of mutations in novel causative genes for epilepsy. One such gene is EEF1A2, encoding a neuromuscular specific translation elongation factor, which has been found to be mutated de novo in five cases of severe epilepsy. We now report on a further seven cases, each with a different mutation, of which five are newly described. Methods New cases were identified and sequenced through the Deciphering Developmental Disabilities project, via direct contact with neurologists or geneticists, or recruited via our website. Results All the mutations cause epilepsy and intellectual disability, but with a much wider range of severity than previously identified. All new cases share specific subtle facial dysmorphic features. Each mutation occurs at an evolutionarily highly conserved amino acid position indicating strong structural or functional selective pressure. Conclusions EEF1A2 should be considered as a causative gene not only in cases of epileptic encephalopathy but also in children with less severe epilepsy and intellectual disability. The emergence of a possible discernible phenotype, a broad nasal bridge, tented upper lip, everted lower lip and downturned corners of the mouth may help in identifying patients with mutations in EEF1A2. PMID:27441201

  18. Lack of effect of brivanib on the pharmacokinetics of midazolam, a CYP3A4 substrate, administered intravenously and orally in healthy participants.

    PubMed

    Syed, Shariq; Clemens, Pamela L; Lathers, Deanne; Kollia, Georgia; Dhar, Arindam; Walters, Ian; Masson, Eric

    2012-06-01

    Brivanib alaninate is the orally available prodrug of brivanib, a dual inhibitor of fibroblast growth factor and vascular endothelial growth factor signaling pathways that is under therapeutic investigation for various malignancies. Brivanib alaninate inhibits CYP3A4 in vitro, and thus there is potential for drug-drug interaction with CYP3A4 substrates, such as midazolam. The present study evaluated pharmacokinetic parameters and safety/tolerability upon coadministration of brivanib alaninate and midazolam. Healthy participants received intravenous (IV) or oral midazolam with and without oral brivanib alaninate. Blood samples for pharmacokinetic analysis were collected up to 12 hours after midazolam and up to 48 hours after brivanib alaninate. Twenty-four participants were administered study drugs; 21 completed the trial. No clinically relevant effect of brivanib alaninate on the overall exposure to midazolam following IV or oral administration was observed. Orally administered brivanib alaninate was generally well tolerated in the presence of IV or oral midazolam. The lack of a pharmacokinetic interaction between brivanib and midazolam indicates that brivanib alaninate does not influence either intestinal or hepatic CYP3A4 and confirms that brivanib alaninate may be safely coadministered with midazolam and other CYP3A4 substrates. PMID:21659627

  19. The APOA1/C3/A4/A5 cluster and markers of allostatic load in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The APOA1/C3/A4/A5 cluster encodes key regulators of plasma lipids. Interactions between dietary factors and single nucleotide polymorphisms (SNPs) in the cluster have been reported. Allostatic load, or physiological dysregulation in response to stress, has been implicated in shaping health disparit...

  20. Concomitant use of selective serotonin reuptake inhibitors with other cytochrome P450 2D6 or 3A4 metabolized medications: how often does it really happen?

    PubMed

    Gregor, K J; Way, K; Young, C H; James, S P

    1997-10-01

    This study retrospectively examines the one-month concomitant use of cytochrome P450 2D6 or 3A4 metabolized medications in 544,309 patients who were also receiving selective serotonin reuptake inhibitors (SSRIs). Overall, 25.53% of SSRI patients experienced concomitant use with at least one of the 33 studied CYP 2D6 or 3A4 metabolized medications. Certain drugs and drug classes were more likely to be used concurrently among SSRI patients (e.g., benzodiazepines, tricyclic antidepressants, calcium channel blockers). Similarly, of the SSRI patients experiencing concomitant use, this concurrent use was twice as likely with cytochrome P450 medications metabolized by the 3A4 isoenzyme as with those metabolized by the 2D6 isoenzyme. Finally, the vast majority (80.9%) of SSRI patients experiencing concomitant use did so with one CYP 2D6 or 3A4 metabolized medication. In sum, concomitant use generally was not extensive and did not appear to be differential among the fluoxetine, paroxetine, or sertraline patient comparison groups. PMID:9387087

  1. Effects of variations in the APOA1/C3/A4/A5 gene cluster on different parameters of postprandial lipid metabolism in healthy young men

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The APOA1/C3/A4/A5 gene cluster encodes important regulators of fasting lipids, but the majority of lipid metabolism takes place in the postprandial state, and knowledge about gene regulation in this state is scarce. With the aim of characterizing possible regulators of lipid metabolism...

  2. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    PubMed Central

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-01-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotics detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet-drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that cautions should be taken for PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. PMID:23707768

  3. A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4.

    PubMed

    Dai, Zi-Ru; Ai, Chun-Zhi; Ge, Guang-Bo; He, Yu-Qi; Wu, Jing-Jing; Wang, Jia-Yue; Man, Hui-Zi; Jia, Yan; Yang, Ling

    2015-01-01

    Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy. PMID:26133240

  4. Effect of Garden Cress Seeds Powder and Its Alcoholic Extract on the Metabolic Activity of CYP2D6 and CYP3A4

    PubMed Central

    Al-Jenoobi, Fahad I.; Al-Thukair, Areej A.; Abbas, Fawkeya A.; Al-Mohizea, Abdullah M.; Alkharfy, Khalid M.; Al-Suwayeh, Saleh A.

    2014-01-01

    The powder and alcoholic extract of dried seeds of garden cress were investigated for their effect on metabolic activity of CYP2D6 and CYP3A4 enzymes. In vitro and clinical studies were conducted on human liver microsomes and healthy human subjects, respectively. Dextromethorphan was used as a common marker for measuring metabolic activity of CYP2D6 and CYP3A4 enzymes. In in vitro studies, microsomes were incubated with NADPH in presence and absence of different concentrations of seeds extract. Clinical investigations were performed in two phases. In phase I, six healthy female volunteers were administered a single dose of dextromethorphan and in phase II volunteers were treated with seeds powder for seven days and dextromethorphan was administered with last dose. The O-demethylated and N-demethylated metabolites of dextromethorphan were measured as dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Observations suggested that garden cress inhibits the formation of DOR and 3-MM metabolites. This inhibition of metabolite level was attributed to the inhibition of CYP2D6 and CYP3A4 activity. Garden cress decreases the level of DOR and 3-MM in urine and significantly increases the urinary metabolic ratio of DEX/DOR and DEX/3-MM. The findings suggested that garden cress seeds powder and ethanolic extract have the potential to interact with CYP2D6 and CYP3A4 substrates. PMID:24711855

  5. A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4

    PubMed Central

    Dai, Zi-Ru; Ai, Chun-Zhi; Ge, Guang-Bo; He, Yu-Qi; Wu, Jing-Jing; Wang, Jia-Yue; Man, Hui-Zi; Jia, Yan; Yang, Ling

    2015-01-01

    Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy. PMID:26133240

  6. Effects of Flavonoids in Lysimachia clethroides Duby on the Activities of Cytochrome P450 CYP2E1 and CYP3A4 in Rat Liver Microsomes.

    PubMed

    Zhang, Zhi-Juan; Xia, Zhao-Yang; Wang, Jin-Mei; Song, Xue-Ting; Wei, Jin-Feng; Kang, Wen-Yi

    2016-01-01

    Incubation systems were established to investigate the effects of quercetin, kaempferol, isoquercitrin and astragalin in Lysimachia clethroides Duby on the activities of CYP2E1 and CYP3A4 in rat liver microsomes in vitro. Probe substrates of 4-nitrophenol and testosterone as well as flavonoids at different concentrations were added to the incubation systems. After incubation, a validated high performance liquid chromatography (HPLC) method was applied to separate and determine the relevant metabolites. The results suggested that kaempferol exhibited a weak inhibition of CYP2E1 activity with an IC50 of 60.26 ± 2.54 μM, while quercetin and kaempferol caused a moderate inhibition of CYP3A4 activity with IC50 values of 18.77 ± 1.69 μM and 32.65 ± 1.32 μM, respectively. Isoquercitrin and astragalin had no effects on the activities of either CYP2E1 or CYP3A4. It could be speculated from these results that the inhibitory effects of quercetin and kaempferol on the activities of CYP2E1 and CYP3A4 could be the mechanisms underlying the hepatoprotective effects of L. clethroides. PMID:27314315

  7. Differential Interactions of Cytochrome P450 3A5 and 3A4 with Chemotherapeutic Agent-Vincristine: A Comparative Molecular Dynamics Study.

    PubMed

    Saba, Nikhat; Bhuyan, Rajabrata; Nandy, Suman Kumar; Seal, Alpana

    2015-01-01

    The chemotherapeutic agent vincristine, used for treatment of acute lymphoblastic leukemia is metabolized preferentially by polymorphic cytochrome P450 3A5 (CYP3A5) with higher clearance rate than cytochrome P450 3A4 (CYP3A4). As a result, CYP3A5 expressers have a reduced amount of vincristine-induced peripheral neuropathy than non-expressers. We modeled the structure of CYP3A5 and its interaction with vincristine, compared with CYP3A4-vincristine complex using molecular docking and simulation studies. This relative study helped us to understand the molecular mechanisms behind the interaction at the atomic level through interaction energy, binding free energy, hydrogen bond and solvent accessible surface area analysis - giving an insight into the binding mode and the main residues involved in this particular interaction. Our results show that the interacting groups get closer in CYP3A5-vincristine complex due to different orientation of vincristine. This leads to higher binding affinity of vincristine towards CYP3A5 compared to CYP3A4 and explains the preferential metabolism of vincristine by CYP3A5. We believe that, the results of the current study will be helpful for future studies on structure-based drug design in this area. PMID:25634447

  8. Constituents of Indonesian medicinal plant Averrhoa bilimbi and their cytochrome P450 3A4 and 2D6 inhibitory activities.

    PubMed

    Auw, Lidyawati; Subehan; Sukrasno; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2015-01-01

    As constituents of Averrhoa bilimbi leaves we identified three new compounds (1-3) together with 12 known ones (4-15); their inhibitory activities on cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) were examined. Among the isolated compounds, the mixture of 1 and 2, and compounds 4 and 9 showed strong inhibition on CYP3A4, but mild or no inhibition on CYP2D6. These compounds revealed the characteristics of 1) time- and concentration-dependent inhibition, 2) requirement of NADPH for the inhibition, 3) no protection by nucleophiles, and 4) suppression of the inhibition by competitive inhibitor. Thus, they are suggested to be mechanism-based inactivators of CYP3A4 and CYP2D6. The kinetic parameters for the inactivation (k(inact) and K(I)) were 0.19 min(-1) and 36.7 μM for the mixture of 1 and 2, 0.126 min(-1) and 10.5 μM for 4, and 0.29 min(-1) and 23.4 μM for 9. PMID:25920220

  9. Mechanism of interactions of α-naphthoflavone with cytochrome P450 3A4 explored with an engineered enzyme bearing a fluorescent probe†

    PubMed Central

    Tsalkova, Tamara N.; Davydova, Nadezhda Y.; Halpert, James R.; Davydov, Dmitri R.

    2008-01-01

    Design of a partially cysteine-depleted C98S/C239S/C377S/C468A cytochrome P450 3A4 mutant designated CYP3A4(C58,C64) allowed site-directed incorporation of thiol-reactive fluorescent probes into α-helix A‥ The site of modification was identified as Cys-64 with the help of CYP3A4(C58) and CYP3A4(C64), each bearing only one accessible cysteine. Changes in the fluorescence of CYP3A4(C58,C64) labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN), 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin (CPM), or monobromobimane (mBBr) were used to study the interactions with bromocriptine (BCT), 1-pyrenebutanol (1-PB), testosterone (TST), and α-naphthoflavone (ANF). Of these substrates only ANF has a specific effect, causing a considerable decrease in fluorescence intensity of BADAN and CPM and increasing the fluorescence of mBBr. This ANF-binding event in the case of BADAN-modified enzyme is characterized by an S50 of 18.2 ± 0.7, compared with the value of 2.2 ± 0.3 for the ANF-induced spin transition, thus revealing an additional low affinity binding site. Studies of the effect of TST, 1-PB, and BCT on the interactions of ANF monitored by changes in fluorescence of CYP3A4(C58,C64)-BADAN or by the ANF-induced spin transition revealed no competition by these substrates. Investigation of the kinetics of fluorescence increase upon H2O2-dependent heme depletion suggests that labeled CYP3A4(C58,C64) is represented by two conformers, one of which has the fluorescence of the BADAN and CPM labels completely quenched, presumably by photoinduced electron transfer from the neighboring Trp-72 and/or Tyr-68 residues. The binding of ANF to the newly discovered binding site appears to affect the interactions of the label with the above residue(s), thus modulating the fraction of the fluorescent conformer. PMID:17198380

  10. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    PubMed

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  11. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

  12. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  13. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

    PubMed

    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation. PMID:24832206

  14. Dynamics of Cytosine Methylation in the Proximal Promoters of CYP3A4 and CYP3A7 in Pediatric and Prenatal Livers.

    PubMed

    Vyhlidal, Carrie A; Bi, Chengpeng; Ye, Shui Qing; Leeder, J Steven

    2016-07-01

    Members of the human CYP3A family of metabolizing enzymes exhibit developmental changes in expression whereby CYP3A7 is expressed in fetal tissues, followed by a transition to expression of CYP3A4 in the first months of life. Despite knowledge about the general pattern of CYP3A activity in human development, the mechanisms that regulate developmental expression remain poorly understood. Epigenetic changes, including cytosine methylation, have been suggested to play a role in the regulation of CYP3A expression. The objective of this study was to investigate changes in cytosine methylation of the CYP3A4 and CYP3A7 genes in human pediatric and prenatal livers. The methylation status of cytosine-phospho-guanine dinucleotides was determined in 16 pediatric liver samples using methyl-seq and confirmed by bisulfite sequencing of 48 pediatric and 34 prenatal liver samples. Samples were separated by age into five groups (prenatal, < 1 year of age, 1.8-6 years, 7-11 years, and 12-17 years). Methyl-seq anaylsis revealed that cytosines in the proximal promoter of CYP3A7 are hypomethylated in neonates compared with adolescents (P < 0.001). In contrast, a cytosine 383 base pair upstream of CYP3A4 is hypermethylated in liver samples from neonates compared with adolescents (P = 0.00001). Developmental changes in methylation of cytosines in the proximal promoters of CYP3A4 and CYP3A7 in pediatric livers were confirmed by bisulfite sequencing. In addition, the methylation status of cytosine in the CYP3A4 and CYP3A7 proximal promoters correlated with changes in developmental expression of mRNA for the two enzymes. PMID:26772622

  15. Enhancement of CYP3A4 Activity in Hep G2 Cells by Lentiviral Transfection of Hepatocyte Nuclear Factor-1 Alpha

    PubMed Central

    Chiang, Tsai-Shin; Yang, Kai-Chiang; Chiou, Ling-Ling; Huang, Guan-Tarn; Lee, Hsuan-Shu

    2014-01-01

    Human hepatoma cell lines are commonly used as alternatives to primary hepatocytes for the study of drug metabolism in vitro. However, the phase I cytochrome P450 (CYP) enzyme activities in these cell lines occur at a much lower level than their corresponding activities in primary hepatocytes, and thus these cell lines may not accurately predict drug metabolism. In the present study, we selected hepatocyte nuclear factor-1 alpha (HNF1α) from six transcriptional regulators for lentiviral transfection into Hep G2 cells to optimally increase their expression of the CYP3A4 enzyme, which is the major CYP enzyme in the human body. We subsequently found that HNF1α-transfected Hep G2 enhanced the CYP3A4 expression in a time- and dose-dependent manner and the activity was noted to increase with time and peaked 7 days. With a multiplicity of infection (MOI) of 100, CYP3A4 expression increased 19-fold and enzyme activity more than doubled at day 7. With higher MOI (1,000 to 3,000), the activity increased 8- to 10-fold; however, it was noted the higher MOI, the higher cell death rate and lower cell survival. Furthermore, the CYP3A4 activity in the HNF1α-transfected cells could be induced by CYP3A4-specific inducer, rifampicin, and metabolized nifedipine in a dose-dependent manner. With an MOI of 3,000, nifedipine-metabolizing activity was 6-fold of control and as high as 66% of primary hepatocytes. In conclusion, forceful delivery of selected transcriptional regulators into human hepatoma cells might be a valuable method to enhance the CYP activity for a more accurate determination of drug metabolism in vitro. PMID:24733486

  16. Overexpression of MMP-7 increases collagen 1A2 in the aging kidney

    PubMed Central

    Ślusarz, Anna; Nichols, LaNita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-01-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis that leads to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however, it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, with over a 500-fold upregulation in 2-year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of protein kinase A (PKA), src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 upregulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src, and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  17. Overexpression of MMP-7 Increases Collagen 1A2 in the Aging Kidney.

    PubMed

    Oelusarz, Anna; Nichols, Lanita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-10-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  18. Analysis of single-nucleotide polymorphisms (SNPs) in human CYP3A4 and CYP3A5 genes: potential implications for the metabolism of HIV drugs

    PubMed Central

    2014-01-01

    Background Drug metabolism via the cytochrome P450 (CYP450) system has emerged as an important determinant in the occurrence of several drug interactions (adverse drug reactions, reduced pharmacological effect, drug toxicities). In particular, CYP3A4 and CYP3A5 (interacting with more than 60% of licensed drugs) exhibit the most individual variations of gene expression, mostly caused by single nucleotide polymorphisms (SNPs) within the regulatory region of the CYP3A4 and CYP3A5 genes which might affect the level of enzyme production. In this study, we sought to improve the performance of sensitive screening for CYP3A polymorphism detection in twenty HIV-1 infected patients undergoing lopinavir/ritonavir (LPV/r) monotherapy. Methods The study was performed by an effective, easy and inexpensive home-made Polymerase Chain Reaction Direct Sequencing approach for analyzing CYP3A4 and CYP3A5 genes which can detect both reported and unreported genetic variants potentially associated with altered or decreased functions of CYP3A4 and CYP3A5 proteins. Proportions and tests of association were used. Results Among the genetic variants considered, CYP3A4*1B (expression of altered function) was only found in 3 patients (15%) and CYP3A5*3 (expression of splicing defect) in 3 other patients (15%). CYP3A5*3 did not appear to be associated with decreased efficacy of LPV/r in any patient, since none of the patients carrying this variant showed virological rebound during LPV/r treatment or low levels of TDM. In contrast, low-level virological rebound was observed in one patient and a low TDM level was found in another; both were carrying CYP3A4*1B. Conclusions Our method exhibited an overall efficiency of 100% (DNA amplification and sequencing in our group of patients). This may contribute to producing innovative results for better understanding the inter-genotypic variability in gene coding for CYP3A, and investigating SNPs as biological markers of individual response to drugs

  19. Mg2+/Mn2+-dependent phosphatase 1A is involved in regulating pregnane X receptor-mediated cytochrome p450 3A4 gene expression.

    PubMed

    Pondugula, Satyanarayana R; Flannery, Patrick C; Apte, Udayan; Babu, Jeganathan Ramesh; Geetha, Thangiah; Rege, Shraddha D; Chen, Taosheng; Abbott, Kodye L

    2015-03-01

    Variations in the expression of human pregnane X receptor (hPXR)-mediated cytochrome p450 3A4 (CYP3A4) in liver can alter therapeutic response to a variety of drugs and may lead to potential adverse drug interactions. We sought to determine whether Mg(2+)/Mn(2+)-dependent phosphatase 1A (PPM1A) regulates hPXR-mediated CYP3A4 expression. PPM1A was found to be coimmunoprecipitated with hPXR. Genetic or pharmacologic activation of PPM1A led to a significant increase in hPXR transactivation of CYP3A4 promoter activity. In contrast, knockdown of endogenous PPM1A not only attenuated hPXR transactivation, but also increased proliferation of HepG2 human liver carcinoma cells, suggesting that PPM1A expression levels regulate hPXR, and that PPM1A expression is regulated in a proliferation-dependent manner. Indeed, PPM1A expression and hPXR transactivation were found to be significantly reduced in subconfluent HepG2 cells compared with confluent HepG2 cells, suggesting that both PPM1A expression and hPXR-mediated CYP3A4 expression may be downregulated in proliferating livers. Elevated PPM1A levels led to attenuation of hPXR inhibition by tumor necrosis factor-α and cyclin-dependent kinase-2, which are known to be upregulated and essential during liver regeneration. In mouse regenerating livers, similar to subconfluent HepG2 cells, expression of both PPM1A and the mouse PXR target gene cyp3a11 was found to be downregulated. Our results show that PPM1A can positively regulate PXR activity by counteracting PXR inhibitory signaling pathways that play a major role in liver regeneration. These results implicate a novel role for PPM1A in regulating hPXR-mediated CYP3A4 expression in hepatocytes and may explain a mechanism for CYP3A repression in regenerating livers. PMID:25561723

  20. [Evaluation of pharmacokinetic interaction of aphobazole with CYP1A2 drug-substrate in experiments].

    PubMed

    Novitskaia, Ia G; Litvin, A A; Viglinskaia, A O; Zherdev, V P

    2013-01-01

    The effect of aphobazole on CYP1A2 (drug-marker caffeine) was studied in rats. Aphobazole was administered orally at doses 5 and 25 mg/kg, caffeine 50 mg/kg. The metabolic ratios (MR) for the caffeine metabolites (theobromine and paraxanthine) were accounted. After aphobazole administration at the effective, anxiolytic dose (5 mg/kg) for 4 days (3 times per day every 3 hours) neither the inhibiting nor the inducing effects on NOD1A2 was revealed. Increasing the aphobazole dose up to 25 mg/kg after 2 days repeated administrations of the drug made it possible to reveal a moderate inducing effect. Longer aphobazole administration (4 days), the inducing effect is amplified. Since the MR values on theobromine and paraxanthine after 2-day administration aphobazole exceed similar values in the control of 2.5 and 3.3 times, respectively. MR values after the 4-days aphobazole administration in dose 25 mg/kg exceed similar values in the control of 4.2 times for theobromine and in 6.1 times for paraxanthine. PMID:24003488

  1. Identification and in silico prediction of metabolites of the model compound, tebufenozide by human CYP3A4 and CYP2C19.

    PubMed

    Shirotani, Naoki; Togawa, Moe; Ikushiro, Shinichi; Sakaki, Toshiyuki; Harada, Toshiyuki; Miyagawa, Hisashi; Matsui, Masayoshi; Nagahori, Hirohisa; Mikata, Kazuki; Nishioka, Kazuhiko; Hirai, Nobuhiro; Akamatsu, Miki

    2015-10-15

    The metabolites of tebufenozide, a model compound, formed by the yeast-expressed human CYP3A4 and CYP2C19 were identified to clarify the substrate recognition mechanism of the human cytochrome P450 (CYP) isozymes. We then determined whether tebufenozide metabolites may be predicted in silico. Hydrogen abstraction energies were calculated with the density functional theory method B3LYP/6-31G(∗). A docking simulation was performed using FRED software. Several alkyl sites of tebufenozide were hydroxylated by CYP3A4 whereas only one site was modified by CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen abstraction energy estimation, respectively. PMID:26404412

  2. Inhibition of the metabolism of brotizolam by erythromycin in humans: in vivo evidence for the involvement of CYP3A4 in brotizolam metabolism

    PubMed Central

    Tokairin, Takaki; Fukasawa, Takashi; Yasui-Furukori, Norio; Aoshima, Toshiaki; Suzuki, Akihito; Inoue, Yoshimasa; Tateishi, Tomonori; Otani, Koichi

    2005-01-01

    Aims To obtain in vivo evidence for the involvement of cytochrome P450 (CYP) 3A4 in the metabolism of brotizolam. Methods Fourteen healthy male volunteers received erythromycin 1200 mg day−1 or placebo for 7 days in a double-blind randomized crossover manner. On the 6th day they received a single oral 0.5-mg dose of brotizolam, and blood samplings were performed for 24 h. Results Erythromycin treatment significantly increased the peak plasma concentration (P < 0.05), total area under the plasma concentration-time curve (P < 0.01), and elimination half-life (P < 0.01) of brotizolam. Conclusions The present study provides in vivo evidence for the involvement of CYP3A4 in brotizolam metabolism. PMID:16042670

  3. Thiazide-like diuretic drug metolazone activates human pregnane X receptor to induce cytochrome 3A4 and multidrug-resistance protein 1

    PubMed Central

    Banerjee, Monimoy; Chen, Taosheng

    2014-01-01

    Human pregnane X receptor (hPXR) regulates the expression of drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) and drug transporters such as multidrug-resistance protein 1 (MDR1). PXR can be modulated by small molecules, including Federal Drug Administration (FDA)–approved drugs, thus altering drug metabolism and causing drug-drug interactions. To determine the role of FDA-approved drugs in PXR-mediated regulation of drug metabolism and clearance, we screened 1481 FDA-approved small-molecule drugs by using a luciferase reporter assay in HEK293T cells and identified the diuretic drug metolazone as an activator of PXR. Our data showed that metolazone activated hPXR-mediated expression of CYP3A4 and MDR1 in human hepatocytes and intestine cells and increased CYP3A4 promoter activity in various cell lines. Mammalian two-hybrid assays showed that hPXR recruits its co-activator SRC-1 upon metolazone binding in HepG2 cells, explaining the mechanism of hPXR activation. To understand the role of other commonly-used diuretics in PXR activation and the structure-activity relationship of metolazone, thiazide and non-thiazide diuretics drugs were also tested but only metolazone activates PXR. To understand the molecular mechanism, docking studies and mutational analysis were carried out and showed that metolazone binds in the ligand-binding pocket and interacts with mostly hydrophobic amino acid residues. This is the first report showing that metolazone activates PXR. Because activation of hPXR might cause drug-drug interactions, metolazone should be used with caution for drug treatment in patients undergoing combination therapy. PMID:25181459

  4. Differential regulation of CYP3A4 promoter activity by a new class of natural product derivatives binding to pregnane X receptor

    PubMed Central

    Banerjee, Monimoy; Chen, Taosheng

    2013-01-01

    The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. The activity of PXR can be controlled by the binding of small molecule agonists or antagonists. Because of its unique ligand binding pocket, PXR binds promiscuously to structurally diverse chemicals. To study the structure-activity relationship, novel modulators for PXR are needed. Here we report the virtual screening of ~25,000 natural product derivatives from the ZINC database using the Molecular Operating Environment docking software tool against the PXR-rifampicin complex x-ray crystal structure. Our screening resulted in identification of compounds based on the lowest S score, which measures Gibbs free energy. Interestingly, we found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. We further investigated the mechanism of binding by analyzing the functional groups that are important for distinguishing agonists from antagonists. The approach we used to identify novel modulators that bind to PXR can be useful for finding novel modulators of PXR. PMID:23928187

  5. Theoretical Characterization of Substrate Access/Exit Channels in the Human Cytochrome P450 3A4 Enzyme: Involvement of Phenylalanine Residues in the Gating Mechanism

    PubMed Central

    2009-01-01

    The human cytochrome P450 3A4 mono-oxygenates ∼50% of all drugs. Its substrates/products enter/leave the active site by access/exit channels. Here, we perform steered molecular dynamics simulations, pulling the products temazepam and testosterone-6βOH out of the P450 3A4 enzyme in order to identify the preferred substrate/product pathways and their gating mechanism. We locate six different egress pathways of products from the active site with different exit preferences for the two products and find that there is more than just one access/exit channel in CYP3A4. The so-called solvent channel manifests the largest opening for both tested products, thereby identifying this channel as a putative substrate channel. Most channels consist of one or two π-stacked phenylalanine residues that serve as gate keepers. The oxidized drug breaks the hydrophobic interactions of the gating residues and forms mainly hydrophobic contacts with the gate. We argue that product exit preferences in P450s are regulated by protein−substrate specificity. PMID:19728720

  6. Determination of midazolam and its metabolite as a probe for cytochrome P450 3A4 phenotype by liquid chromatography-mass spectrometry.

    PubMed

    Kanazawa, Hideko; Okada, Akiko; Igarashi, Eri; Higaki, Megumu; Miyabe, Takako; Sano, Tadashi; Nishimura, Ryouhei

    2004-03-26

    This study demonstrated the analysis of midazolam and its metabolites by liquid chromatography-mass spectrometry (LC-MS) with a sonic spray ionization (SSI) interface. The analytical column was a YMC-Pak Pro C18 (50 mm x 2.0 mm i.d.) using 10 mM ammonium acetate (pH 4.8)-methanol (1:1) at a flow rate of 0.2 ml min(-1). The drift voltage was 100 V. The sampling aperture was heated at 110 degrees C and the shield temperature was 230 degrees C. The lower limits for the detection of midazolam and 1'-hydroxymidazolam were 26.3 and 112.76 pg injected, respectively. The calibration curves for midazolam and 1'-hydroxymidazolam were linear in the range of 0.1-5 microg ml(-1). Within-day relative standard deviations was less than 7%. The method was applied to the determination of midazolam in monkey plasma, and the analysis of midazolam and its metabolites in an in vitro study with recombinant cytochrome P450 (CYP) 3A4. This method is sufficiently sensitive and useful to elucidate the kinetics of midazolam metabolite formation. We also investigated the effect of propofol on the metabolism of midazolam using recombinant CYP3A4. Propofol competitively inhibited the metabolism of midazolam to 1'-hydroxymidazolam by CYP3A4. PMID:15058585

  7. Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries

    PubMed Central

    Peng, Hwei-Ming; Auchus, Richard J.

    2014-01-01

    Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23–42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl] > 50 mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type > E48G/E49G > D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4. PMID:24256945

  8. Two surfaces of cytochrome b5 with major and minor contributions to CYP3A4-catalyzed steroid and nifedipine oxygenation chemistries.

    PubMed

    Peng, Hwei-Ming; Auchus, Richard J

    2014-01-01

    Conserved human cytochrome b5 (b5) residues D58 and D65 are critical for interactions with CYP2E1 and CYP2C19, whereas E48 and E49 are essential for stimulating the 17,20-lyase activity of CYP17A1. Here, we show that b5 mutations E48G, E49G, D58G, and D65G have reduced capacity to stimulate CYP3A4-catalyzed progesterone and testosterone 6β-hydroxylation or nifedipine oxidation. The b5 double mutation D58G/D65G fails to stimulate these reactions, similar to CYP2E1 and CYP2C19, whereas mutation E48G/E49G retains 23-42% of wild-type stimulation. Neither mutation impairs the activity stimulation of wild-type b5, nor does mutation D58G/D65G impair the partial stimulation of mutations E48G or E48G/E49G. For assays reconstituted with a single phospholipid, phosphatidyl serine afforded the highest testosterone 6β-hydroxylase activity with wild-type b5 but the poorest activity with b5 mutation E48G/E49G, and the activity stimulation of mutation E48G/E49G was lost at [NaCl]>50mM. Cross-linking of CYP3A4 and b5 decreased in the order wild-type>E48G/E49G>D58G/D65G and varied with phospholipid. We conclude that two b5 acidic surfaces, primarily the domain including residues D58-D65, participate in the stimulation of CYP3A4 activities. Our data suggest that a minor population of CYP3A4 molecules remains sensitive to b5 mutation E48G/E49G, consistent with phospholipid-dependent conformational heterogeneity of CYP3A4. PMID:24256945

  9. Impact of Tetrahydropalmatine on the Pharmacokinetics of Probe Drugs for CYP1A2, 2D6 and 3A Isoenzymes in Beagle Dogs.

    PubMed

    Zhao, Yong; Liang, Aihua; Zhang, Yushi; Li, Chunying; Yi, Yan; Nilsen, Odd Georg

    2016-06-01

    Tetrahydropalmatine (Tet) exhibit multiple pharmacological activities and is used frequently by clinical practitioners. In this study, we evaluate the in vivo effects of single and repeated oral Tet administrations on CYP1A2, 2D6 and 3A activities in six beagle dogs in a randomized, controlled, open-label, crossover study. A cocktail approach, with dosages of the probe drugs caffeine (3.0 mg/kg), metoprolol (2.33 mg/kg) and midazolam (0.45 mg/kg), was used to measure cytochrome P450 (CYP) metabolic activities. The cocktail was administered orally as a single dose (12 mg/kg) 1 day prior to and 4 days after repeated oral Tet administrations (12 mg/kg three times daily). The probe drugs and their metabolites in plasma were quantified simultaneously by a validated HPLC technique, and non-compartmental parameters were used to evaluate metabolic variables for assessment of CYP inhibition or induction. Tet had no or minor impact on the pharmacokinetics and metabolism of the probe drugs caffeine and metoprolol, CYP1A2 and CYP2D6 substrates, respectively. However, Tet increased AUC0-24 h and decreased AUCratio(0-24 h) (1-hydroxymidazolam/midazolam ratio) for midazolam statistically significant, both in single or multiple dosing of Tet, with up to 39 or 57% increase for AUC0-24 h and 29% or 22 decrease for AUCratio(0-24 h), respectively, in line with previous in vitro findings for its CYP3A4 inhibition. The extensive use of Tet and herbal medicines containing Tet makes Tet a candidate for further evaluation of CYP3A-mediated herb-drug interactions. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990021

  10. USE OF CYP1A2(-/-) KNOCKOUT AND CYP1A2(+/+) C57BL/6N PARENTAL STRAINS OF MICE TO COMPARE METABOLISM OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

    EPA Science Inventory

    USE OF CYP1A2 (-/-) KNOCKOUT AND CYP1A2 (+/+) C57BL/6N PARENTAL STRAINS OF MICE TO COMPARE METABOLISM OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD). J J Diliberto1 and H Hakk2. 1USEPA ORD, NHEERL, ETD, PKB, Research Triangle Park, NC, USA; 2USDA-ARS, BRL, Fargo, ND, USA. Spons...

  11. Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies, linkage disequilibrium and influence on caffeine metabolism

    PubMed Central

    Sachse, Christoph; Bhambra, Upinder; Smith, Gillian; Lightfoot, Tracy J; Barrett, Jennifer H; Scollay, Jenna; Garner, R Colin; Boobis, Alan R; Wolf, C Roland; Gooderham, Nigel J

    2003-01-01

    Aim Several single nucleotide polymorphisms (SNPs) of the cytochrome P450 enzyme 1A2 gene (CYP1A2) have been reported. Here, frequencies, linkage disequilibrium and phenotypic consequences of six SNPs are described. Methods From genomic DNA, 114 British Caucasians (49 colorectal cancer cases and 65 controls) were genotyped for the CYP1A2 polymorphisms −3858G→A (allele CYP1A2*1C), −2464T→delT (CYP1A2*1D), −740T→G (CYP1A2*1E and *1G), −164A→C (CYP1A2*1F), 63C→G (CYP1A2*2), and 1545T→C (alleles CYP1A2*1B, *1G, *1H and *3), using polymerase chain reaction–restriction fragment length polymorphism assays. All patients and controls were phenotyped for CYP1A2 by h.p.l.c. analysis of urinary caffeine metabolites. Results In 114 samples, the most frequent CYP1A2 SNPs were 1545T→C (38.2% of tested chromosomes), −164A→C (CYP1A2*1F, 33.3%) and −2464T→delT (CYP1A2*1D, 4.82%). The SNPs were in linkage disequilibrium: the most frequent constellations were found to be −3858G/−2464T/−740T/−164A/63C/1545T (61.8%), −3858G/−2464T/−740T/−164C/63C/1545C (33.3%), and −3858G/−2464delT/−740T/−164A/63C/1545C (3.51%), with no significant frequency differences between cases and controls. In the phenotype analysis, lower caffeine metabolic ratios were detected in cases than in controls. This was significant in smokers (n = 14, P = 0.020), and in a subgroup of 15 matched case-control pairs (P = 0.007), but it was not significant in nonsmokers (n = 100, P = 0.39). There was no detectable association between CYP1A2 genotype and caffeine phenotype. Conclusions (i) CYP1A2 polymorphisms are in linkage disequilibrium. Therefore, only −164A→C (CYP1A2*1F) and −2464T→delT (CYP1A2*1D) need to be analysed in the routine assessment of CYP1A2 genotype; (ii) in vivo CYP1A2 activity is lower in colorectal cancer patients than in controls, and (iii) CYP1A2 genotype had no effect on phenotype (based on the caffeine metabolite ratio). However, this

  12. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  13. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  14. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  15. Defining CYP3A4 structural responses to substrate binding. Raman spectroscopic studies of a nanodisc-incorporated mammalian Cytochrome P450

    PubMed Central

    Mak, Piotr J.; Denisov, Ilia G.; Grinkova, Yelena V.; Sligar, Stephen G.; Kincaid, James R.

    2011-01-01

    Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates; bromocriptine (BC), erythromycin (ERY) and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While, ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome. PMID:21207936

  16. PYRETHROID INSECTICIDES: ISOFORM-DEPENDENT HYDROLYSIS, INDUCTION OF CYTOCHROME P450 3A4 AND EVIDENCE ON THE INVOLVEMENT OF THE PREGNANE X RECEPTOR

    PubMed Central

    Yang, Dongfang; Wang, Xiliang; Chen, Yi-tzai; Deng, Ruitang; Yan, Bingfang

    2009-01-01

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity. PMID:19249324

  17. Comparative inhibitory potential of selected dietary bioactive polyphenols, phytosterols on CYP3A4 and CYP2D6 with fluorometric high-throughput screening.

    PubMed

    Vijayakumar, Thangavel Mahalingam; Kumar, Ramasamy Mohan; Agrawal, Aruna; Dubey, Govind Prasad; Ilango, Kaliappan

    2015-07-01

    Cytochrome P450 (CYP450) inhibition by the bioactive molecules of dietary supplements or herbal products leading to greater potential for toxicity of co-administered drugs. The present study was aimed to compare the inhibitory potential of selected common dietary bioactive molecules (Gallic acid, Ellagic acid, β-Sitosterol, Stigmasterol, Quercetin and Rutin) on CYP3A4 and CYP2D6 to assess safety through its inhibitory potency and to predict interaction potential with co-administered drugs. CYP450-CO complex assay was carried out for all the selected dietary bioactive molecules in isolated rat microsomes. CYP450 concentration of the rat liver microsome was found to be 0.474 nmol/mg protein, quercetin in DMSO has shown maximum inhibition on CYP450 (51.02 ± 1.24 %) but less when compared with positive control (79.02 ± 1.61 %). In high throughput fluorometric assay, IC50 value of quercetin (49.08 ± 1.02-54.36 ± 0.85 μg/ml) and gallic acid (78.46 ± 1.32-83.84 ± 1.06 μg/ml) was lower than other bioactive compounds on CYP3A4 and CYP2D6 respectively but it was higher than positive controls (06.28 ± 1.76-07.74 ± 1.32 μg/ml). In comparison of in vitro inhibitory potential on CYP3A4 and CYP2D6, consumption of food or herbal or dietary supplements containing quercetin and gallic acid without any limitation should be carefully considered when narrow therapeutic drugs are administered together. PMID:26139922

  18. Pyrethroid insecticides: Isoform-dependent hydrolysis, induction of cytochrome P450 3A4 and evidence on the involvement of the pregnane X receptor

    SciTech Connect

    Yang Dongfang; Wang Xiliang; Chen Yitzai; Deng Ruitang; Yan Bingfang

    2009-05-15

    Pyrethroids account for more than one-third of the insecticides currently marketed in the world. In mammals, these insecticides undergo extensive metabolism by carboxylesterases and cytochrome P450s (CYPs). In addition, some pyrethroids are found to induce the expression of CYPs. The aim of this study was to determine whether pyrethroids induce carboxylesterases and CYP3A4, and whether the induction is correlated inversely with their hydrolysis. Human liver microsomes were pooled and tested for the hydrolysis of 11 pyrethroids. All pyrethroids were hydrolyzed by the pooled microsomes, but the hydrolytic rates varied by as many as 14 fold. Some pyrethroids such as bioresmethrin were preferably hydrolyzed by carboxylesterase HCE1, whereas others such as bifenthrin preferably by HCE2. In primary human hepatocytes, all pyrethroids except tetramethrin significantly induced CYP3A4. In contrast, insignificant changes were detected on the expression of carboxylesterases. The induction of CYP3A4 was confirmed in multiple cell lines including HepG2, Hop92 and LS180. Overall, the magnitude of the induction was correlated inversely with the rates of hydrolysis, but positively with the activation of the pregnane X receptor (PXR). Transfection of a carboxylesterase markedly decreased the activation of PXR, and the decrease was in agreement with carboxylesterase-based preference for hydrolysis. In addition, human PXR variants as well as rat PXR differed from human PXR (wild-type) in responding to certain pyrethroids (e.g., lambda-cyhalothrin), suggesting that induction of PXR target genes by these pyrethroids varies depending on polymorphic variants and the PXR species identity.

  19. In vitro metabolic interactions between black cohosh (Cimicifuga racemosa) and tamoxifen via inhibition of cytochromes P450 2D6 and 3A4.

    PubMed

    Li, Jinghu; Gödecke, Tanja; Chen, Shao-Nong; Imai, Ayano; Lankin, David C; Farnsworth, Norman R; Pauli, Guido F; van Breemen, Richard B; Nikolić, Dejan

    2011-08-01

    Women who experience hot flashes as a side effect of tamoxifen (TAM) therapy often try botanical remedies such as black cohosh to alleviate these symptoms. Since pharmacological activity of TAM is dependent on the metabolic conversion into active metabolites by the action of cytochromes P450 2D6 (CYP2D6) and 3A4, the objective of this study was to evaluate whether black cohosh extracts can inhibit formation of active TAM metabolites and possibly reduce its clinical efficacy. At 50 μg/mL, a 75% ethanolic extract of black cohosh inhibited formation of 4-hydroxy- TAM by 66.3%, N-desmethyl TAM by 74.6% and α-hydroxy TAM by 80.3%. In addition, using midazolam and dextromethorphan as probe substrates, this extract inhibited CYP3A4 and CYP2D6 with IC(50) values of 16.5 and 50.1 μg/mL, respectively. Eight triterpene glycosides were identified as competitive CYP3A4 inhibitors with IC(50) values ranging from 2.3-5.1 µM, while the alkaloids protopine and allocryptopine were identified as competitive CYP2D6 inhibitors with K(i) values of 78 and 122 nM, respectively. The results of this study suggests that co-administration of black cohosh with TAM might interfere with the clinical efficacy of this drug. However, additional clinical studies are needed to determine the clinical significance of these in vitro results. PMID:21827327

  20. A food contaminant ochratoxin A suppresses pregnane X receptor (PXR)-mediated CYP3A4 induction in primary cultures of human hepatocytes.

    PubMed

    Doricakova, Aneta; Vrzal, Radim

    2015-11-01

    Ochratoxin A (OCHA) is a mycotoxin, which can be found in food such as coffee, wine, cereals, meat, nuts. Since it is absorbed via gastrointestinal tract, it is reasonable to anticipate that the liver will be the first organ to which OCHA comes into the contact before systemic circulation. Many xenobiotics are metabolically modified after the passage of the liver to biologically more active substances, sometimes with more harmful activity. Promoting own metabolism is often achieved via transcriptional regulation of biotransformation enzymes through ligand-activated transcription factors. Pregnane X receptor (PXR) belongs to such a group of regulators and it was demonstrated to be activated by many compounds of synthetic as well as natural origin. Our intention was to investigate if OCHA is capable of activating the PXR with consequent induction of PXR-regulated CYP3A4 gene. We found that OCHA does not activate PXR but displays antagonist-like behavior when combined with rifampicin (RIF) in gene reporter assay in human embryonal kidney cells (Hek293T). It was very weak inducer of CYP3A4 mRNA in primary cultures of human hepatocytes and it antagonized RIF-mediated CYP3A4 induction of mRNA as well as protein. In addition, it caused the decline of PXR protein as well as mRNA which was faster than that with actinomycin D, a transcription inhibitor. Since we found that OCHA induced the expression of miR-148a, which was described to regulate PXR expression, we conclude that antagonist-like behavior of OCHA is not due to the antagonism itself but due to the downregulation of PXR gene expression. Herein we provide important findings which bring a piece of puzzle into the understanding of mechanism of toxic action of ochratoxin A. PMID:26341324

  1. AhrdCyp1a2(−/−) mice show increased susceptibility to PCB-induced developmental neurotoxicity

    PubMed Central

    Curran, Christine Perdan; Altenhofen, Emily; Ashworth, Amy; Brown, Austin; Kamau-Cheggeh, Cellestine; Curran, Melinda; Evans, Amber; Floyd, Rikki; Fowler, Jocelyn; Garber, Helen; Hays, Breann; Kraemer, Sarah; Lang, Anna; Mynhier, Andrea; Samuels, Ashton; Strohmaier, Carly

    2012-01-01

    Polychlorinated biphenyls (PCBs) are developmental neurotoxicants that produce cognitive and behavioral changes in children exposed during gestation and lactation. Coplanar PCBs bind the aryl hydrocarbon receptor (AHR) and can be sequestered in liver by cytochrome P450 1A2 (CYP1A2). The AHR is a ligand-activated transcription factor which increases expression of the CYP1 family, including CYP1A2. Our previous work examining genetic susceptibility to developmental PCB neurotoxicity showed that AhrbCyp1a2(−/−) mice with the high-affinity Ahrb allele and lacking CYP1A2 were most susceptible while AhrbCyp1a2(+/+) and poor-affinity AhrdCyp1a2(+/+) mice were resistant. To follow up, a fourth line of mice was generated with the AhrdCyp1a2(−/−) genotype and compared with the background strain AhrbCyp1a2(+/+). Dams received a PCB mixture or the corn oil vehicle at gestational day 10 (GD10) and postnatal day 5 (PND5). Offspring were tested at PND60 in open field locomotor, acoustic startle with pre-pulse inhibition (PPI), novel object recognition and Morris water maze. Locomotor activity was increased in PCB-treated AhrbCyp1a2(+/+) mice, but no differences were seen in control v. PCB-treated AhrdCyp1a2(−/−) mice. PCB-treated AhrdCyp1a2(−/−) mice had a higher baseline startle response and significantly reduced pre-pulse inhibition at the 74dB level compared with corn oil-treated controls (P<0.05). PCB-treated AhrdCyp1a2(−/−) mice had impairments in novel objective recognition (P<0.05) and during all three hidden platform phases of Morris water maze (P<0.01). Combined with our previous findings, these results indicate Cyp1a2 genotype is more important in susceptibility to PCB-induced deficits in learning and memory, but Ahr genotype appears more important when assessing acoustic startle-PPI and locomotor activity. PMID:22935098

  2. Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

    SciTech Connect

    Sun, Yue; Du, Chengli; Wang, Bo; Zhang, Yanling; Liu, Xiaoyan; Ren, Guoping

    2014-07-18

    Highlights: • The expression of eEF1A2 is up-regulated in prostate cancer tissues. • Suppression of eEF1A2 inhibits the proliferation and promotes apoptosis. • Inhibition of eEF1A2 enhances the expression of apoptotic relevant proteins. • The expressions of eEF1A2 and cleavage-caspase3 are inversely correlated. - Abstract: Background: eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development. Methods: We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis. Results: Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues. Conclusion: Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.

  3. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    PubMed

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  4. A Cytochrome P450 3A4 Biosensor Based on Generation 4.0 PAMAM Dendrimers for the Detection of Caffeine.

    PubMed

    Müller, Michael; Agarwal, Neha; Kim, Jungtae

    2016-01-01

    Cytochromes P450 (CYP, P450) are a large family of heme-active-site proteins involved in many catalytic processes, including steroidogenesis. In humans, four primary enzymes are involved in the metabolism of almost all xenobiotics. Among these enzymes, CYP3A4 is responsible for the inactivation of the majority of used drugs which makes this enzyme an interesting target for many fields of research, especially pharmaceutical research. Since the late 1970s, attempts have been made to construct and develop electrochemical sensors for the determination of substrates. This paper is concerned with the establishment of such a CYP3A4-containing biosensor. The sensor was constructed by adsorption of alternating layers of sub-nanometer gold particle-modified PAMAM (poly-amido-amine) dendrimers of generation 4.0, along with the enzyme by a layer-by-layer assembly technique. Atomic force microscopy (AFM), quartz crystal microbalance (QCM), and Fourier-transformed infrared spectroscopy (FTIR) were employed to elucidate the sensor assembly. Additionally, the biosensor was tested by cyclic voltammetry using caffeine as a substrate. PMID:27548239

  5. Effects of EPHX1 and CYP3A4*22 genetic polymorphisms on carbamazepine metabolism and drug response among Tunisian epileptic patients.

    PubMed

    Chbili, Chahra; Fathallah, Neila; Laouani, Aicha; Nouira, Manel; Hassine, Anis; Ben Amor, Sana; Ben Ammou, Sofiene; Ben Salem, Chaker; Saguem, Saad

    2016-03-01

    The aim of this study was to evaluate the impact of polymorphisms in the EPHX1 (c.416A > G, c.337T > C) and CYP3A4*22 genes involved in carbamazepine (CBZ) metabolism and pharmacoresistance among 118 Tunisian patients with epilepsy under maintenance dose of CBZ. These genetic polymorphisms were analyzed by PCR-RFLP. Associations between plasma CBZ concentration, CBZ-E concentration, maintenance doses and metabolic ratio (CBZ-E:CBZ, CBZ-D:CBZ-E) were analyzed with each polymorphism. Both variants of EPHX1 c.416A > G and c.337T > C are significantly associated with higher metabolic ratio CBZ-E:CBZ and seem to decrease the activity of the epoxide hydrolase. The CYP3A4*22 variant allele is significantly associated with lower CBZ-D:CBZ-E ratio and seems also to be associated with less activity of the cytochrome. Our data suggest that certain polymorphisms of metabolizing enzyme genes could influence inter-individual variability of CBZ metabolism. PMID:27276192

  6. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6

    PubMed Central

    Ke, A; Barter, Z; Rowland‐Yeo, K

    2016-01-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug‐drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0–1.7‐fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  7. Synthetic and natural compounds that interact with human cytochrome P450 1A2 and implications in drug development.

    PubMed

    Wang, Bo; Zhou, Shu-Feng

    2009-01-01

    Human cytochrome P450 1A2 (CYP1A2) is one of the major CYPs in the liver ( approximately 13%) and metabolizes about 20% of clinically used drugs. CYP1A2 is a 515-residue protein with a molecular mass of 58,294 Dal. The recently published crystal structure of CYP1A2 in complex with alpha-naphthoflavone has showed a rather compact active site with a relatively small volume of the cavity of 375 A(3), which is 44.2% and 49.3% larger than that of CYP2A6 (260 A(3)) and CYP2E1 (190 A(3)), respectively. A series of residues in the substrate recognition regions of CYP1A2 (e.g. Arg108, Thr124, Thr223, Glu225, Phe226, Lys250, Arg251, Lys253, Asn312, Asp313, Glu318, Thr319, Asp320, Thr321, Val322, Leu382, Thr385, and Ile386) have been shown to play important roles in ligand-enzyme binding based on site-directed mutagenesis and homology modeling studies. Typical CYP1A2 substrates generally contain planar ring that can fit the narrow and planar active site of the enzyme, such as propranolol, clozapine, guanabenz, flutamide, imatinib, thalidomide, carbamazepine, lidocaine, theophylline, tacrine, tizanidine, zolpidem, riluzole, zileuton, and leflunomide. CYP1A2 is one of the major enzymes that bioactivate a number of procarcinogens including polycyclic aromatic hydrocarbons (e.g., benzo[a]pyrene), heterocyclic aromatic amines/amides (e.g. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), mycotoxins (e.g. aflatoxin B(1)) and some natural compounds such as aristolochic acids present in several Chinese herbal medicines. This enzyme also metabolizes several important endogenous compounds including retinols, melatonin, steroids, uroporphyrinogen and arachidonic acids. Like many of other CYPs, CYP1A2 is subject to induction and inhibition by a number of compounds. In particular, several therapeutic drugs including antofloxacin, carbamazepine, dihydralazine, furafylline, isoniazid, rofecoxib, clorgyline, thiabendazole, and zileuton are mechanism-based inhibitors of CYP1A2. Reversible and

  8. Quantitative Assessment of the Influence of Cytochrome P450 1A2 Gene Polymorphism and Colorectal Cancer Risk

    PubMed Central

    Rewuti, Abudouaini; Ma, Yu-Shui; Wang, Xiao-Feng; Xia, Qing; Fu, Da; Han, Yu-Song

    2013-01-01

    Cytochrome P450 1A2 (CYP1A2) encodes a member of the cytochrome P450 superfamily of enzymes, which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of colorectal cancer (CRC). The CYP1A2*C (rs2069514) and CYP1A2*F (rs762551) polymorphism are two of the most commonly studied polymorphisms of the gene for their association with risk of CRC, but the results are conflicting. To derive a more precise estimation of the relationship between CYP1A2 and genetic risk of CRC, we performed a comprehensive meta-analysis which included 7088 cases and 7568 controls from 12 published case-control studies. In a combined analysis, the summary per-allele odds ratio for CRC was 0.91 (95% CI: 0.83–1.00, P = 0.04), and 0.91 (95% CI: 0.68–1.22, P = 0.53), for CYP1A2 *F and *C allele, respectively. In the subgroup analysis by ethnicity, significant associations were found in Asians for CYP1A2*F and CYP1A2*C, while no significant associations were detected among Caucasian populations. Similar results were also observed using dominant genetic model. Potential sources of heterogeneity were explored by subgroup analysis and meta-regression. No significant heterogeneity was detected in most of comparisons. This meta-analysis suggests that the CYP1A2 *F and *C polymorphism is a protective factor against CRC among Asians. PMID:23951174

  9. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease

    NASA Astrophysics Data System (ADS)

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-01

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, 1H, 13C, 119Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310 K were 2.51 × 107 and 3.09 × 105, respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309.

  10. Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease.

    PubMed

    Wei, Ying; Niu, Lin; Liu, Xinxin; Zhou, Hongyan; Dong, Hongzhou; Kong, Depeng; Li, Yunlan; Li, Qingshan

    2016-06-15

    A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, (1)H, (13)C, (119)Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants Kb of CYP3A4 with compound at 298 and 310K were 2.51×10(7) and 3.09×10(5), respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309. PMID:27049867

  11. Bone mineral properties in growing Col1a2(+/G610C) mice, an animal model of osteogenesis imperfecta.

    PubMed

    Masci, Marco; Wang, Min; Imbert, Laurianne; Barnes, Aileen M; Spevak, Lyudmila; Lukashova, Lyudmila; Huang, Yihe; Ma, Yan; Marini, Joan C; Jacobsen, Christina M; Warman, Matthew L; Boskey, Adele L

    2016-06-01

    The Col1a2(+/G610C) knock-in mouse, models osteogenesis imperfecta in a large old order Amish family (OOA) with type IV OI, caused by a G-to-T transversion at nucleotide 2098, which alters the gly-610 codon in the triple-helical domain of the α2(I) chain of type I collagen. Mineral and matrix properties of the long bones and vertebrae of male Col1a2(+/G610C) and their wild-type controls (Col1a2(+/+)), were characterized to gain insight into the role of α2-chain collagen mutations in mineralization. Additionally, we examined the rescuability of the composition by sclerostin inhibition initiated by crossing Col1a2(+/G610C) with an LRP(+/A214V) high bone mass allele. At age 10-days, vertebrae and tibia showed few alterations by micro-CT or Fourier transform infrared imaging (FTIRI). At 2-months-of-age, Col1a2(+/G610C) tibias had 13% fewer secondary trabeculae than Col1a2(+/+), these were thinner (11%) and more widely spaced (20%) than those of Col1a2(+/+) mice. Vertebrae of Col1a2(+/G610C) mice at 2-months also had lower bone volume fraction (38%), trabecular number (13%), thickness (13%) and connectivity density (32%) compared to Col1(a2+/+). The cortical bone of Col1a2(+/G610C) tibias at 2-months had 3% higher tissue mineral density compared to Col1a2(+/+); Col1a2(+/G610C) vertebrae had lower cortical thickness (29%), bone area (37%) and polar moment of inertia (38%) relative to Col1a2(+/+). FTIRI analysis, which provides information on bone chemical composition at ~7μm-spatial resolution, showed tibias at 10-days did not differ between genotypes. Comparing identical bone types in Col1a2(+/G610C) to Col1a2(+/+) at 2-months-of-age, tibias showed higher mineral-to-matrix ratio in trabeculae (17%) and cortices (31%). and in vertebral cortices (28%). Collagen maturity was 42% higher at 10-days-of-age in Col1a2(+/G610C) vertebral trabeculae and in 2-month tibial cortices (12%), vertebral trabeculae (42%) and vertebral cortices (12%). Higher acid-phosphate substitution

  12. Investigation of the effect of the uneven distribution of CYP3A4 and P-glycoprotein in the intestine on the barrier function against xenobiotics: a simulation study.

    PubMed

    Watanabe, Takao; Maeda, Kazuya; Nakai, Chikako; Sugiyama, Yuichi

    2013-09-01

    CYP3A4 and P-glycoprotein (P-gp) have similar substrate specificities and work together to form an intestinal absorption barrier against xenobiotics. Previous reports have indicated that CYP3A4 expression decreases gradually, whereas P-gp expression increases, from the upper to lower small intestine. The physiological rationale for this uneven distribution of CYP3A4 and P-gp as a barrier against xenobiotics has not been determined. To clarify the effect of these distribution patterns on barrier function, we constructed a mathematical model that included passive membrane permeation, P-gp-mediated apical efflux, and CYP3A4-mediated metabolism, and we simulated the effects of these distribution patterns on the fraction absorbed of co-substrates without changing their overall activities. The simulation showed that the physiological distribution patterns of both CYP3A4 and P-gp result in the lowest fraction absorbed, but not for drugs with low CYP3A4 and high P-gp-mediated clearances. These results suggest that the distribution pattern of CYP3A4 is especially important for the barrier function. On the other hand, physiological distribution pattern of P-gp exerts the maximum barrier function for dual good substrates for P-gp and CYP3A4, but even distribution of P-gp mostly suppresses the intestinal absorption of good P-gp, but poor CYP3A4 substrates. PMID:23754337

  13. Application of CYP3A4 in vitro data to predict clinical drug–drug interactions; predictions of compounds as objects of interaction

    PubMed Central

    Youdim, Kuresh A; Zayed, Aref; Dickins, Maurice; Phipps, Alex; Griffiths, Michelle; Darekar, Amanda; Hyland, Ruth; Fahmi, Odette; Hurst, Susan; Plowchalk, David R; Cook, Jack; Guo, Feng; Obach, R Scott

    2008-01-01

    AIMS The aim of this study was to explore and optimize the in vitro and in silico approaches used for predicting clinical DDIs. A data set containing clinical information on the interaction of 20 Pfizer compounds with ketoconazole was used to assess the success of the techniques. METHODS The study calculated the fraction and the rate of metabolism of 20 Pfizer compounds via each cytochrome P450. Two approaches were used to determine fraction metabolized (fm); 1) by measuring substrate loss in human liver microsomes (HLM) in the presence and absence of specific chemical inhibitors and 2) by measuring substrate loss in individual cDNA expressed P450s (also referred to as recombinant P450s (rhCYP)) The fractions metabolized via each CYP were used to predict the drug–drug interaction due to CYP3A4 inhibition by ketoconazole using the modelling and simulation software SIMCYP®. RESULTS When in vitro data were generated using Gentest supersomes, 85% of predictions were within two-fold of the observed clinical interaction. Using PanVera baculosomes, 70% of predictions were predicted within two-fold. In contrast using chemical inhibitors the accuracy was lower, predicting only 37% of compounds within two-fold of the clinical value. Poorly predicted compounds were found to either be metabolically stable and/or have high microsomal protein binding. The use of equilibrium dialysis to generate accurate protein binding measurements was especially important for highly bound drugs. CONCLUSIONS The current study demonstrated that the use of rhCYPs with SIMCYP® provides a robust in vitro system for predicting the likelihood and magnitude of changes in clinical exposure of compounds as a consequence of CYP3A4 inhibition by a concomitantly administered drug. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Numerous retrospective analyses have shown the utility of in vitro systems for predicting potential drug–drug interactions (DDIs). Prediction of DDIs from in vitro data is commonly

  14. Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel

    PubMed Central

    Kus, Tulay; Aktas, Gokmen; Kalender, Mehmet Emin; Demiryurek, Abdullah Tuncay; Ulasli, Mustafa; Oztuzcu, Serdar; Sevinc, Alper; Kul, Seval; Camci, Celaletdin

    2016-01-01

    Background Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. Methods From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m2 every 3 weeks for four cycles, or IV 80 mg/m2 weekly for 12 cycles, and IV 100 mg/m2 docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. Results Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172–6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033–4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001–3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. Conclusion ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy. PMID:27574448

  15. Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system☆

    PubMed Central

    Lu, Huiyuan; Ma, Jun; Liu, Nian; Wang, Shoulin

    2010-01-01

    Objective CYP1A2 and NADPH-CYP450 oxidoreductase (POR) were expressed in the baculovirus/Spodoptera frugiperda (sf9) system. The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR. Methods The heme precursors [δ-Aminolaevulinic Acid (5-ALA), Fe3+ and hemin] were introduced into the system to evaluate their effects on the expression of CYP1A2, POR and their co-expression. All the proteins were identified using immunoblotting, CO-difference spectroscopy, or cytochrome c assay. Results In the present study, functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system, and both of them showed high activities. Co-addition of 5-ALA and Fe3+ significantly improved expression of CYP1A2 by about 50% compared with the addition of 5-ALA, Fe3+ or hemin alone. Either co-addition of 5-ALA and Fe3+ or addition of 5-ALA or Fe3+ alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control (no addition). However, unlike CYP1A2, there was no difference between the co-addition and addition of these heme precursors alone. Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR, with a 3:1 ratio of BvCYP1A2 / BvPOR significantly increasing their co-expression. Surprisingly, the addition of 0.1 mM 5-ALA or Fe3+ alone, but not their co-addition, could significantly improve the CYP1A2 and POR co-expression (P < 0.05). Conclusion 5-ALA and Fe3+ increased the expression of CYP1A2 and POR in a baculovirus/sf9 system, but the pattern of their expression was different between their expression alone and co-expression. PMID:23554636

  16. CYP1A2 rs762551 polymorphism contributes to risk of lung cancer: a meta-analysis.

    PubMed

    Ma, Zheng; Guo, Wei; Gong, Taiqian; Niu, Hui-Jun; Wang, Ru-Wen; Jiang, Yao-Guang

    2014-03-01

    Previous studies proposed that CYP1A2 rs762551 polymorphism might be associated with risk of lung cancer by influencing the function of CYP1A2. However, previous studies on the association between CYP1A2 rs762551 polymorphism and risk of lung cancer reported inconsistent findings. We performed a meta-analysis of the published case-control studies to assess the association between CYP1A2 rs762551 polymorphism and risk of lung cancer. PubMed and Embase were searched to identify relevant studies on the association between CYP1A2 rs762551 polymorphism and risk of lung cancer, and seven studies with a total of 3,320 subjects were finally included into the meta-analysis. The pooled odds ratio (OR) and 95 % confidence interval (95%CI) was calculated to evaluate the association. Meta-analysis of total studies showed that CYP1A2 rs762551 polymorphism contributed to risk of lung cancer under all four genetic models (C versus A: OR = 1.26, 95%CI 1.13 to 1.40, P < 0.001; CC versus AA: OR = 1.61, 95%CI 1.28 to 2.04, P < 0.001; CC versus AA/AC: OR = 1.52, 95%CI 1.11 to 2.09, P = 0.009; CC/AC versus AA: OR = 1.28, 95%CI 1.10 to 1.48, P = 0.001). Subgroup analysis based on ethnicity further suggested that CYP1A2 rs762551 polymorphism was associated with risk of lung cancer in Caucasians. These results from the meta-analysis suggest that CYP1A2 rs762551 polymorphism contributes to risk of lung cancer. PMID:24293373

  17. Expression of Organic Anion Transporting Polypeptide 1A2 in Red Blood Cells and Its Potential Impact on Antimalarial Therapy.

    PubMed

    Hubeny, Andrea; Keiser, Markus; Oswald, Stefan; Jedlitschky, Gabriele; Kroemer, Heyo K; Siegmund, Werner; Grube, Markus

    2016-10-01

    Important antimalarial drugs, including quinolines, act against blood schizonts by interfering with hemoglobin metabolism. To reach their site of action, these compounds have to cross the plasma membrane of red blood cells (RBCs). Organic cation transporters (OCTs) and organic anion transporting polypeptides (OATPs) are important uptake transporters and interesting candidates for local drug transport. We therefore studied their interaction with antimalarial compounds (quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate) and characterized the expression of OATP1A2 and OATP2B1 in RBCs. Competition assays using transporter-overexpressing Madin-Darby canine kidney (MDCKII) cells and the model substrate estrone-3-sulfate identified quinine and chloroquine as potent inhibitors of OATP1A2 function (IC50 quinine: 0.7 ± 1.2 µM; chloroquine: 1.0 ± 1.5 µM), but no or only moderate effects were observed for OATP2B1. Subsequently, quinine was identified as a substrate of OATP1A2 (Km 23.4 µM). The OATP1A2-mediated uptake was sensitive to the OATP1A2-specific inhibitor naringin. Both OATPs were expressed in human RBCs, and ex vivo transport studies demonstrated naringin-sensitive accumulation of quinine in these cells (60 pmol versus 38 pmol/5 × 10(5) RBCs). Additional transport studies using OCT1-3 and organic cation transporter novel type 1 (OCTN1) indicated only significant quinine uptake by OCT1, which was not detected in RBCs. In conclusion, our data demonstrate expression of OATP2B1 and OATP1A2 in RBCs as well as OATP1A2-mediated uptake of quinine. Therefore, modulation of OATP1A2 function may affect quinine uptake into erythrocytes. PMID:27504015

  18. Identification of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with no evidence of CYP3A4 time-dependent inhibition and improved aqueous solubility.

    PubMed

    Zak, Mark; Liederer, Bianca M; Sampath, Deepak; Yuen, Po-Wai; Bair, Kenneth W; Baumeister, Timm; Buckmelter, Alexandre J; Clodfelter, Karl H; Cheng, Eric; Crocker, Lisa; Fu, Bang; Han, Bingsong; Li, Guangkun; Ho, Yen-Ching; Lin, Jian; Liu, Xiongcai; Ly, Justin; O'Brien, Thomas; Reynolds, Dominic J; Skelton, Nicholas; Smith, Chase C; Tay, Suzanne; Wang, Weiru; Wang, Zhongguo; Xiao, Yang; Zhang, Lei; Zhao, Guiling; Zheng, Xiaozhang; Dragovich, Peter S

    2015-02-01

    Herein we report the optimization efforts to ameliorate the potent CYP3A4 time-dependent inhibition (TDI) and low aqueous solubility exhibited by a previously identified lead compound from our NAMPT inhibitor program (1, GNE-617). Metabolite identification studies pinpointed the imidazopyridine moiety present in 1 as the likely source of the TDI signal, and replacement with other bicyclic systems was found to reduce or eliminate the TDI finding. A strategy of reducing the number of aromatic rings and/or lowering cLogD7.4 was then employed to significantly improve aqueous solubility. These efforts culminated in the discovery of 42, a compound with no evidence of TDI, improved aqueous solubility, and robust efficacy in tumor xenograft studies. PMID:25556090

  19. NblA1/A2-Dependent Homeostasis of Amino Acid Pools during Nitrogen Starvation in Synechocystis sp. PCC 6803

    PubMed Central

    Kiyota, Hiroshi; Hirai, Masami Yokota; Ikeuchi, Masahiko

    2014-01-01

    Nutrient balance is important for photosynthetic growth and biomass production in microalgae. Here, we investigated and compared metabolic responses of amino acid pools to nitrogen and sulfur starvation in a unicellular model cyanobacterium, Synechocystis sp. PCC 6803, and its mutant nblA1/A2. It is known that NblA1/A2-dependent and -independent breakdown of abundant photosynthetic phycobiliproteins and other cellular proteins supply nutrients to the organism. However, the contribution of the NblA1/A2-dependent nutrient supply to amino acid pool homeostasis has not been studied. Our study demonstrates that changes in the pool size of many amino acids during nitrogen starvation can be categorized as NblA1/A2-dependent (Gln, Glu, glutathione, Gly, Ile, Leu, Met, Phe, Pro, Ser, Thr, Tyr and Val) and NblA1/A2-independent (Ala, Asn, Lys, and Trp). We also report unique changes in amino acid pool sizes during sulfur starvation in wild type and the mutant and found a generally marked increase in the Lys pool in cyanobacteria during nutrient starvation. In conclusion, the NblA1/A2-dependent protein turnover contributes to the maintenance of many amino acid pools during nitrogen starvation. PMID:24983765

  20. ALTERATION IN CYTOCHROME P450 3A4 ACTIVITY AS MEASURED BY A URINE CORTISOL ASSAY IN HIV-1-INFECTED PREGNANT WOMEN AND RELATIONSHIP TO ANTIRETROVIRAL PHARMACOKINETICS

    PubMed Central

    Aweeka, Francesca T.; Hu, Chengcheng; Huang, Liusheng; Best, Brookie M.; Stek, Alice; Lizak, Patricia; Burchett, Sandra K.; Read, Jennifer S.; Watts, Heather; Mirochnick, Mark; Capparelli, Edmund V.

    2014-01-01

    Objectives Pregnancy results in physiological changes altering the pharmacokinetics of drugs metabolized by cytochrome p450 3A4. The urinary ratio of 6-β hydroxycortisol to cortisol (6βHF:F) is a marker of CYP3A4 induction. We sought to evaluate its change in antiretroviral (ARV) treated HIV-1-infected women and to relate this change to ARV pharmacokinetics. Methods Women receiving various ARV had pharmacokinetic evaluations during third trimester pregnancy (>30 weeks) and postpartum with determination of 6βHF:F carried out on the same days. Wilcoxon signed rank test compared the ratio antepartum to postpartum. The relationship between the change in ratio to the change in pharmacokinetics was done using Kendall’s tau. Results 6βHF:F ratios were available for 107 women antepartum with 54 having postpartum values. The ratio was higher antepartum (p=0.033) [median comparison 1.35 (95% CI: 1.01, 1.81]. For 71 women taking a protease inhibitor (PI), the antepartum versus postpartum 6βHF:F comparison was marginally significant (p=0.058). When relating the change in the 6βHF:F ratio to the change in the dose-adjusted ARV AUC antepartum to postpartum, the 35 subjects in the LPV/r arms demonstrated an inverse relationship (p=0.125), albeit this correlation did not reach statistical significance. Conclusions A 35% increase in the urinary 6βHF:F ratio was measured during late pregnancy compared to postpartum, indicating CYP3A induction occurs during pregnancy. The trend to an inverse relationship between the change in the 6βHF:F ratio and the change in the LPV AUC antepartum versus postpartum suggests CYP3A induction may be one mechanism behind altered LPV exposure during pregnancy. PMID:25407158

  1. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  2. Inhibitory effects of pomelo on the metabolism of tacrolimus and the activities of CYP3A4 and P-glycoprotein.

    PubMed

    Egashira, Kanoko; Ohtani, Hisakazu; Itoh, Suwako; Koyabu, Noriko; Tsujimoto, Masayuki; Murakami, Hideyasu; Sawada, Yasufumi

    2004-08-01

    We recently reported a case of increase in the blood level of tacrolimus following intake of pomelo in a renal transplant recipient. To clarify the mechanism of this increase in the blood level of tacrolimus, we investigated the effect of pomelo juice extract on the activities of CYP3A4 and P-glycoprotein, in comparison with that of extract of grapefruit juice (GFJ). The 10% ethyl acetate extracts of the juice of three pomelos of different origins (Banpeiyu, pomelo I; Hirado Buntan, pomelo II; and Tosa Buntan, pomelo III) and GFJ significantly inhibited 6beta-hydroxylation of testosterone in human liver microsomes by 76.4, 67.2, 37.5, and 83.9%, respectively. The extract of pomelo I was as potent as that of GFJ. The metabolism of tacrolimus itself was also inhibited by the extract of pomelo I, as well as that of GFJ. Furthermore, the inhibition of both 6beta-hydroxylation of testosterone and metabolism of tacrolimus by pomelo I and GFJ was preincubation time-dependent. On the other hand, the extract of pomelo I had little effect on the transcellular transport of tacrolimus or [(3)H]digoxin across a monolayer of LLC-GA5-COL150 cells (a porcine kidney epithelial cell line, LLC-PK1, transfected with human MDR1 cDNA and overexpressing human P-glycoprotein). In conclusion, pomelo constituents inhibit the activity of CYP3A4 and may thereby produce an increase in the blood level of tacrolimus. PMID:15258108

  3. Proximal Roux-en-Y Gastric Bypass Alters Drug Absorption Pattern But Not Systemic Exposure of CYP3A4 and P-glycoprotein Substrates

    PubMed Central

    Chan, Lingtak-Neander; Lin, Yvonne S.; Tay-Sontheimer, Jessica C.; Trawick, Dorothy; Oelschlager, Brant K.; Flum, David R.; Patton, Kristen K.; Shen, Danny D.; Horn, John R.

    2015-01-01

    Study Objectives To evaluate the effect of Roux-en-Y gastric bypass surgery (RYGB) on the pharmacokinetics of midazolam (a CYP3A4 substrate) and digoxin (a P-glycoprotein substrate). Design Prospective, nonblinded, longitudinal, single-dose pharmacokinetic study in three phases: presurgery baseline and postoperative assessments at 3 and 12 months. Patients Twelve obese patients meeting current standards for bariatric surgery. Measurements and Main Results At each study visit, patients received a single dose of oral digoxin and midazolam at 8 a.m. Blood samples were collected at regular intervals for 24 hours after dosing. Continuous 12-lead electrocardiogram (EKG), heart rate, blood pressure, and respiratory rate were monitored, and pharmacokinetic parameters from the three visits were compared. The peak plasma concentration (Cmax) of midazolam increased by 66% and 71% at 3- and 12-month post-RYGB (p=0.017 and p=0.001, respectively), whereas the median time to peak concentration (Tmax) was reduced by 50%. The mean Cmax for 1′-hydroxymidazolam increased by 87% and 80% at 3 and 12 months (p=0.001 and p<0.001, respectively). However, neither the area under the concentration-time curve (AUC) for midazolam nor the metabolite-to-parent AUC ratio changed significantly over time. For digoxin, the median Tmax decreased from 40 minutes at baseline to 30 and 20 minutes at 3 and 12 months, respectively. The mean AUC for digoxin, heart rate, and EKG patterns were similar across the three study phases. Conclusion Contemporary proximal RYGB increases the rate of drug absorption without significantly changing the overall exposure to midazolam and digoxin. The Cmax of a CYP3A4 substrate with a high extraction ratio was substantially increased after RYGB. PMID:25757445

  4. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

    PubMed

    Mazzari, Andre L D A; Milton, Flora; Frangos, Samantha; Carvalho, Ana C B; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  5. Effects of strong CYP2D6 and 3A4 inhibitors, paroxetine and ketoconazole, on the pharmacokinetics and cardiovascular safety of tamsulosin

    PubMed Central

    Troost, Joachim; Tatami, Shinji; Tsuda, Yasuhiro; Mattheus, Michaela; Mehlburger, Ludwig; Wein, Martina; Michel, Martin C

    2011-01-01

    AIM To determine the effect of the strong CYP2D6 inhibitor paroxetine and strong CYP3A4 inhibitor ketoconazole on the pharmacokinetics and safety (orthostatic challenge) of tamsulosin. METHODS Two open-label, randomized, two-way crossover studies were conducted in healthy male volunteers (extensive CYP2D6 metabolizers). RESULTS Co-administration of multiple oral doses of 20 mg paroxetine once daily with a single oral dose of the 0.4 mg tamsulosin HCl capsule increased the adjusted geometric mean (gMean) values of Cmax and AUC(0,∞) of tamsulosin by factors of 1.34 (90% CI 1.21, 1.49) and 1.64 (90% CI 1.44, 1.85), respectively, and increased the terminal half-life (t1/2) of tamsulosin HCl from 11.4 h to 15.3 h. Co-administration of multiple oral doses of 400 mg ketoconazole once dailywith a single oral dose of the 0.4 mg tamsulosin increased the gMean values of Cmax and AUC(0,∞) of tamsulosin by a factor of 2.20 (90% CI 1.96, 2.45) and 2.80 (90% CI 2.56, 3.07), respectively. The terminal half-life was slightly increased from 10.5 h to 11.8 h. These pharmacokinetic changes were not accompanied by clinically significant alterations of haemodynamic responses during orthostatic stress testing. CONCLUSION The exposure to tamsulosin is increased upon co-administration of strong CYP2D6 inhibitors and even more so of strong 3A4 inhibitors, but neither PK alteration was accompanied by clinically significant haemodynamic changes during orthostatic stress testing. PMID:21496064

  6. Assessment of a Candidate Marker Constituent Predictive of a Dietary Substance–Drug Interaction: Case Study with Grapefruit Juice and CYP3A4 Drug Substrates

    PubMed Central

    Ainslie, Garrett R.; Wolf, Kristina K.; Li, Yingxin; Connolly, Elizabeth A.; Scarlett, Yolanda V.; Hull, J. Heyward

    2014-01-01

    Dietary substances, including herbal products and citrus juices, can perpetrate interactions with conventional medications. Regulatory guidances for dietary substance–drug interaction assessment are lacking. This deficiency is due in part to challenges unique to dietary substances, a lack of requisite human-derived data, and limited jurisdiction. An in vitro–in vivo extrapolation (IVIVE) approach to help address some of these hurdles was evaluated using the exemplar dietary substance grapefruit juice (GFJ), the candidate marker constituent 6′,7′-dihydroxybergamottin (DHB), and the purported victim drug loperamide. First, the GFJ-loperamide interaction was assessed in 16 healthy volunteers. Loperamide (16 mg) was administered with 240 ml of water or GFJ; plasma was collected from 0 to 72 hours. Relative to water, GFJ increased the geometric mean loperamide area under the plasma concentration–time curve (AUC) significantly (1.7-fold). Second, the mechanism-based inhibition kinetics for DHB were recovered using human intestinal microsomes and the index CYP3A4 reaction, loperamide N-desmethylation (KI [concentration needed to achieve one-half kinact], 5.0 ± 0.9 µM; kinact [maximum inactivation rate constant], 0.38 ± 0.02 minute−1). These parameters were incorporated into a mechanistic static model, which predicted a 1.6-fold increase in loperamide AUC. Third, the successful IVIVE prompted further application to 15 previously reported GFJ-drug interaction studies selected according to predefined criteria. Twelve of the interactions were predicted to within the 25% predefined criterion. Results suggest that DHB could be used to predict the CYP3A4-mediated effect of GFJ. This time- and cost-effective IVIVE approach could be applied to other dietary substance–drug interactions to help prioritize new and existing drugs for more advanced (dynamic) modeling and simulation and clinical assessment. PMID:25253884

  7. Regulation of cytochrome P450 3A4 by small vault RNAb derived from the non-coding vault RNA1 of multidrug resistance-linked vault particle.

    PubMed

    Meng, Chunjie; Wei, Zhiyun; Zhang, Yiting; Yan, Liang; He, Hang; Zhang, Lirong; Xing, Qinghe

    2016-07-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and intestine, contributing to the metabolism of >60% of all pharmaceuticals. The expression levels of hepatic CYP3A4 show great inter‑individual variation. However, the detailed regulatory mechanism of CYP3A4 expression has remained largely elusive. It has been reported that the non‑coding RNA small vault (sv)RNAb targets the 3' untranslated region (3'UTR) of CYP3A4 in MCF7 cells. However, to date, the role of svRNAb has not been examined in human liver tissue and hepatic cell lines such as HepG2, which was the aim of the present study. Polymerase chain reaction analysis indicated that the expression of CYP3A4 was significantly different within a study cohort (n=19). In addition, a significant negative correlation was observed between svRNAb and CYP3A4 expression in human liver tissue samples. Furthermore, a luciferase assay on HepG2 cells verified that svRNAb directly targets CYP3A4 and regulates the expression of CYP3A4 by interacting with the validated binding sites of the CYP3A4 3'UTR. The results provided insight into the variation of the expression of CYP3A4 among individuals and provided a novel method for the adjustment of personalized drug treatment. Furthermore, the present study provided a mechanism of the regulatory role of svRNAb in multidrug‑resistant cells. PMID:27177257

  8. The isolation of minor-occurring furanocoumarins in grapefruit and analysis of their inhibition of cyp 3a4 and p-glycoprotein transport of talinolol from caco-2 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of grapefruit juice on the disposition of certain prescription drugs in humans have been mainly attributed to the inhibition of intestinal cytochrome P450 3A4 (CYP 3A4) by linear furanocoumarins. A number of the main furanocoumarins in grapefruit juice have been identified and analyzed ...

  9. Dynamically simulating the interaction of midazolam and the CYP3A4 inhibitor itraconazole using individual coupled whole-body physiologically-based pharmacokinetic (WB-PBPK) models

    PubMed Central

    Vossen, Michaela; Sevestre, Michael; Niederalt, Christoph; Jang, In-Jin; Willmann, Stefan; Edginton, Andrea N

    2007-01-01

    Background Drug-drug interactions resulting from the inhibition of an enzymatic process can have serious implications for clinical drug therapy. Quantification of the drugs internal exposure increase upon administration with an inhibitor requires understanding to avoid the drug reaching toxic thresholds. In this study, we aim to predict the effect of the CYP3A4 inhibitors, itraconazole (ITZ) and its primary metabolite, hydroxyitraconazole (OH-ITZ) on the pharmacokinetics of the anesthetic, midazolam (MDZ) and its metabolites, 1' hydroxymidazolam (1OH-MDZ) and 1' hydroxymidazolam glucuronide (1OH-MDZ-Glu) using mechanistic whole body physiologically-based pharmacokinetic simulation models. The model is build on MDZ, 1OH-MDZ and 1OH-MDZ-Glu plasma concentration time data experimentally determined in 19 CYP3A5 genotyped adult male individuals, who received MDZ intravenously in a basal state. The model is then used to predict MDZ, 1OH-MDZ and 1OH-MDZ-Glu concentrations in an CYP3A-inhibited state following ITZ administration. Results For the basal state model, three linked WB-PBPK models (MDZ, 1OH-MDZ, 1OH-MDZ-Glu) for each individual were elimination optimized that resulted in MDZ and metabolite plasma concentration time curves that matched individual observed clinical data. In vivo Km and Vmax optimized values for MDZ hydroxylation were similar to literature based in vitro measures. With the addition of the ITZ/OH-ITZ model to each individual coupled MDZ + metabolite model, the plasma concentration time curves were predicted to greatly increase the exposure of MDZ as well as to both increase exposure and significantly alter the plasma concentration time curves of the MDZ metabolites in comparison to the basal state curves. As compared to the observed clinical data, the inhibited state curves were generally well described although the simulated concentrations tended to exceed the experimental data between approximately 6 to 12 hours following MDZ administration. This

  10. Identification of inhibitory component in cinnamon--O-methoxycinnamaldehyde inhibits CYP1A2 and CYP2E1-.

    PubMed

    Hasegawa, Atsushi; Yoshino, Masaki; Nakamura, Hiroyoshi; Ishii, Itsuko; Watanabe, Toshiko; Kiuchi, Masahiro; Ishikawa, Tsutomu; Ohmori, Shigeru; Kitada, Mitsukazu

    2002-01-01

    The Cinnamomi Cortex and Ephedra Herba were found to more strongly inhibit aminopyrine N-demethylation in rat liver microsomes compared to other constituents included in Sho-seiryu-to. The component inhibiting drug oxidations catalyzed by CYP1A2 and CYP2E1 was isolated from Cinnamomi Cortex, and was identified as o-methoxycinnamaldehyde (OMCA). When phenacetin and 4-nitrophenol were used as probe substrates for CYP1A2 and CYP2E1, respectively, the OMCA was shown to be a competitive inhibitor against CYP1A2 while it was a mixed type inhibitor against CYP2E1. The inhibitory effect of OMCA on 4-nitrophenol 2-hydroxylation (K(i)=6.3 microM) was somewhat potent compared to that observed on phenacetin O-deethylation (K(i)=13.7 microM) in rat liver microsomes. PMID:15618674

  11. Meta-analysis of correlation between the CYP1A2 -3860 G > A polymorphism and lung cancer risk.

    PubMed

    Ren, J; He, B Z; Zhang, T S; Lu, S P; Yan, T

    2016-01-01

    The aim of this meta-analysis was to assess the association between a polymorphism (-3860 G > A) in the cytochrome P450 1A2 (CYP1A2) gene and lung cancer susceptibility. Relevant studies were retrieved from the PubMed and EMBase databases, and additionally evaluated for conformance with the inclusion criteria. The odds ratios (ORs) and their 95% confidence intervals (95%CIs) in all selected studies were used to assess the relationship between the CYP1A2 -3860 G > A polymorphism and lung cancer risk. The data was pooled using Stata v.11. Six studies, comprising 1168 lung cancer patients and 1598 controls, were included in this meta-analysis. We found no correlation between the CYP1A2 -3860 G > A polymorphism and lung cancer risk in any of the models (AA vs GG: OR = 4.79, 95%CI = 0.03-702.67; GA vs GG: OR = 1.33, 95%CI = 0.74-2.39; dominant model: OR = 1.41, 95%CI = 0.69-2.90; recessive model: OR = 4.07, 95%CI = 0.04-368.35). Moreover, we observed no statistically significant association between CYP1A2 -3860 G > A and lung cancer susceptibility when stratified by the ethnicity of the sample populations, sample size, and study quality, except in a low-quality study. Our findings indicated that the -3860 G > A polymorphism in CYP1A2 might not be a risk factor for lung cancer. PMID:27323197

  12. Variable inhibitory effect of herbal supplements of different brands on human P450 CYP1A2

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2012-01-01

    Herbal supplements are not governed by the same regulations as prescription drugs, we hypothesize that the content of their active ingredients may vary largely among different manufacturers. This may produce variable therapeutic outcomes. This study aims to examine this hypothesis on commonly used herbal supplements among cancer patients. CYP1A2 has been implicated in the activation of many carcinogens and alteration in its activity may be a mechanism associated with the protective effect of herbal products. Activity of human CYP1A2 was used to determine the effect of four herbal supplements of different brands, namely, black cohosh (BC), ginseng, grape seed extract (GSE) and green tea extract (GTE). The herbal content was extracted with methanol, and extract aliquots were used to determine their effect on CYP1A2. Human liver microsomes, the CYP1A2 probe (7-ethoxyresorufin) and NADPH in buffer were incubated with and without herbal extract. Metabolite (resorufin) formation was monitored by HPLC. Seven BC products caused a mild inhibition of CYP1A2, ranging from 2.4 % by GNC Plus to 21.9 % by Nature's Resource. Among nine ginseng products tested, the inhibitory effect varied from 4.2 % by Imperial to 44.6 % by Solarays. The effect of nine GSE brands also varied, ranging from 1.7 % (Country Life) to 26.5 % (Veg Life). Of twelve GTE products, the inhibitory effect varied from 2.9 % by Henry's to 46.6 % by GNC Plus. It appears that the inhibition of selected herbal supplements on CYP1A2 activity varies considerably among different brands of the products. This may be due to variations in the herbal products' active ingredients content. PMID:27298605

  13. Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1.

    PubMed

    Bakari, Sana; Lembrouk, Mehdi; Sourd, Laura; Ousalem, Fares; André, François; Orlowski, Stéphane; Delaforge, Marcel; Frelet-Barrand, Annie

    2016-04-01

    Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies. PMID:26961909

  14. RS-predictor: a new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4.

    PubMed

    Zaretzki, Jed; Bergeron, Charles; Rydberg, Patrik; Huang, Tao-wei; Bennett, Kristin P; Breneman, Curt M

    2011-07-25

    This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as "metabolophores": A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent (O-dealkylation vs N-oxidation vs Csp(3) hydroxylation, etc.), isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared. PMID:21528931

  15. Molecular and crystal structures of some novel derivatives of 3-aryl-7-arylidene-3,3a,4,5,6,7-hexahydroindazoles

    SciTech Connect

    Abakumov, V. V.; Shishkina, S. V.; Zubatyuk, R. I.; Gella, I. M.; Pivnenko, N. S.; Kutulya, L. A. Shishkin, O. V.

    2007-03-15

    The stereochemical aspects of the interaction of 2,6-bis(arylidene)-cyclohexanone and 2,6-bis(arylidene)-3-methylcyclohexanone with arylhydrazine (aryl is phenyl or 4-nitrophenyl) and methylhydrazine are investigated using X-ray diffraction analysis. The molecular structure of the 3-aryl-7-arylidene-3,3a,4,5,6,7-hexahydroindazoles synthesized is determined by X-ray diffraction analysis for the first time. It is established that the stereochemistry of the products of the interaction between the cyclohexanone derivatives and arylhydrazines depends on the electronic nature of the substituent in the aryl group. Two regioisomeric products with different positions of the methyl group in the cyclohexane ring with respect to the arylidene fragment are synthesized by the reaction of 2,6-bis(4-methoxybenzylidene)-3-methylcyclohexanone with methylhydrazine. The influence of the substituents at the nitrogen atom of the pyrazoline ring on the intramolecular electronic interactions and the geometry of the heterocycle in the compounds under investigation is discussed.

  16. RS-Predictor: A new tool for predicting sites of cytochrome P450-mediated metabolism applied to CYP 3A4

    PubMed Central

    Zaretzki, Jed; Bergeron, Charles; Rydberg, Patrik; Huang, Tao-wei; Bennett, Kristin P.; Breneman, Curt M.

    2011-01-01

    This article describes RegioSelectivity-Predictor (RS-Predictor), a new in silico method for generating predictive models of P450-mediated metabolism for drug-like compounds. Within this method, potential sites of metabolism (SOMs) are represented as “metabolophores”: A concept that describes the hierarchical combination of topological and quantum chemical descriptors needed to represent the reactivity of potential metabolic reaction sites. RS-Predictor modeling involves the use of metabolophore descriptors together with multiple-instance ranking (MIRank) to generate an optimized descriptor weight vector that encodes regioselectivity trends across all cases in a training set. The resulting pathway-independent,i isozyme-specific regioselectivity model may be used to predict potential metabolic liabilities. In the present work, cross-validated RS-Predictor models were generated for a set of 394 substrates of CYP 3A4 as a proof-of-principle for the method. Rank aggregation was then employed to merge independently generated predictions for each substrate into a single consensus prediction. The resulting consensus RS-Predictor models were shown to reliably identify at least one observed site of metabolism in the top two rank-positions on 78% of the substrates. Comparisons between RS-Predictor and previously described regioselectivity prediction methods reveal new insights into how in silico metabolite prediction methods should be compared. PMID:21528931

  17. Haplotypes in the APOA1-C3-A4-A5 gene cluster affect plasma lipids in both humans and baboons

    SciTech Connect

    Wang, Qian-fei; Liu, Xin; O'Connell, Jeff; Peng, Ze; Krauss, Ronald M.; Rainwater, David L.; VandeBerg, John L.; Rubin, Edward M.; Cheng, Jan-Fang; Pennacchio, Len A.

    2003-09-15

    Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human QTL genes in a highly controlled non-human primate model.

  18. Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies.

    PubMed

    Carreño, Oriel; Corominas, Roser; Serra, Selma Angèlica; Sintas, Cèlia; Fernández-Castillo, Noèlia; Vila-Pueyo, Marta; Toma, Claudio; Gené, Gemma G; Pons, Roser; Llaneza, Miguel; Sobrido, María-Jesús; Grinberg, Daniel; Valverde, Miguel Ángel; Fernández-Fernández, José Manuel; Macaya, Alfons; Cormand, Bru

    2013-11-01

    Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing. PMID:24498617

  19. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    PubMed

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-07-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. PMID:26771455

  20. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of these standards can be inspected at the Federal... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System...

  1. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  2. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  3. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  4. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  5. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of these standards can be inspected at the Federal... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System...

  6. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  7. Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies

    PubMed Central

    Carreño, Oriel; Corominas, Roser; Serra, Selma Angèlica; Sintas, Cèlia; Fernández-Castillo, Noèlia; Vila-Pueyo, Marta; Toma, Claudio; Gené, Gemma G; Pons, Roser; Llaneza, Miguel; Sobrido, María-Jesús; Grinberg, Daniel; Valverde, Miguel Ángel; Fernández-Fernández, José Manuel; Macaya, Alfons; Cormand, Bru

    2013-01-01

    Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing. PMID:24498617

  8. Suppression of Hepatic Cyp1a2 by Total Ginsenosides in Lipopolysaccharide-Treated Mice and Primary Mouse Hepatocytes.

    PubMed

    Sun, Haiyan; Yan, Yijing; Xu, Chenshu; Wan, Hongxia; Liu, Dong

    2016-03-23

    The roots of Panax ginseng (ginseng) have been extensively used in traditional Chinese medicine. However, herb-drug interactions between ginseng and other co-administered drugs are not fully understood concerning the effect of ginseng on drug metabolism and clearance. The current study aimed to elucidate the effect of total ginsenosides, a typical ginseng extract, on the regulation of Cyp1a2, a key enzyme to regulate drug metabolism under the normal and inflammatory conditions in mice. Female C57BL/6J mice treated with vehicle and lipopolysaccharide (LPS) were intragastrically administered ginseng extract for 7 days before hepatic P450 expression was analyzed. Primary mouse hepatocytes were also employed to further explore the effects of total ginsenosides on Cyp1a2 expression. The results showed that total ginsenosides in P. ginseng extract exhibited a concentration-dependent suppression on Cyp1a2 mRNA and protein level in both mice and primary mouse hepatocytes. Notably, the inhibitory effects of total ginsenosides on Cyp1a2 mRNA and protein expression were further enhanced following LPS treatment. Therefore, future research is warranted to investigate the role of ginsenosides in the regulation of hepatic CYP450s. Moreover, consumption of ginseng as food or supplement should be monitored for patients on combinational therapy, especially those with inflammatory diseases. PMID:26923348

  9. Drug-metabolising enzymes are down-regulated by hypoxia in differentiated human hepatoma HepaRG cells: HIF-1alpha involvement in CYP3A4 repression.

    PubMed

    Legendre, Claire; Hori, Tamaki; Loyer, Pascal; Aninat, Caroline; Ishida, Seiichi; Glaise, Denise; Lucas-Clerc, Catherine; Boudjema, Karim; Guguen-Guillouzo, Christiane; Corlu, Anne; Morel, Fabrice

    2009-11-01

    Weak blood irrigation within solid tumours including hepatocellular carcinomas (HCCs) plays an important role in resistance to anticancer drugs by decreasing accessibility of cytotoxic agents to tumour cells. Reduced oxygen levels, or hypoxia, also contribute to drug resistance because many anticancer drugs require molecular oxygen to be cytotoxic. Our aim was to develop a new in vitro model mimicking hypoxic cells within HCCs in order to further explore the molecular responses to hypoxia, including regulation of drug-metabolising enzymes (DMEs) expression. For this purpose, we used the highly differentiated human hepatoma HepaRG cells cultured under either normoxic or hypoxic (24h at 1% O(2)) conditions. Gene and protein expressions were investigated by quantitative PCR and immunoblotting, respectively. We showed that HepaRG cells adapt to prolonged moderate hypoxia by a switch from aerobic to anaerobic glycolysis and a repression of critical genes involved in amino acid, lipid and ethanol metabolisms. Importantly, expression of several DMEs (particularly cytochromes P450 (CYPs) and phase II enzymes) and xenosensors (CAR, PXR and AhR) was down-regulated and CYPs activities (using testosterone and paclitaxel as substrates) were decreased during hypoxia. In addition, a new role for HIF-1alpha in the repression of CYP3A4 is demonstrated in cells treated with chemical inducers of HIF-1alpha, cobalt chloride or desferrioxamine, and by transfecting untreated HepaRG cells with HIF-1alpha expression vector. In conclusion, HepaRG cells cultured under hypoxia might mimic metabolic changes occurring within poorly irrigated differentiated HCCs. Furthermore, hypoxia down-regulates hepatic DMEs, a phenomenon that might compromise chemotherapy effectiveness in HCC treatment. Thus, HepaRG cells might represent a new in vitro model to test anticancer agents in hypoxic versus normoxic conditions. PMID:19695866

  10. Effect of coadministration of ketoconazole, a strong CYP3A4 inhibitor, on pharmacokinetics and tolerability of motesanib diphosphate (AMG 706) in patients with advanced solid tumors.

    PubMed

    Lorusso, Patricia; Heath, Elisabeth I; McGreivy, Jesse; Sun, Yu-Nien; Melara, Rebeca; Yan, Lucy; Malburg, Lisa; Ingram, Megan; Wiezorek, Jeffrey; Chen, Li; Pilat, Mary Jo

    2008-10-01

    Motesanib diphosphate is a novel angiogenesis inhibitor selectively targeting vascular endothelial growth factor receptors 1, 2, and 3; platelet-derived growth factor receptor and stem cell factor receptor. The purpose of this phase 1b, drug-drug interaction study was to investigate the effect of ketoconazole, a strong inhibitor of the cytochrome P450 3A4 isoenzyme, on the pharmacokinetics and tolerability of motesanib diphosphate. Fourteen patients with advanced solid tumors refractory to standard treatment were enrolled and received motesanib diphosphate 50 mg once daily from day 1 through 15. Patients were randomized to receive a single oral dose of ketoconazole 400 mg either on day 8 (Sequence 1; n = 7) or day 15 (Sequence 2; n = 7), while pharmacokinetic samples were collected. After completion of this part (day 16), 13 patients received an escalated once-daily dose of motesanib diphosphate 125 mg. Evaluable pharmacokinetic data (n = 12) suggest that ketoconazole modestly increased motesanib exposure. The motesanib area under the concentration-time curve (AUC) from 0 to 24 h increased by 86% (90% CI, 1.50-2.29; P < 0.001) and the maximum plasma concentration (C (max)) by 35% (90% CI, 1.12-1.64; P = 0.02), compared with motesanib diphosphate administration alone. The tolerability profile (with or without ketoconazole coadministration) was consistent with that from other motesanib diphosphate monotherapy studies. Treatment-related adverse events were mild to moderate and commonly included fatigue (50% of patients), hypertension (43%), diarrhea (21%), dizziness (14%), paresthesia (14%), and vomiting (14%). Hypertension was the most common related grade 3 event (21%). No grade 4 or 5 treatment-related adverse events occurred. PMID:18574557

  11. Dietary effect on mixed function P450 1A2 activity assayed by estimation of caffeine metabolism in man.

    PubMed

    Kall, M A; Clausen, J

    1995-10-01

    Two studies were performed in order to evaluate cytochrome P450 1A2 mediated caffeine metabolism during different nutritional conditions. 1. In the first study, 23 healthy male non-smokers, mean age 25, changed from a customary mixed diet to a standard diet in 6 days. The 6 day's standard diet was based on bread, potatoes, rice and boiled meat. Thus, broccoli, cabbage and other cruciferous vegetables, spinach, leeks, onion, parsley, grapefruit, toasted bread, fried and charcoal grilled food, smoked fish and meat, ham and sausages were avoided. 2. In the second study, 33 healthy non-smoking subjects, 24 men and nine women mean age 25 years, volunteered. The study was designed to compare a customary home dietary period with the 6 day period of low dietary P450 induction and with a 5 day supplementary dietary period, i.e. ingestion of known dietary inducers. None of the women were using oral contraceptives or were pregnant during the experimental period. In the period of diet supplementation, the volunteers received charcoal grilled hamburger as a supplement to the standard low induction diet for lunch for 5 days. The hamburgers were made with 150 g beef (18-20% fat) and were grilled on charcoal for 10 min on each side until they were 'well done'. In the present study P450 1A2 activity was estimated from the caffeine metabolic ratio, the so-called CYP 1A2 index:(AFMU + 1-MX + 1-MU/ 17 -DMU) of the caffeine metabolites formed after oral ingestion of 200 mg caffeine. Urine was collected 4-8 h after caffeine ingestion in study 1 and 5 h after caffeine ingestion in study 2. In study 1 the CYP 1A2 index decreased from 4.28 +/- 0.98 in the customary home dietary period to 3.87 +/- 0.69 in the standard dietary period corresponding to 10.6% (P < 0.06) decrease in the CYP 1A2 index. In study 2 the CYP 1A2 index decreased from 4.47 +/- 1.76 in the customary home dietary period to 3.90 +/- 1.12 in the standard dietary period corresponding to a 14.6% decrease (P < 0.2) in P450 1A

  12. Piezoelectric crystal microbalance measurements of enthalpy of sublimation of C2-C9 dicarboxylic acids

    NASA Astrophysics Data System (ADS)

    Dirri, F.; Palomba, E.; Longobardo, A.; Zampetti, E.

    2015-07-01

    We present here a novel experimental setup able to measure the enthalpy of sublimation of a given compound by means of Piezoelectric Crystal Microbalances (PCM). This experiment was performed in the TG-Lab facility in IAPS-INAF, dedicated to the development of TGA sensors for space measurements, such as detection of organic and non-organic volatile species and refractory materials in planetary environments. In order to study physical-chemical processes concerning the Volatile Organic Compounds (VOC) present in atmospheric environments, the setup has been tested on Dicarboxylic acids. Acids with low molecular weight are among the components of organic fraction of particulate matter in the atmosphere, coming from different sources (biogenic and anthropogenic). Considering their relative abundance, it is useful to consider Dicarboxylic acid as "markers" to define the biogenic or anthropogenic origin of the aerosol, thus obtaining some information of the emission sources. In this work, a temperature controlled effusion cell was used to sublimate VOC, creating a molecular flux that was collimated onto a cold PCM. The VOC re-condensed onto the PCM quartz crystal allowing the determination of the deposition rate. From the measurements of deposition rates, it was possible to infer the enthalpy of sublimation of Adipic acid, i.e. Δ Hsub: 141.6 ± 0.8 kJ mol-1, Succinic acid, i.e. 113.3 ± 1.3 kJ mol-1, Oxalic acid, i.e. 62.5 ± 3.1 kJ mol-1 and Azelaic acid, i.e. 124.2 ± 1.2 kJ mol-1 (weight average values). The results obtained are in very good agreement with literature within 10 % for the Adipic, Succinic and Oxalic acid.

  13. Piezoelectric crystal microbalance measurements of enthalpy of sublimation of C2-C9 dicarboxylic acids

    NASA Astrophysics Data System (ADS)

    Dirri, F.; Palomba, E.; Longobardo, A.; Zampetti, E.

    2016-02-01

    We present here a novel experimental set-up that is able to measure the enthalpy of sublimation of a given compound by means of piezoelectric crystal microbalances (PCMs). The PCM sensors have already been used for space measurements, such as for the detection of organic and non-organic volatile species and refractory materials in planetary environments. In Earth atmospherics applications, PCMs can be also used to obtain some physical-chemical processes concerning the volatile organic compounds (VOCs) present in atmospheric environments. The experimental set-up has been developed and tested on dicarboxylic acids. In this work, a temperature-controlled effusion cell was used to sublimate VOC, creating a molecular flux that was collimated onto a cold PCM. The VOC recondensed onto the PCM quartz crystal, allowing the determination of the deposition rate. From the measurements of deposition rates, it has been possible to infer the enthalpy of sublimation of adipic acid, i.e. ΔHsub : 141.6 ± 0.8 kJ mol-1, succinic acid, i.e. 113.3 ± 1.3 kJ mol-1, oxalic acid, i.e. 62.5 ± 3.1 kJ mol-1, and azelaic acid, i.e. 124.2 ± 1.2 kJ mol-1. The results obtained show an accuracy of 1 % for succinic, adipic, and azelaic acid and within 5 % for oxalic acid and are in very good agreement with previous works (within 6 % for adipic, succinic, and oxalic acid and within 11 % or larger for azelaic acid).

  14. Gas-Phase Reactions of Atomic Gold Cations with Linear Alkanes (C2-C9).

    PubMed

    Zhang, Ting; Li, Zi-Yu; Zhang, Mei-Qi; He, Sheng-Gui

    2016-06-30

    To develop proper ionization methods for alkanes, the reactivity of bare or ligated transition metal ions toward alkanes has attracted increasing interests. In this study, the reactions of the gold cations with linear alkanes from ethane up to nonane (CnH2n+2, n = 2-9) under mild conditions have been characterized by mass spectrometry and density functional theory calculations. When reacting with Au(+), small alkanes (n = 2-6) were confirmed to follow specific reaction channels of dehydrogenation for ethane and hydride transfer for others to generate product ions characteristic of the original alkanes, which indicates that Au(+) can act as a reagent ion to ionize alkanes from ethane to n-hexane. Strong dependence of the chain length of alkanes was observed for the rate constants and reaction efficiencies. Extensive fragmentation took place for larger alkanes (n > 6). Theoretical results show that the fragmentation induced by the hydride transfer occurs after the release of AuH. Moreover, the fragmentation of n-heptane was successfully avoided when the reaction took place in a high-pressure reactor. This implies that Au(+) is a potential reagent ion to ionize linear and even the branched alkanes. PMID:27266670

  15. VKORC1 and CYP2C9 Gene Polymorphisms and Warfarin Management

    ClinicalTrials.gov

    2009-09-02

    Atrial Fibrillation; Cardiac Thrombus; Deep Vein Thrombosis; Pulmonary Embolism; Heart Valve Replacement (Mechanical or Biological With AF); Cardiomyopathy (Ischemic or Dilated); Peripheral Vascular Disease

  16. In vitro inhibitory effect of piperlonguminine isolated from Piper longum on human cytochrome P450 1A2.

    PubMed

    Song, Min; Hwang, Jae Yun; Lee, Min Young; Jee, Jun-Goo; Lee, You Mie; Bae, Jong-Sup; Kim, Jeong Ah; Lee, Seung Ho; Lee, Sangkyu

    2014-08-01

    Piperlonguminine (PL), a major alkaloid isolated from Piper longum fruits, shows several biological activities including anti-tumor, anti-hyperlipidemic and anti-inflammatory effects. Although there have been studies of the biological effects of PL, the potential drug-interaction effect of PL following evaluation of inhibitory effects of cytochrome P450 (CYP) activities was not investigated. Here, to investigate the inhibitory effects of PL on the activities of CYP isoforms, CYP inhibition assays were conducted using a cocktail of probe substrates in pooled human liver microsome (HLMs) and human recombinant cDNA-expressed CYP. PL strongly inhibited CYP1A2-mediated phenacetin O-deethylation with an IC50 value of 8.8 μM, as NADPH-independent inhibition, while other CYPs were not significantly inhibited. A Lineweaver-Burk plot resulted in the inhibition mechanism of PL being divided into two different modes, reversible competitive inhibition in a low concentration range of 0-16 μM with a Ki value of 1.39 μM and uncompetitive inhibitory behavior at a high concentration range of 16-40 μM. In addition, PL only decreased CYP 1A2-catalyzed phenacetin O-deethylase activity with IC50 values of 10.0 μM in human recombinant cDNA-expressed 1A2, not 1A1. Overall, this is the first investigation of potential herb-drug interactions associated with PL conducted by identifying the competitive inhibitory effects of PL on CYP1A2 in HLMs. PMID:24194261

  17. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2.

    PubMed

    Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M; Backes, Wayne L

    2015-12-01

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. PMID:26423927

  18. A1/A2-Diamino-Substituted Pillar[5]arene-Based Acid-Base-Responsive Host-Guest System.

    PubMed

    Hu, Wei-Bo; Hu, Wen-Jing; Zhao, Xiao-Li; Liu, Yahu A; Li, Jiu-Sheng; Jiang, Biao; Wen, Ke

    2016-05-01

    An acid-base-responsive supramolecular host-guest system based on a planarly chiral A1/A2-diamino-substituted pillar[5]arene (1)/imidazolium ion recognition motif was created. The pillar[4]arene[1]diaminobenzene 1 can bring an electron-deficient imidazolium cation into its cylindrically shaped cavity under neutral or basic conditions and release it under acidic conditions. PMID:27088317

  19. The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding.

    PubMed

    Parker, D'Andra N; Tasneem, Subia; Farndale, Richard W; Bihan, Dominique; Sadler, J Evan; Sebastian, Silvie; de Groot, Philip G; Hayward, Catherine P M

    2016-07-01

    Multimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbα binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates. PMID:27052467

  20. Effects of caffeine intake on the pharmacokinetics of melatonin, a probe drug for CYP1A2 activity

    PubMed Central

    Härtter, Sebastian; Nordmark, Anna; Rose, Dirk-Matthias; Bertilsson, Leif; Tybring, Gunnel; Laine, Kari

    2003-01-01

    Aims The aim of this study was to assess the influence of concomitant caffeine intake on the pharmacokinetics of oral melatonin, a probe drug for CYP1A2 activity. Methods Twelve healthy subjects, six smokers and six nonsmokers, were given melatonin (6 mg) either alone or in combination with caffeine (3 × 200 mg). Blood samples for the analysis of melatonin or caffeine and paraxanthine were taken from 1 h before until 6 h after intake of melatonin. Subjects were genotyped with respect to the CYP1A2*1F (C734A) polymorphism. Results When caffeine was coadministered the Cmax and AUC of melatonin were increased on average by 142% (P = 0.001, confidence interval on the difference 44, 80%) and 120% (P < 0.001, confidence interval on the difference 63, 178%), respectively. The inhibitory effect of caffeine was more pronounced in nonsmokers and in individuals with the *1F/*1F genotype. Conclusion The results of this study revealed a pronounced effect of caffeine on the bioavailability of orally given melatonin, most probably due to inhibition of CYP1A2 activity. PMID:14616429

  1. Association study of polymorphisms in the excitatory amino acid transporter 2 gene (SLC1A2) with schizophrenia

    PubMed Central

    Deng, Xiangdong; Shibata, Hiroki; Ninomiya, Hideaki; Tashiro, Nobutada; Iwata, Nakao; Ozaki, Norio; Fukumaki, Yasuyuki

    2004-01-01

    Background The glutamatergic dysfunction hypothesis of schizophrenia suggests that genes involved in glutametergic transmission are candidates for schizophrenic susceptibility genes. We have been performing systematic association studies of schizophrenia with the glutamate receptor and transporter genes. In this study we report an association study of the excitatory amino acid transporter 2 gene, SLC1A2 with schizophrenia. Methods We genotyped 100 Japanese schizophrenics and 100 controls recruited from the Kyushu area for 11 single nucleotide polymorphism (SNP) markers distributed in the SLC1A2 region using the direct sequencing and pyrosequencing methods, and examined allele, genotype and haplotype association with schizophrenia.The positive finding observed in the Kyushu samples was re-examined using 100 Japanese schizophrenics and 100 controls recruited from the Aichi area. Results We found significant differences in genotype and allele frequencies of SNP2 between cases and controls (P = 0.013 and 0.008, respectively). After Bonferroni corrections, the two significant differences disappeared. We tested haplotype associations for all possible combinations of SNP pairs. SNP2 showed significant haplotype associations with the disease (P = 9.4 × 10-5, P = 0.0052 with Bonferroni correction, at the lowest) in 8 combinations. Moreover, the significant haplotype association of SNP2-SNP7 was replicated in the cumulative analysis of our two sample sets. Conclusion We concluded that at least one susceptibility locus for schizophrenia is probably located within or nearby SLC1A2 in the Japanese population. PMID:15296513

  2. Sequence variants at CYP1A1–CYP1A2 and AHR associate with coffee consumption

    PubMed Central

    Sulem, Patrick; Gudbjartsson, Daniel F.; Geller, Frank; Prokopenko, Inga; Feenstra, Bjarke; Aben, Katja K.H.; Franke, Barbara; den Heijer, Martin; Kovacs, Peter; Stumvoll, Michael; Mägi, Reedik; Yanek, Lisa R.; Becker, Lewis C.; Boyd, Heather A.; Stacey, Simon N.; Walters, G. Bragi; Jonasdottir, Adalbjorg; Thorleifsson, Gudmar; Holm, Hilma; Gudjonsson, Sigurjon A.; Rafnar, Thorunn; Björnsdottir, Gyda; Becker, Diane M.; Melbye, Mads; Kong, Augustine; Tönjes, Anke; Thorgeirsson, Thorgeir; Thorsteinsdottir, Unnur; Kiemeney, Lambertus A.; Stefansson, Kari

    2011-01-01

    Coffee is the most commonly used stimulant and caffeine is its main psychoactive ingredient. The heritability of coffee consumption has been estimated at around 50%. We performed a meta-analysis of four genome-wide association studies of coffee consumption among coffee drinkers from Iceland (n = 2680), the Netherlands (n = 2791), the Sorbs Slavonic population isolate in Germany (n = 771) and the USA (n = 369) using both directly genotyped and imputed single nucleotide polymorphisms (SNPs) (2.5 million SNPs). SNPs at the two most significant loci were also genotyped in a sample set from Iceland (n = 2430) and a Danish sample set consisting of pregnant women (n = 1620). Combining all data, two sequence variants significantly associated with increased coffee consumption: rs2472297-T located between CYP1A1 and CYP1A2 at 15q24 (P = 5.4 · 10−14) and rs6968865-T near aryl hydrocarbon receptor (AHR) at 7p21 (P = 2.3 · 10−11). An effect of ∼0.2 cups a day per allele was observed for both SNPs. CYP1A2 is the main caffeine metabolizing enzyme and is also involved in drug metabolism. AHR detects xenobiotics, such as polycyclic aryl hydrocarbons found in roasted coffee, and induces transcription of CYP1A1 and CYP1A2. The association of these SNPs with coffee consumption was present in both smokers and non-smokers. PMID:21357676

  3. Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters.

    PubMed

    Vermeer, Lydia M M; Isringhausen, Caleb D; Ogilvie, Brian W; Buckley, David B

    2016-03-01

    Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics. PMID:26668209

  4. Amine-free melanin-concentrating hormone receptor 1 antagonists: Novel 1-(1H-benzimidazol-6-yl)pyridin-2(1H)-one derivatives and design to avoid CYP3A4 time-dependent inhibition.

    PubMed

    Igawa, Hideyuki; Takahashi, Masashi; Shirasaki, Mikio; Kakegawa, Keiko; Kina, Asato; Ikoma, Minoru; Aida, Jumpei; Yasuma, Tsuneo; Okuda, Shoki; Kawata, Yayoi; Noguchi, Toshihiro; Yamamoto, Syunsuke; Fujioka, Yasushi; Kundu, Mrinalkanti; Khamrai, Uttam; Nakayama, Masaharu; Nagisa, Yasutaka; Kasai, Shizuo; Maekawa, Tsuyoshi

    2016-06-01

    Melanin-concentrating hormone (MCH) is an attractive target for antiobesity agents, and numerous drug discovery programs are dedicated to finding small-molecule MCH receptor 1 (MCHR1) antagonists. We recently reported novel pyridine-2(1H)-ones as aliphatic amine-free MCHR1 antagonists that structurally featured an imidazo[1,2-a]pyridine-based bicyclic motif. To investigate imidazopyridine variants with lower basicity and less potential to inhibit cytochrome P450 3A4 (CYP3A4), we designed pyridine-2(1H)-ones bearing various less basic bicyclic motifs. Among these, a lead compound 6a bearing a 1H-benzimidazole motif showed comparable binding affinity to MCHR1 to the corresponding imidazopyridine derivative 1. Optimization of 6a afforded a series of potent thiophene derivatives (6q-u); however, most of these were found to cause time-dependent inhibition (TDI) of CYP3A4. As bioactivation of thiophenes to form sulfoxide or epoxide species was considered to be a major cause of CYP3A4 TDI, we introduced electron withdrawing groups on the thiophene and found that a CF3 group on the ring or a Cl adjacent to the sulfur atom helped prevent CYP3A4 TDI. Consequently, 4-[(5-chlorothiophen-2-yl)methoxy]-1-(2-cyclopropyl-1-methyl-1H-benzimidazol-6-yl)pyridin-2(1H)-one (6s) was identified as a potent MCHR1 antagonist without the risk of CYP3A4 TDI, which exhibited a promising safety profile including low CYP3A4 inhibition and exerted significant antiobesity effects in diet-induced obese F344 rats. PMID:27112449

  5. Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

    PubMed

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J; Liu, Yi; Burlingame, A L; Correia, Maria Almira

    2015-02-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  6. Kinetics of electron transfer in the complex of cytochrome P450 3A4 with the flavin domain of cytochrome P450BM-3 as evidence of functional heterogeneity of the heme protein

    PubMed Central

    Fernando, Harshica; Halpert, James R.; Davydov, Dmitri R.

    2008-01-01

    We used a rapid scanning stop-flow technique to study the kinetics of reduction of cytochrome P450 3A4 (CYP3A4) by the flavin domain of cytochrome P450-BM3 (BMR), which was shown to form a stoichiometric complex (KD = 0.48 µM) with CYP3A4. In the absence of substrates only about 50% of CYP3A4 was able to accept electrons from BMR. Whereas the high-spin fraction was completely reducible, the reducibility of the low-spin fraction did not exceed 42%. Among four substrates tested (testosterone, 1-pyrenebutanol, bromocriptine, or α-naphthoflavone (ANF)) only ANF is capable of increasing the reducibility of the low-spin fraction to 75%. Our results demonstrate that the pool of CYP3A4 is heterogeneous, and not all P450 is competent for electron transfer in the complex with reductase. The increase in the reducibility of the enzyme in the presence of ANF may represent an important element of the mechanism of action of this activator. PMID:18086551

  7. ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease

    PubMed Central

    Friedrich, Thomas; Tavraz, Neslihan N.; Junghans, Cornelia

    2016-01-01

    Mutations in four genes have been identified in familial hemiplegic migraine (FHM), from which CACNA1A (FHM type 1) and SCN1A (FHM type 3) code for neuronal voltage-gated calcium or sodium channels, respectively, while ATP1A2 (FHM type 2) encodes the α2 isoform of the Na+,K+-ATPase's catalytic subunit, thus classifying FHM primarily as an ion channel/ion transporter pathology. FHM type 4 is attributed to mutations in the PRRT2 gene, which encodes a proline-rich transmembrane protein of as yet unknown function. The Na+,K+-ATPase maintains the physiological gradients for Na+ and K+ ions and is, therefore, critical for the activity of ion channels and transporters involved neuronal excitability, neurotransmitter uptake or Ca2+ signaling. Strikingly diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations which frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities, but some mutations are truly deleterious for enzyme function and thus cause full haploinsufficiency. Here, we summarize structural and functional data about the Na+,K+-ATPase available to date and an overview is provided about the particular properties of the α2 isoform that explain its physiological relevance in electrically excitable tissues. In addition, current concepts about the neurobiology of migraine, the correlations between primary brain dysfunction and mechanisms of headache pain generation are described, together with insights gained recently from modeling approaches in computational neuroscience. Then, a survey is given about ATP1A2 mutations implicated in migraine cases as documented in the literature with focus on mutations that were described to completely destroy enzyme function, or lead to misfolded or mistargeted protein in particular model cell lines. We also discuss whether or not there are correlations between these most severe mutational effects and clinical phenotypes. Finally, perspectives

  8. ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease.

    PubMed

    Friedrich, Thomas; Tavraz, Neslihan N; Junghans, Cornelia

    2016-01-01

    Mutations in four genes have been identified in familial hemiplegic migraine (FHM), from which CACNA1A (FHM type 1) and SCN1A (FHM type 3) code for neuronal voltage-gated calcium or sodium channels, respectively, while ATP1A2 (FHM type 2) encodes the α2 isoform of the Na(+),K(+)-ATPase's catalytic subunit, thus classifying FHM primarily as an ion channel/ion transporter pathology. FHM type 4 is attributed to mutations in the PRRT2 gene, which encodes a proline-rich transmembrane protein of as yet unknown function. The Na(+),K(+)-ATPase maintains the physiological gradients for Na(+) and K(+) ions and is, therefore, critical for the activity of ion channels and transporters involved neuronal excitability, neurotransmitter uptake or Ca(2+) signaling. Strikingly diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations which frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities, but some mutations are truly deleterious for enzyme function and thus cause full haploinsufficiency. Here, we summarize structural and functional data about the Na(+),K(+)-ATPase available to date and an overview is provided about the particular properties of the α2 isoform that explain its physiological relevance in electrically excitable tissues. In addition, current concepts about the neurobiology of migraine, the correlations between primary brain dysfunction and mechanisms of headache pain generation are described, together with insights gained recently from modeling approaches in computational neuroscience. Then, a survey is given about ATP1A2 mutations implicated in migraine cases as documented in the literature with focus on mutations that were described to completely destroy enzyme function, or lead to misfolded or mistargeted protein in particular model cell lines. We also discuss whether or not there are correlations between these most severe mutational effects and clinical phenotypes

  9. Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

    PubMed

    Sato, Kenjiro; Takahagi, Hiroki; Kubo, Osamu; Hidaka, Kousuke; Yoshikawa, Takeshi; Kamaura, Masahiro; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Maki, Toshiyuki; Take, Kazumi; Takekawa, Shiro; Kitazaki, Tomoyuki; Maekawa, Tsuyoshi

    2015-08-01

    Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts. PMID:26100443

  10. Adaptations for the Oxidation of Polycyclic Aromatic Hydrocarbons Exhibited By the Structure of Human 450 1a2

    SciTech Connect

    Sansen, S.; Yano, J.K.; Reynald, R.L.; Schoch, G.A.; Griffin, K.J.; Stout, C.D.; Johnson, E.F.

    2007-07-12

    Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor alpha-naphthoflavone, determined to a resolution of 1.95 A. alpha-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members.

  11. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  12. Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers

    PubMed Central

    Simon, T; Becquemont, L; Hamon, B; Nouyrigat, E; Chodjania, Y; Poirier, J M; Funck-Brentano, C; Jaillon, P

    2001-01-01

    Aims To assess the age-associated changes over time of plasma paraxanthine/caffeine (PAX/CAF) ratios used as a probe for CYP1A2 activity. Methods Intraindividual and interindividual variabilities in PAX/CAF ratio were compared by phenotyping with caffeine, 16 young and 16 elderly healthy subjects on five occasions. Results PAX/CAF ratio variability was comparable regardless of age (intraindividual CV: 17.6 ± 6% and 16.2 ± 5.9%, interindividual CV: 48.1 ± 2.9% and 42.7 ± 3.6% in young and elderly, respectively). The PAX/CAF ratio was lower in elderly than in young subjects (95% CI for the difference: 0.004, 0.32) but the difference was not significant in nonsmokers compared separately. Conclusions The variability over time of the PAX/CAF ratio is not influenced by age. PMID:11736870

  13. Effects of cytokines on CYP3A4 expression and reversal of the effects by anti-cytokine agents in the three-dimensionally cultured human hepatoma cell line FLC-4.

    PubMed

    Mimura, Hanaka; Kobayashi, Kaoru; Xu, Linxiaoqing; Hashimoto, Mari; Ejiri, Yoko; Hosoda, Masaya; Chiba, Kan

    2015-02-01

    The expression of hepatic cytochrome P450 (CYP) enzymes is altered under pathological conditions with increased levels of cytokines. In this study, we analyzed the effects of cytokines (interleukin [IL]-1β, IL-6 and tumor necrosis factor α) on the expression of CYP3A4 using newly introduced three-dimensionally cultured human hepatocarcinoma FLC-4 cells. The mRNA level of CYP3A4 was significantly decreased by IL-1β, IL-6 and tumor necrosis factor-α. Formation of α-hydroxytriazolam catalyzed by CYP3A was decreased by IL-1β and IL-6. Pre-treatment with IL-6 enhanced the cytotoxic effects of gefitinib and paclitaxel. In addition, tocilizumab and IL-1 receptor antagonist restored the decreased expression of CYP3A4 mRNA by IL-6 and IL-1β, respectively. These results obtained by using three-dimensionally cultured FLC-4 cells are consistent with results obtained by using primary human hepatocytes and results of clinical studies. Therefore, three-dimensionally cultured FLC-4 cell system may be a promising cellular tool to assess the effects of cytokines on CYP3A4 expression. PMID:25760537

  14. Hyperconjugation with lone pair of morpholine nitrogen stabilizes transition state for phenyl hydroxylation in CYP3A4 metabolism of ( S)- N-[1-(3-morpholin-4-yl phenyl) ethyl]-3-phenylacrylamide

    NASA Astrophysics Data System (ADS)

    Shaikh, Abdul Rajjak; Broclawik, Ewa; Ismael, Mohamed; Tsuboi, Hideyuki; Koyama, Michihisa; Kubo, Momoji; Del Carpio, Carlos A.; Miyamoto, Akira

    2006-02-01

    Using quantum chemical modelling we describe a novel effect in the mechanism of CYP3A4 metabolism for the arene substrate with o-substituent yielding a lone pair donation to conjugate π system; this will compensate for the loss of aromaticity on formation of the tetrahedral complex and lower the rate-determining energy barrier.

  15. Variable Bone Fragility Associated With an Amish COL1A2 Variant and a Knock-in Mouse Model

    PubMed Central

    Daley, Ethan; Streeten, Elizabeth A; Sorkin, John D; Kuznetsova, Natalia; Shapses, Sue A; Carleton, Stephanie M; Shuldiner, Alan R; Marini, Joan C; Phillips, Charlotte L; Goldstein, Steven A; Leikin, Sergey; McBride, Daniel J

    2010-01-01

    Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z-scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F1 lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI. © 2010 American Society for Bone and Mineral Research PMID:19594296

  16. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation

    PubMed Central

    Capoferri, Luigi; Verkade-Vreeker, Marlies C. A.; Buitenhuis, Danny; Commandeur, Jan N. M.; Pastor, Manuel; Vermeulen, Nico P. E.; Geerke, Daan P.

    2015-01-01

    Prediction of human Cytochrome P450 (CYP) binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD) simulations and Linear Interaction Energy (LIE) theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE) of 4.1 kJ mol-1 and a standard error in prediction (SDEP) in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units). PMID:26551865

  17. Modulatory effects of extracts of vinegar-baked Radix Bupleuri and saikosaponins on the activity of cytochrome P450 enzymes in vitro.

    PubMed

    Yu, Tongya; Chen, Xianzhi; Wang, Yinjie; Zhao, Ruizhi; Mao, Shirui

    2014-10-01

    1. In this article, the modulatory effects of extracts from vinegar-baked Radix Bupleuri (VBRB) and saikosaponins on the activity of CYP1A2, CYP2C9 and CYP3A4 were investigated in vitro. 2. Microsomal in vitro incubation method was utilized to simulate metabolic reaction under physiological environment by incubating the marker with liver microsomes in the absence or presence of VBRB and saikosaponins. The contents of 4-acetamidophenol, 6β-hydroxyltestosterone and 4-hydroxydiclofenac, the metabolites of phenacetin, testosterone and diclofenac, which were selected as specific probe drugs of CYP1A2, CYP2C9 and CYP3A4, respectively, were analyzed by high-performance liquid chromatography. 3. The production of the metabolites was incubation time dependent. The modulatory effects of different VBRB extracts and saikosaponins on CYP isoforms increased with concentration. Among all the extracts studied, BC1 has a strong inhibition effect compared to the three CYP isoforms tested, while the others have only significant inhibition on the activity of CYP2C9. 4. This in vitro study demonstrated that various extracts of VBRB tested in this study have negligible potential to interfere with CYP1A2- and CYP3A4-metabolized drugs; risk of herb-drug interaction might occur when VBRB is concurrently taken with CYP2C9 substrates. PMID:24779639

  18. Studies on the Role of Metabolic Activation in Tyrosine Kinase Inhibitor–Dependent Hepatotoxicity: Induction of CYP3A4 Enhances the Cytotoxicity of Lapatinib in HepaRG Cells

    PubMed Central

    Hardy, Klarissa D.; Wahlin, Michelle D.; Papageorgiou, Ioannis; Unadkat, Jashvant D.; Nelson, Sidney D.

    2014-01-01

    Idiosyncratic hepatotoxicity has been associated with the oral tyrosine kinase inhibitor lapatinib, which is used in metastatic breast cancer therapy. Lapatinib is extensively metabolized by cytochrome P450 3A4/5 to yield an O-debenzylated metabolite, which can undergo further oxidation to a reactive quinone imine. A recent clinical study reported that concomitant use of lapatinib with dexamethasone increased the incidence of hepatotoxicity in metastatic breast cancer patients treated with lapatinib, and so we hypothesized that induction of CYP3A enhances the bioactivation of lapatinib to reactive intermediates that contribute to hepatotoxicity. Therefore, we examined the effect of CYP3A4 induction on the cytotoxicity and metabolism of lapatinib in the HepaRG human hepatic cell line. Differentiated HepaRG cells were pretreated with dexamethasone (100 μM) or the prototypical CYP3A4 inducer rifampicin (4 μM) for 72 hours, followed by incubation with lapatinib (0–100 μM) for 24 hours. Cell viability was monitored using WST-1 assays, and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry. Induction of CYP3A4 by dexamethasone or rifampicin enhanced lapatinib-induced cytotoxicity, compared with treatment with lapatinib alone. A direct comparison of the cytotoxicity of lapatinib versus O-debenzylated lapatinib demonstrated that the O-debenzylated metabolite was significantly more cytotoxic than lapatinib itself. Furthermore, pretreatment with 25 μM l-buthionine sulfoximine to deplete intracellular glutathione markedly enhanced lapatinib cytotoxicity. Cytotoxicity was correlated with increased formation of O-debenzylated lapatinib and cysteine adducts of the putative quinone imine intermediate. Collectively, these data suggest that CYP3A4 induction potentiates lapatinib-induced hepatotoxicity via increased reactive metabolite formation. PMID:24191259

  19. Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.

    PubMed

    Palma, Bernardo Brito; Moutinho, Daniela; Urban, Philippe; Rueff, José; Kranendonk, Michel

    2016-08-01

    Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level. PMID:27476332

  20. Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

    PubMed

    Lonsdale, Richard; Fort, Rachel M; Rydberg, Patrik; Harvey, Jeremy N; Mulholland, Adrian J

    2016-06-20

    The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism. PMID:27064685

  1. 13C-methacetin breath test reproducibility study reveals persistent CYP1A2 stimulation on repeat examinations

    PubMed Central

    Kasicka-Jonderko, Anna; Nita, Anna; Jonderko, Krzysztof; Kamińska, Magdalena; Błońska-Fajfrowska, Barbara

    2011-01-01

    AIM: To find the most reproducible quantitative parameter of a standard 13C-methacetin breath test (13C-MBT). METHODS: Twenty healthy volunteers (10 female, 10 male) underwent the 13C-MBT after intake of 75 mg 13C-methacetin p.o. on three occasions. Short- and medium-term reproducibility was assessed with paired examinations taken at an interval of 2 and 18 d (medians), respectively. RESULTS: The reproducibility of the 1-h cumulative 13C recovery (AUC0-60), characterized by a coefficient of variation of 10%, appeared to be considerably better than the reproducibility of the maximum momentary 13C recovery or the time of reaching it. Remarkably, as opposed to the short gap between consecutive examinations, the capacity of the liver to handle 13C-methacetin increased slightly but