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Sample records for cyp3a enzyme activity

  1. 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.

  2. Expression and Activity of CYP3A Enzymes in the Liver of Piglets Fed Dairy- or Soy-Based Formula in Comparison to Breast Feeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have published previous data showing that feeding soy protein isolate, the major protein source in soy-infant formula, to rats during early development results in increased expression and activity of the major liver enzyme involved in breakdown and removal of pediatric medications, CYP3A. This s...

  3. In vitro and pharmacophore insights into CYP3A enzymes.

    PubMed

    Ekins, Sean; Stresser, David M; Williams, J Andrew

    2003-04-01

    The cytochrome P450 3A (CYP3A) enzymes have a major role in the metabolism of drugs in humans. Their wide substrate specificity and induction by a vast array of structurally diverse compounds presents the possibility of metabolic drug-drug interactions. Understanding the enzymes themselves is crucial. Over the past decade, this has occurred mostly with in vitro studies, although more recent approaches incorporate computational models to predict CYP inhibition and substrate potential. The three-dimensional displacement, or pharmacophore, of chemical features in space that are derived from inhibition data have produced pharmacophores for CYP3A4, CYP3A5 and CYP3A7, and provide new insights into ligand binding for each enzyme. PMID:12707001

  4. 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

  5. Inhibitory effects of fruit juices on CYP3A activity.

    PubMed

    Kim, Hyunmi; Yoon, Yune-Jung; Shon, Ji-Hong; Cha, In-June; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2006-04-01

    There have been very limited reports on the effects of commercial fruit juices on human CYP3A activity. Therefore, the inhibitory effects of readily available commercial fruit juices on midazolam 1'-hydroxylase activity, a marker of CYP3A, were evaluated in pooled human liver microsomes. The fruit juices investigated were black raspberry, black mulberry, plum, and wild grape. White grapefruit, pomegranate, and orange juice were used as positive and negative controls. The black mulberry juice showed the most potent inhibition of CYP3A except for grapefruit juice. The inhibition depended on the amount of a fruit juice added to the incubation mixture. The inhibitory potential of human CYP3A was in the order: grapefruit > black mulberry > wild grape > pomegranate > black raspberry. The IC(50) values of all fruit juices tested were reduced after preincubation with microsomes in the presence of the NADPH-generating system, suggesting that a mechanism-based inhibitory component was present in these fruit juices, as in the case of grapefruit. The results suggest that, like grapefruit juice, commercial fruit juices also have the potential to inhibit CYP3A-catalzyed midazolam 1'-hydroxylation. Therefore, in vivo studies investigating the interactions between fruit juices such as black mulberry and wild grape and CYP3A substrates are necessary to determine whether inhibition of CYP3A activity by fruit juices is clinically relevant. PMID:16415112

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. CYP3A Specifically Catalyzes 1β-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.

    PubMed

    Hayes, Martin A; Li, Xue-Qing; Grönberg, Gunnar; Diczfalusy, Ulf; Andersson, Tommy B

    2016-09-01

    The endogenous bile acid metabolite 1β-hydroxy-deoxycholic acid (1β-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1β-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1β-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1β-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1β-OH-DCA formation. This indicated that 1β-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1β-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1β-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1β-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1β-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1β-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies. PMID:27402728

  12. Modulation of CYP3a expression and activity in mice models of type 1 and type 2 diabetes

    PubMed Central

    Patoine, Dany; Petit, Michaël; Pilote, Sylvie; Picard, Frédéric; Drolet, Benoit; Simard, Chantale

    2014-01-01

    CYP3A4, the most abundant cytochrome P450 enzyme in the human liver and small intestine, is responsible for the metabolism of about 50% of all marketed drugs. Numerous pathophysiological factors, such as diabetes and obesity, were shown to affect CYP3A activity. Evidences suggest that drug disposition is altered in type 1 (T1D) and type 2 diabetes (T2D). The objective was to evaluate the effect of T1D and T2D on hepatic and intestinal CYP3a drug-metabolizing activity/expression in mice. Hepatic and intestinal microsomes were prepared from streptozotocin-induced T1D, db/db T2D and control mice. Domperidone was selected as a probe substrate for CYP3a and formation of five of its metabolites was evaluated using high performance liquid chromatography. Hepatic CYP3a protein and mRNA expression were assessed by Western blot and reverse-transcription quantitative polymerase chain reaction respectively. Hepatic microsomal CYP3a activity was significantly increased in both T1D and T2D groups versus control group. Intestinal CYP3a activity was also significantly increased in both T1D and T2D groups. Moreover, significant increases of both hepatic CYP3a mRNAs and protein expression were observed in both T1D and T2D groups versus control group. Additional experiments with testosterone further validated the increased activity of CYP3a under the effect of both T1D and T2D. Although differences exist in the pathophysiological insults associated with T1D and T2D, our results suggest that these two distinct diseases may have the same modulating effect on the regulation of CYP3a, ultimately leading to variability in drug response, ranging from lack of effect to life-threatening toxicity. PMID:25505621

  13. Metabolic pathways of inhaled glucocorticoids by the CYP3A enzymes.

    PubMed

    Moore, Chad D; Roberts, Jessica K; Orton, Christopher R; Murai, Takahiro; Fidler, Trevor P; Reilly, Christopher A; Ward, Robert M; Yost, Garold S

    2013-02-01

    Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ(6)-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ(6)-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography-mass spectrometry and NMR analysis. PMID:23143891

  14. Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

    PubMed Central

    Moore, Chad D.; Roberts, Jessica K.; Orton, Christopher R.; Murai, Takahiro; Fidler, Trevor P.; Reilly, Christopher A.; Ward, Robert M.

    2013-01-01

    Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and Δ6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of Δ6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis. PMID:23143891

  15. The distribution and gender difference of CYP3A activity in Chinese subjects

    PubMed Central

    Zhu, Bing; Liu, Zhao-Qian; Chen, Guo-Lin; Chen, Xiao-Ping; Ou-Yang, Dong-Sheng; Wang, Lian-Sheng; Huang, Song-Lin; Tan, Zhi-Rong; Zhou, Hong-Hao

    2003-01-01

    Aims To investigate the distribution of CYP3A activity in the Chinese population, and to test for gender-related differences in CYP3A activity. Methods Using midazolam as a probe drug, CYP3A activity in 202 Chinese healthy subjects (104 men) was measured by plasma 1′-hydroxymidazolam:midazolam (1′-OH-MDZ:MDZ) ratio at 1 h after oral administration of 7.5 mg midazolam. The different phases of the menstrual cycle including preovulatory, ovulatory and luteal phases of 66 women phenotyped with midazolam were recorded. The concentrations of 1′-OH-MDZ and MDZ in plasma were measured by HPLC Results A 13-fold variation of CYP3A activity (log1′-OH-MDZ:MDZ: range −0.949–0.203) was shown. The CYP3A activity was normally distributed as indicated by the frequency distribution histogram, the probit plot and the Kolmogorov–Smirnov test (P > 0.05). The CYP3A activity of women was higher than that of men (median: −0.36 vs −0.43, P < 0.05; 95% CI for difference: −0.127, −0.012). There was a significant difference in CYP3A activity between the three phases of the menstrual cycle. The activity was highest in the preovulatory phase and decreased sequentially in the ovulatory and luteal phases (P < 0.05). Conclusions A normal distribution of CYP3A activity was observed in the Chinese population. The CYP3A activity is higher in female subjects than in males. CYP3A activity differed across the phases of the menstrual cycle. PMID:12630976

  16. 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

  17. Pinelliae rhizoma, a toxic chinese herb, can significantly inhibit CYP3A activity in rats.

    PubMed

    Wu, Jinjun; Cheng, Zaixing; He, Shugui; Shi, Jian; Liu, Shuqiang; Zhang, Guiyu; Zhu, Lijun; Liu, Liang; Liu, Zhongqiu; Lin, Na; Lu, Linlin

    2015-01-01

    Raw Pinelliae Rhizoma (RPR) is a representative toxic herb that is widely used for eliminating phlegm or treating cough and vomiting. Given its irritant toxicity, its processed products, including Pinelliae Rhizoma Praeparatum (PRP) and Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRPZA), are more commonly applied and administered concomitantly with other chemical drugs, such as cough medications. This study aimed to investigate the effects of RPR, PRP, and PRPZA on CYP3A activity. Testosterone (Tes) and buspirone (BP) were used as specific probe substrates ex vivo and in vivo, respectively. CYP3A activity was determined by the metabolite formation ratios from the substrates. Ex vivo results show that the metabolite formation ratios from Tes significantly decreased, indicating that RPR, PRP, and PRPZA could inhibit CYP3A activity in rats. CYP3A protein and mRNA levels were determined to explore the underlying mechanism. These levels showed marked and consistent down-regulation with CYP3A activity. A significant decrease in metabolite formation ratios from BP was also found in PRPZA group in vivo, implying that PRPZA could inhibit CYP3A activity. Conclusively, co-administration of PR with other CYP3A-metabolizing drugs may cause drug-drug interactions. Clinical use of PR-related formulae should be monitored carefully to avoid adverse interactions. PMID:25574821

  18. 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

  19. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro. PMID:15155547

  20. The effects of CYP2D6 and CYP3A activities on the pharmacokinetics of immediate release oxycodone

    PubMed Central

    Samer, CF; Daali, Y; Wagner, M; Hopfgartner, G; Eap, CB; Rebsamen, MC; Rossier, MF; Hochstrasser, D; Dayer, P; Desmeules, JA

    2010-01-01

    Background and purpose: There is high interindividual variability in the activity of drug-metabolizing enzymes catalysing the oxidation of oxycodone [cytochrome P450 (CYP) 2D6 and 3A], due to genetic polymorphisms and/or drug–drug interactions. The effects of CYP2D6 and/or CYP3A activity modulation on the pharmacokinetics of oxycodone remains poorly explored. Experimental approach: A randomized crossover double-blind placebo-controlled study was performed with 10 healthy volunteers genotyped for CYP2D6 [six extensive (EM), two deficient (PM/IM) and two ultrarapid metabolizers (UM)]. The volunteers randomly received on five different occasions: oxycodone 0.2 mg·kg−1 and placebo; oxycodone and quinidine (CYP2D6 inhibitor); oxycodone and ketoconazole (CYP3A inhibitor); oxycodone and quinidine+ketoconazole; placebo. Blood samples for plasma concentrations of oxycodone and metabolites (oxymorphone, noroxycodone and noroxymorphone) were collected for 24 h after dosing. Phenotyping for CYP2D6 (with dextromethorphan) and CYP3A (with midazolam) were assessed at each session. Key results: CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs and Cmax (−0.71 < Spearman correlation coefficient ρs < −0.92). Oxymorphone Cmax was 62% and 75% lower in PM than EM and UM. Noroxymorphone Cmax reduction was even more pronounced (90%). In UM, oxymorphone and noroxymorphone concentrations increased whereas noroxycodone exposure was halved. Blocking CYP2D6 (with quinidine) reduced oxymorphone and noroxymorphone Cmax by 40% and 80%, and increased noroxycodone AUC∞ by 70%. Blocking CYP3A4 (with ketoconazole) tripled oxymorphone AUC∞ and reduced noroxycodone and noroxymorphone AUCs by 80%. Shunting to CYP2D6 pathway was observed after CYP3A4 inhibition. Conclusions and implications: Drug–drug interactions via CYP2D6 and CYP3A affected oxycodone pharmacokinetics and its magnitude depended on CYP2D6 genotype. PMID:20590587

  1. Labeled Content of Two Furanocoumarins in Dietary Supplements Correlates with neither Actual Content nor CYP3A Inhibitory Activity

    PubMed Central

    VanderMolen, Karen M.; Ainslie, Garrett R.; Paine, Mary F.; Oberlies, Nicholas H.

    2014-01-01

    Dietary supplements are a multi-billion dollar business, with yearly profit increases. Allegedly safe, these supplements are marketed to a variety of niches, encompassing claims from immune support to weight loss. Six sports nutrition supplements were acquired that were labeled to contain the furanocoumarin(s) bergamottin and/or 6′,7′-dihydroxybergamottin (DHB), both of which are potent irreversible inhibitors of the prominent drug metabolizing enzyme cytochrome P450 3A (CYP3A). Both furanocoumarins are typically present in grapefruit juice, which has been shown to inhibit intestinal CYP3A, perpetrating an increase in the systemic exposure of certain concomitant ‘victim’ drugs. The acquired supplements were analyzed using ultra-performance liquid chromatography coupled to both a photodiode array (PDA) detector and a triple quadrupole mass spectrometer (MS). Contrary to the product labeling, four of the supplements contained no detectable quantities of either furanocoumarin (LOD 0.060 μg/capsule), while two of the supplements contained minimal amounts (one contained 12.13 (± 0.23) μg bergamottin and 65.51 (± 0.64) μg DHB per capsule; the other contained 2.705 (± 0.069) μg bergamottin per capsule and no detectable quantities of DHB). A CYP3A inhibition bioassay was used to assess whether the actual content of the furanocoumarins correlated with CYP3A inhibitory activity. Despite the low amounts of bergamottin and DHB, CYP3A inhibition by the supplements was greater than could be accounted for by the two furanocoumarins. The additional activity suggests the presence of other potent or highly abundant CYP3A inhibitors. PMID:24951959

  2. Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

    PubMed Central

    Dong, Pei-pei; Fang, Zhong-ze; Zhang, Yan-yan; Ge, Guang-bo; Mao, Yu-xi; Zhu, Liang-liang; Qu, Yan-qing; Li, Wei; Wang, Li-ming; Liu, Chang-xiao; Yang, Ling

    2011-01-01

    Aim: To ascertain the effects of erlotinib on CYP3A, to investigate the amplitude and kinetics of erlotinib-mediated inhibition of seven major CYP isoforms in human liver microsomes (HLMs) for evaluating the magnitude of erlotinib in drug-drug interaction in vivo. Methods: The activities of 7 major CYP isoforms (CYP1A2, CYP2A6, CYP3A, CYP2C9, CYP2D6, CYP2C8, and CYP2E1) were assessed in HLMs using HPLC or UFLC analysis. A two-step incubation method was used to examine the time-dependent inhibition of erlotinib on CYP3A. Results: The activity of CYP2C8 was inhibited with an IC50 value of 6.17±2.0 μmol/L. Erlotinib stimulated the midazolam 1′-hydroxy reaction, but inhibited the formation of 6β-hydroxytestosterone and oxidized nifedipine. Inhibition of CYP3A by erlotinib was substrate-dependent: the IC50 values for inhibiting testosterone 6β-hydroxylation and nifedipine metabolism were 31.3±8.0 and 20.5±5.3 μmol/L, respectively. Erlotinib also exhibited the time-dependent inhibition on CYP3A, regardless of the probe substrate used: the value of KI and kinact were 6.3 μmol/L and 0.035 min−1 for midazolam; 9.0 μmol/L and 0.045 min−1 for testosterone; and 10.1 μmol/L and 0.058 min−1 for nifedipine. Conclusion: The inhibition of CYP3A by erlotinib was substrate-dependent, while its time-dependent inhibition on CYP3A was substrate-independent. The time-dependent inhibition of CYP3A may be a possible cause of drug-drug interaction, suggesting that attention should be paid to the evaluation of erlotinib's safety, especially in the context of combination therapy. PMID:21372830

  3. Hepatic and intestinal CYP3A expression and activity in broilers.

    PubMed

    Osselaere, A; De Bock, L; Eeckhaut, V; De Backer, P; Van Bocxlaer, J; Boussery, K; Croubels, S

    2013-12-01

    Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT-PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra-acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity. PMID:23330986

  4. 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

  5. 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.

  6. Role of furanocoumarin derivatives on grapefruit juice-mediated inhibition of human CYP3A activity.

    PubMed

    Guo, L Q; Fukuda, K; Ohta, T; Yamazoe, Y

    2000-07-01

    With juices of grapefruit and related fruits, possible relationships between contents of six different furanocoumarins and extents of inhibition of microsomal CYP3A activity have been studied in vitro. Microsomal CYP3A-mediated testosterone 6beta-hydroxylation was inhibited by the addition of a fruit juice (2.5%, v/v) from eight different grapefruit sources, two sweeties, three pomelos, and one sour orange, whereas no clear inhibition was observed with two sweet orange juices. The inhibitory component in grapefruit juice resides mainly in the precipitate rather than in the supernatant after centrifugation. Higher amounts of (R)-6',7'-dihydroxybergamottin (DHB) were distributed in the supernatant, whereas GF-I-1, GF-I-2, GF-I-4, and the newly isolated GF-I-5 and GF-I-6 were detected predominantly in the precipitate. Mixing of five representative furanocoumarins at their detectable levels in grapefruit juice reproduced roughly the inhibitory potencies of grapefruit juice, but omission of any of the components resulted in decreased potencies. These results suggested that all the major furanocoumarins contributed to the CYP3A inhibitory properties of grapefruit juice. Furthermore, all six furanocoumarins showed stronger CYP3A inhibitory potencies after preincubation in the presence of NADPH, suggesting that both competitive and mechanism-based inhibition occur in a grapefruit juices-drug interaction. PMID:10859150

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

    PubMed Central

    Samer, CF; Daali, Y; Wagner, M; Hopfgartner, G; Eap, CB; Rebsamen, MC; Rossier, MF; Hochstrasser, D; Dayer, P; Desmeules, JA

    2010-01-01

    Background and purpose: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug–drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. Experimental approach: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg·kg−1) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. Key results: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone Cmax was correlated with SPT assessment (ρS= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. Conclusions and implications: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism. PMID:20590588

  12. 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

  13. Inhibitory effects of citrus fruits on cytochrome P450 3A (CYP3A) activity in humans.

    PubMed

    Fujita, Ken-Ichi; Hidaka, Muneaki; Takamura, Norito; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Yamaguchi, Masatoshi; Ikenoue, Tsuyomu; Arimori, Kazuhiko

    2003-09-01

    The capacities of citrus fruits to inhibit midazolam 1'-hydroxylase activity of cytochrome P450 3A (CYP3A) expressed in human liver microsomes were evaluated. Eight citrus fruits such as ama-natsu, banpeiyu, Dekopon, hassaku, hyuga-natsu, completely matured kinkan (Tamatama), takaoka-buntan and unshu-mikan were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and grapefruit juice (white, Tropicana-Kirin). The addition of a fruit juice prepared from banpeiyu, hassaku, takaoka-buntan or Tamatama caused the inhibition of the microsomal CYP3A activity. The inhibition depended on the amount of a fruit juice added to the incubation mixture (2.5 and 5.0%, v/v). The fruit juice from banpeiyu showed the most potent inhibition of CYP3A. The addition of a banpeiyu juice (5.0%, v/v) resulted in the inhibition of midazolam 1'-hydroxylase activity to about 20% of control without a fruit juice. The elongation of the preincubation period of a fruit juice from banpeiyu (5.0%, v/v) with the microsomal fraction (5 to 15 min) led to the enhancement of the CYP3A inhibition (5% of control). Thus, we discovered ingredients of banpeiyu to be inhibitor(s) or mechanism-based inhibitor(s) of human CYP3A activity, but the inhibitory effects of them were somewhat lower than those of grapefruit. PMID:12951492

  14. 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

  15. 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

  16. Midazolam microdose to determine systemic and pre-systemic metabolic CYP3A activity in humans

    PubMed Central

    Hohmann, Nicolas; Kocheise, Franziska; Carls, Alexandra; Burhenne, Jürgen; Haefeli, Walter E; Mikus, Gerd

    2015-01-01

    Aim We aimed to establish a method to assess systemic and pre-systemic cytochrome P450 (CYP) 3A activity using ineffective microgram doses of midazolam. Methods In an open, one sequence, crossover study, 16 healthy participants received intravenous and oral midazolam at microgram (0.001 mg intravenous and 0.003 mg oral) and regular milligram (1 mg intravenous and 3 mg oral) doses to assess the linearity of plasma and urine pharmacokinetics. Results Dose-normalized AUC and Cmax were 37.1 ng ml−1 h [95% CI 35.5, 40.6] and 39.1 ng ml−1 [95% CI 30.4, 50.2] for the microdose and 39.0 ng ml−1 h [95% CI 36.1, 42.1] and 37.1 ng ml−1 [95% CI 26.9, 51.3] for the milligram dose. CLmet was 253 ml min−1 [95% CI 201, 318] vs. 278 ml min−1 [95% CI 248, 311] for intravenous doses and 1880 ml min−1 [95% CI 1590, 2230] vs. 2050 ml min−1 [95% CI 1720, 2450] for oral doses. Oral bioavailability of a midazolam microdose was 23.4% [95% CI 20.0, 27.3] vs. 20.9% [95% CI 17.1, 25.5] after the regular dose. Hepatic and gut extraction ratios for microgram doses were 0.44 [95% CI 0.39, 0.49] and 0.53 [95% CI 0.45, 0.63] and compared well with those for milligram doses (0.43 [95% CI 0.37, 0.49] and 0.61 [95% CI 0.53, 0.70]). Conclusion The pharmacokinetics of an intravenous midazolam microdose is linear to the applied regular doses and can be used to assess safely systemic CYP3A activity and, in combination with oral microdoses, pre-systemic CYP3A activity. PMID:25588320

  17. 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

  18. Experimental Adjustment on Drug Interactions through Intestinal CYP3A Activity in Rat: Impacts of Kampo Medicines Repeat Administered

    PubMed Central

    Kinoshita, Natsumi; Yamaguchi, Yuriko; Hou, Xiao-Long; Takahashi, Kyoko; Takahashi, Koichi

    2011-01-01

    To provide the information that is necessary for making the proper use of kampo medicines, we have proposed the adequate methodology focused on the following issues: (i) kampo medicines emphasize the effects produced by the combination of herbal drugs rather than the individual effect of any single herb and (ii) Intestinal CYP3A has become a key factor for the bioavailability of orally administrated drugs. In the present study, we investigated both the in vivo and in vitro effects of Saireito and Hochuekkito (kampo formulas) on CYP3A activities. From our study, oral pre-treatment with Saireito or Hochuekkito did not affect the pharmacokinetics of nifedipine after intravenous administration to rats. When nifedipine was administered to rat intrajejunum, a significant decrease of AUC was showed by pre-treatment with both kampo formulas. Saireito pre-treatment led to 80% decrease in Cmax of nifedipine. Saireito caused significant increases in both protein expression and metabolic activity of CYP3A in intestinal microsome, whereas it had no effect on CYP3A in hepatic microsome. Our result also showed that this affect of Saireito can be gone by wash-out with 1 week. These findings demonstrated that Saireito may induce CYP3A activity of intestine but not of liver in rats. When resources for research are limited, well-designed scientific studies except clinical trials also have many advantages. PMID:19884115

  19. Experimental Adjustment on Drug Interactions through Intestinal CYP3A Activity in Rat: Impacts of Kampo Medicines Repeat Administered.

    PubMed

    Kinoshita, Natsumi; Yamaguchi, Yuriko; Hou, Xiao-Long; Takahashi, Kyoko; Takahashi, Koichi

    2011-01-01

    TO PROVIDE THE INFORMATION THAT IS NECESSARY FOR MAKING THE PROPER USE OF KAMPO MEDICINES, WE HAVE PROPOSED THE ADEQUATE METHODOLOGY FOCUSED ON THE FOLLOWING ISSUES: (i) kampo medicines emphasize the effects produced by the combination of herbal drugs rather than the individual effect of any single herb and (ii) Intestinal CYP3A has become a key factor for the bioavailability of orally administrated drugs. In the present study, we investigated both the in vivo and in vitro effects of Saireito and Hochuekkito (kampo formulas) on CYP3A activities. From our study, oral pre-treatment with Saireito or Hochuekkito did not affect the pharmacokinetics of nifedipine after intravenous administration to rats. When nifedipine was administered to rat intrajejunum, a significant decrease of AUC was showed by pre-treatment with both kampo formulas. Saireito pre-treatment led to 80% decrease in C(max) of nifedipine. Saireito caused significant increases in both protein expression and metabolic activity of CYP3A in intestinal microsome, whereas it had no effect on CYP3A in hepatic microsome. Our result also showed that this affect of Saireito can be gone by wash-out with 1 week. These findings demonstrated that Saireito may induce CYP3A activity of intestine but not of liver in rats. When resources for research are limited, well-designed scientific studies except clinical trials also have many advantages. PMID:19884115

  20. Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes.

    PubMed

    Zhang, Shimin; Pillai, Venkateswaran C; Mada, Sripal Reddy; Strom, Steve; Venkataramanan, Raman

    2012-05-01

    Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6β-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6β-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6β-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole. PMID:22106961

  1. 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.

  2. 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

  3. CYP3A5 mediates bioactivation and cytotoxicity of tetrandrine.

    PubMed

    Tian, Ye; Shen, Shuijie; Jiang, Yan; Shen, Qi; Zeng, Su; Zheng, Jiang

    2016-07-01

    Tetrandrine is a diaryl ether-type bisbenzylisoquinoline alkaloid and has shown multiple pharmacological activities. Our early work demonstrated that tetrandrine produced acute pulmonary toxicity and that tetrandrine was biotransformed to a quinone methide-derived metabolite mediated by CYP3A enzymes. The formation of the reactive intermediate is suggested to be responsible for the pulmonary toxicity induced by tetrandrine. In the present study, a WI-38-based Cyp3a5 transgenic cell line (WI-38/Cyp3a5) was established to investigate the role of CYP3A5 in tetrandrine-induced cytotoxicity. The transgenic cells were found to be more susceptible to the cytotoxicity of tetrandrine than the wild-type cells (WI-38/Vector). WI-38/Cyp3a5 cells showed higher cellular ROS levels, higher LDH activities in culture media, but lower cellular GSH contents than those observed in WI-38/Vector cells after exposure to tetrandrine. And severer apoptosis were observed in WI-38/Cyp3a5 cells after treatment with tetrandrine: WI-38/Cyp3a5 cells had higher proportion of early and late apoptotic cells, higher expression levels of caspase-3, but lower level of Bcl-2 than WI-38/Vector cells. This study provided strong evidence that CYP3A5 participated in tetrandrine-induced cytotoxicity. PMID:26302866

  4. 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

  5. Effects of chlorpyrifos on the transcription of CYP3A cDNA, activity of acetylcholinesterase, and oxidative stress response of goldfish (Carassius auratus).

    PubMed

    Ma, Junguo; Liu, Yang; Niu, Daichun; Li, Xiaoyu

    2015-04-01

    Chlorpyrifos (CPF) is the widely used organophosphate pesticide in agriculture throughout the world. It has been found that CPF is relatively safe to human but highly toxic to fish. In this study, acute toxicity of CPF on goldfish was determined and then the transcription of goldfish cytochrome P450 (CYP) 3A was evaluated after 96 h of CPF exposure at concentrations of 15.3 [1/10 50% lethal concentration (LC50 )] or 51 μg L(-1) (1/3 LC50 ) of CPF. Meanwhile, the enzymatic activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT), total antioxidant activity (T-AOC), and the contents of malondialdehyde (MDA) in the liver or brain of goldfish were also determined. The results of acute toxicity testing showed that the 96-h LC50 of CPF to the goldfish was 153 μg L(-1) . Moreover, a length sequence of 1243 bp CYP3A cDNA encoding for 413 amino acids from goldfish liver was cloned. Polymerase chain reaction results reveal that CPF exposure downregulates CYP 3A transcription in goldfish liver, suggesting that goldfish CYP 3A may be not involved in CPF bioactivation. Finally, the results of biochemical assays indicate that 96 h of CPF exposure remarkably inhibits AChE activity in fish liver or brain, alters hepatic antioxidant enzyme activities, decreases brain T-AOC, and causes lipid peroxidation in fish liver. These results suggest that oxidative stress might be involved in CPF toxicity on goldfish. PMID:24190793

  6. 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

  7. 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

  8. Influence of Panax ginseng on Cytochrome P450 (CYP)3A and P-glycoprotein (Pgp) Activity in Healthy Subjects

    PubMed Central

    Malati, Christine Y.; Robertson, Sarah M.; Hunt, Jennifer D.; Chairez, Cheryl; Alfaro, Raul M.; Kovacs, Joseph A.; Penzak, Scott R.

    2012-01-01

    A number of herbal preparations have been shown to interact with prescription medications secondary to modulation of cytochrome P450 (CYP) and/or P-glycoprotein (P-gp). The purpose of this study was to determine the influence of Panax ginseng on CYP3A and P-gp function using the probe substrates midazolam and fexofenadine, respectively. Twelve healthy subjects (8 males) completed this open label, single sequence pharmacokinetic study. Healthy volunteers received single oral doses of midazolam 8 mg and fexofenadine 120 mg, before and after 28 days of P. ginseng 500 mg twice daily. Midazolam and fexofenadine pharmacokinetic parameter values were calculated and compared pre-and post P. ginseng administration. Geometric mean ratios (post-ginseng/pre-ginseng) for midazolam area under the concentration vs. time curve from zero to infinity (AUC0-∞), half life (T1/2), and maximum concentration (Cmax) were significantly reduced at 0.66 (0.55 – 0.78), 0.71 (0.53 – 0.90), and 0.74 (0.56 – 0.93), respectively. Conversely, fexofenadine pharmacokinetics were unaltered by P. ginseng administration. Based on these results, Panax ginseng appeared to induce CYP3A activity in the liver and possibly the gastrointestinal tract. Patients taking Panax ginseng in combination with CYP3A substrates with narrow therapeutic ranges should be monitored closely for adequate therapeutic response to the substrate medication. PMID:21646440

  9. Supplementation with goldenseal (Hydrastis canadensis), but not kava kava (Piper methysticum), inhibits human CYP3A activity in vivo.

    PubMed

    Gurley, B J; Swain, A; Hubbard, M A; Hartsfield, F; Thaden, J; Williams, D K; Gentry, W B; Tong, Y

    2008-01-01

    The effects of goldenseal (Hydrastis canadensis) and kava kava (Piper methysticum) supplementation on human CYP3A activity were evaluated using midazolam (MDZ) as a phenotypic probe. Sixteen healthy volunteers were randomly assigned to receive either goldenseal or kava kava for 14 days. Each supplementation phase was followed by a 30-day washout period. MDZ (8 mg, per os) was administered before and after each phase, and pharmacokinetic parameters were determined using standard non-compartmental methods. Comparisons of pre- and post-supplementation MDZ pharmacokinetic parameters revealed significant inhibition of CYP3A by goldenseal (AUC(0-infinity), 107.9+/-43.3 vs 175.3+/-74.8 ng x h/ml; Cl/F/kg, 1.26+/-0.59 vs 0.81+/-0.45 l/h/kg; T(1/2), 2.01+/-0.42 vs 3.15+/-1.12 h; Cmax, 50.6+/-26.9 vs 71.2+/-50.5 ng/ml). MDZ disposition was not affected by kava kava supplementation. These findings suggest that significant herb-drug interactions may result from the concomitant ingestion of goldenseal and CYP3A substrates. PMID:17495878

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. Cecropin B Represses CYP3A29 Expression through Activation of the TLR2/4-NF-κB/PXR Signaling Pathway

    PubMed Central

    Zhou, Xiaoqiao; Li, Xiaowen; Wang, Xiliang; Jin, Xiue; Shi, Deshi; Wang, Jun; Bi, Dingren

    2016-01-01

    Cecropins are peptide antibiotics used as drugs and feed additives. Cecropin B can inhibit the expression of CYP3A29, but the underlying mechanisms remain unclear. The present study was designed to determine the mechanisms responsible for the effects of cecropin B on CYP3A29 expression, focusing on the Toll-like receptors (TLRs) and NF-κB pathways. Our results indicated that the CYP3A29 expression was inhibited by cecropin B, which was regulated by pregnane X receptor (PXR) in a time- and dose-dependent manner. Cecropin B-induced NF-κB activation played a pivotal role in the suppression of CYP3A29 through disrupting the association of the PXR/retinoid X receptor alpha (RXR-α) complex with DNA sequences. NF-κB p65 directly interacted with the DNA-binding domain of PXR, suppressed its expression, and inhibited its transactivation, leading to the downregulation of the PXR-regulated CYP3A29 expression. Furthermore, cecropin B activated pig liver cells by interacting with TLRs 2 and 4, which modulated NF-κB-mediated signaling pathways. In conclusion, cecropin B inhibited the expression of CYP3A29 in a TLR/NF-κB/PXR-dependent manner, which should be considered in future development of cecropins and other antimicrobial peptides. PMID:27296244

  16. Cecropin B Represses CYP3A29 Expression through Activation of the TLR2/4-NF-κB/PXR Signaling Pathway.

    PubMed

    Zhou, Xiaoqiao; Li, Xiaowen; Wang, Xiliang; Jin, Xiue; Shi, Deshi; Wang, Jun; Bi, Dingren

    2016-01-01

    Cecropins are peptide antibiotics used as drugs and feed additives. Cecropin B can inhibit the expression of CYP3A29, but the underlying mechanisms remain unclear. The present study was designed to determine the mechanisms responsible for the effects of cecropin B on CYP3A29 expression, focusing on the Toll-like receptors (TLRs) and NF-κB pathways. Our results indicated that the CYP3A29 expression was inhibited by cecropin B, which was regulated by pregnane X receptor (PXR) in a time- and dose-dependent manner. Cecropin B-induced NF-κB activation played a pivotal role in the suppression of CYP3A29 through disrupting the association of the PXR/retinoid X receptor alpha (RXR-α) complex with DNA sequences. NF-κB p65 directly interacted with the DNA-binding domain of PXR, suppressed its expression, and inhibited its transactivation, leading to the downregulation of the PXR-regulated CYP3A29 expression. Furthermore, cecropin B activated pig liver cells by interacting with TLRs 2 and 4, which modulated NF-κB-mediated signaling pathways. In conclusion, cecropin B inhibited the expression of CYP3A29 in a TLR/NF-κB/PXR-dependent manner, which should be considered in future development of cecropins and other antimicrobial peptides. PMID:27296244

  17. 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.

  18. Cytochrome P450 CYP3A in marsupials: cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Crittenden, Tamara A; Marchant, Ceilidh L; Stupans, Ieva; McKinnon, Ross A

    2011-11-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP

  19. 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

  20. Thalidomide-induced limb abnormalities in a humanized CYP3A mouse model.

    PubMed

    Kazuki, Yasuhiro; Akita, Masaharu; Kobayashi, Kaoru; Osaki, Mitsuhiko; Satoh, Daisuke; Ohta, Ryo; Abe, Satoshi; Takehara, Shoko; Kazuki, Kanako; Yamazaki, Hiroshi; Kamataki, Tetsuya; Oshimura, Mitsuo

    2016-01-01

    Thalidomide is a teratogen in humans but not in rodents. It causes multiple birth defects including malformations of limbs, ears, and other organs. However, the species-specific mechanism of thalidomide teratogenicity is not completely understood. Reproduction of the human teratogenicity of thalidomide in rodents has previously failed because of the lack of a model reflecting human drug metabolism. In addition, because the maternal metabolic effect cannot be eliminated, the migration of unchanged thalidomide to embryos is suppressed, and the metabolic activation is insufficient to develop teratogenicity. Previously, we generated transchromosomic mice containing a human cytochrome P450 (CYP) 3A cluster in which the endogenous mouse Cyp3a genes were deleted. Here, we determined whether human CYP3A or mouse Cyp3a enzyme expression was related to the species difference in a whole embryo culture system using humanized CYP3A mouse embryos. Thalidomide-treated embryos with the human CYP3A gene cluster showed limb abnormalities, and human CYP3A was expressed in the placenta, suggesting that human CYP3A in the placenta may contribute to the teratogenicity of thalidomide. These data suggest that the humanized CYP3A mouse is a useful model to predict embryonic toxicity in humans. PMID:26903378

  1. Thalidomide-induced limb abnormalities in a humanized CYP3A mouse model

    PubMed Central

    Kazuki, Yasuhiro; Akita, Masaharu; Kobayashi, Kaoru; Osaki, Mitsuhiko; Satoh, Daisuke; Ohta, Ryo; Abe, Satoshi; Takehara, Shoko; Kazuki, Kanako; Yamazaki, Hiroshi; Kamataki, Tetsuya; Oshimura, Mitsuo

    2016-01-01

    Thalidomide is a teratogen in humans but not in rodents. It causes multiple birth defects including malformations of limbs, ears, and other organs. However, the species-specific mechanism of thalidomide teratogenicity is not completely understood. Reproduction of the human teratogenicity of thalidomide in rodents has previously failed because of the lack of a model reflecting human drug metabolism. In addition, because the maternal metabolic effect cannot be eliminated, the migration of unchanged thalidomide to embryos is suppressed, and the metabolic activation is insufficient to develop teratogenicity. Previously, we generated transchromosomic mice containing a human cytochrome P450 (CYP) 3A cluster in which the endogenous mouse Cyp3a genes were deleted. Here, we determined whether human CYP3A or mouse Cyp3a enzyme expression was related to the species difference in a whole embryo culture system using humanized CYP3A mouse embryos. Thalidomide-treated embryos with the human CYP3A gene cluster showed limb abnormalities, and human CYP3A was expressed in the placenta, suggesting that human CYP3A in the placenta may contribute to the teratogenicity of thalidomide. These data suggest that the humanized CYP3A mouse is a useful model to predict embryonic toxicity in humans. PMID:26903378

  2. 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

  3. SOY PROTEIN ISOLATE INDUCES CYP3A1 AND CYP3A2 IN PREPUBERTAL RATS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feeding soy diets has been shown to induce cytochrome P450s in gene family CYP3A in Sprague-Dawley rat liver. We compared expression of CYP3A enzymes on PND33 rats fed casein or soy protein isolate (SPI+)-based AIN-93G diets continuously from gestational day 4 through PND 33 or the diets were switc...

  4. CYP3A in horse intestines.

    PubMed

    Tydén, Eva; Olsén, Lena; Tallkvist, Jonas; Larsson, Pia; Tjälve, Hans

    2004-12-01

    The intestinal enterocytes provide the initial site for cytochrome P450 (CYP)-mediated metabolism of orally absorbed xenobiotics. In man and some animal species, the CYP3A subfamily is highly expressed in the intestines and considered to be important in the first-pass metabolism of drugs and other xenobiotics. The aim of the present study was to investigate the mRNA expression, immunohistochemical localization and catalytic activity of CYP3A in the intestines of horse. Real-time RT-PCR analyses showed that the highest CYP3A mRNA expression was present in the duodenum with a decreasing level towards jejunum, ileum, cecum, and colon. The CYP3A mRNA expression in the liver was similar as in the anterior part of the jejunum, but about 4.5 times lower than in the anterior part of the duodenum. Immunohistochemistry showed CYP3A immunoreactivity in the cytoplasm of the enterocytes, which decreased distally along the intestinal tract. CYP3A-dependent metabolic activity rose slightly from the anterior to the distal part of the duodenum and the anterior part of the jejunum and then declined to the middle and distal parts of the jejunum and the ileum, cecum, and colon. Our results suggest that CYP3A in the small intestine plays a major role in first-pass metabolism and may affect bioavailability and therapeutic efficiency of some orally administrated drugs in horse. PMID:15541751

  5. 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

  6. 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

  7. 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

  8. Recommendations on the Development of a Bioanalytical Assay for 4β-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.

    PubMed

    Aubry, Anne-Françoise; Dean, Brian; Diczfalusy, Ulf; Goodenough, Angela; Iffland, André; McLeod, James; Weng, Naidong; Yang, Ziping

    2016-09-01

    The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development. PMID:27350147

  9. Downregulation of Mouse Hepatic CYP3A Protein by 3-Methylcholanthrene Does Not Require Cytochrome P450-Dependent Metabolism

    PubMed Central

    Lee, Chunja; Ding, Xinxin

    2013-01-01

    The aryl hydrocarbon receptor (AHR)–dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity. PMID:23846873

  10. Influence of Panax ginseng on cytochrome P450 (CYP)3A and P-glycoprotein (P-gp) activity in healthy participants.

    PubMed

    Malati, Christine Y; Robertson, Sarah M; Hunt, Jennifer D; Chairez, Cheryl; Alfaro, Raul M; Kovacs, Joseph A; Penzak, Scott R

    2012-06-01

    A number of herbal preparations have been shown to interact with prescription medications secondary to modulation of cytochrome P450 (CYP) and/or P-glycoprotein (P-gp). The purpose of this study was to determine the influence of Panax ginseng on CYP3A and P-gp function using the probe substrates midazolam and fexofenadine, respectively. Twelve healthy participants (8 men) completed this open-label, single-sequence pharmacokinetic study. Healthy volunteers received single oral doses of midazolam 8 mg and fexofenadine 120 mg, before and after 28 days of P ginseng 500 mg twice daily. Midazolam and fexofenadine pharmacokinetic parameter values were calculated and compared before and after P ginseng administration. Geometric mean ratios (postginseng/preginseng) for midazolam area under the concentration-time curve from zero to infinity (AUC(0-∞)), half-life (t(1/2)), and maximum concentration (C(max)) were significantly reduced at 0.66 (0.55-0.78), 0.71 (0.53-0.90), and 0.74 (0.56-0.93), respectively. Conversely, fexofenadine pharmacokinetics were unaltered by P ginseng administration. Based on these results, P ginseng appeared to induce CYP3A activity in the liver and possibly the gastrointestinal tract. Patients taking P ginseng in combination with CYP3A substrates with narrow therapeutic ranges should be monitored closely for adequate therapeutic response to the substrate medication. PMID:21646440

  11. In vitro effects of the citrus flavonoids diosmin, naringenin and naringin on the hepatic drug-metabolizing CYP3A enzyme in human, pig, mouse and fish.

    PubMed

    Burkina, Viktoriia; Zlabek, Vladimir; Halsne, Ruth; Ropstad, Erik; Zamaratskaia, Galia

    2016-06-15

    Flavonoids are known to have effects on cytochrome P450 enzymatic activity. However, little effort has been made to examine species differences and the relevance of studies on mammalian and fish microsomes so that extrapolations can be made to humans. Therefore, the effects of several naturally occurring flavonoids on the activity of CYP3A-dependent 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylase (BFCOD) were evaluated in human, pig, mouse, and juvenile rainbow trout sources of hepatic microsomes. Each was exposed to three concentrations (1, 10, and 100μM) of diosmin, naringin, and naringenin. Naringenin competitively inhibited BFCOD activity (Ki values were 24.6μM in human, 15.6μM in pig, and 19.6μM in mouse microsomes). In fish, BFCOD activity was inhibited in a noncompetitive manner (Ki=7μM). Neither diosmin nor naringenin affected BFCOD activity in hepatic microsomes from the studied model organisms. These results suggest that dietary flavonoids potentially inhibit the metabolism of clinical drugs. PMID:27107807

  12. Modeling chemical interaction profiles: I. Spectral data-activity relationship and structure-activity relationship models for inhibitors and non-inhibitors of cytochrome P450 CYP3A4 and CYP2D6 isozymes.

    PubMed

    McPhail, Brooks; Tie, Yunfeng; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Valerio, Luis G; Fuscoe, James C; Tong, Weida; Buzatu, Dan A; Wilkes, Jon G; Fowler, Bruce A; Demchuk, Eugene; Beger, Richard D

    2012-01-01

    An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals--drugs, pesticides, and environmental pollutants--interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D ¹³C and 1D ¹⁵N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D ¹³C-NMR and ¹⁵N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

  13. Contribution of three CYP3A isoforms to metabolism of R- and S-warfarin.

    PubMed

    Jones, Drew R; Kim, So-Young; Boysen, Gunnar; Yun, Chul-Ho; Miller, Grover P

    2010-12-01

    Effective coumadin (R/S-warfarin) therapy is complicated by inter-individual variability in metabolism. Recent studies have demonstrated that CYP3A isoforms likely contribute to patient responses and clinical outcomes. Despite a significant focus on CYP3A4, little is known about CYP3A5 and CYP3A7 metabolism of warfarin. Based on our studies, recombinant CYP3A4, CYP3A5 and CYP3A7 metabolized R- and S-warfarin to 10- and 4'-hydroxywarfarin with efficiencies that depended on the individual enzymes. For R-warfarin, CYP3A4, CYP3A7, and CYP3A5 demonstrated decreasing preference for 10-hydroxylation over 4'-hydroxylation. By contrast, there was no regioselectivity toward S-warfarin. While all enzymes preferentially metabolized R-warfarin, CYP3A4 was the most efficient at metabolizing all reactions. Individuals, namely African-Americans and children, with higher relative levels of CYP3A5 and/or CYP3A7, respectively, compared to CYP3A4 may metabolize warfarin less efficiently and thus may require lower doses and be at risk for adverse drug-drug interactions related to the contributions of the respective enzymes. PMID:20615193

  14. Cytochrome P450 CYP3A in marsupials: cloning and identification of the first CYP3A subfamily member, isoform 3A70 from Eastern gray kangaroo (Macropus giganteus).

    PubMed

    El-Merhibi, Adaweyah; Ngo, Suong N T; Marchant, Ceilidh L; Height, Tamara A; Stupans, Ieva; McKinnon, Ross A

    2012-09-15

    Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials. PMID:22759518

  15. Mechanism of ritonavir changes in methadone pharmacokinetics and pharmacodynamics: II. Ritonavir effects on CYP3A and P-glycoprotein activities.

    PubMed

    Kharasch, E D; Bedynek, P S; Walker, A; Whittington, D; Hoffer, C

    2008-10-01

    Ritonavir diminishes methadone plasma concentrations, an effect attributed to CYP3A induction, but the actual mechanisms are unknown. We determined short-term (2-day) and steady-state (2-week) ritonavir effects on intestinal and hepatic CYP3A4/5 (probed with intravenous (IV) and oral alfentanil (ALF) and with miosis) and P-glycoprotein (P-gp) (fexofenadine), and on methadone pharmacokinetics and pharmacodynamics in healthy volunteers. Acute ritonavir increased the area under the concentration-time curve (AUC)(0-infinity)/dose ratio (ritonavir/control) for oral ALF 25-fold. Steady-state ritonavir increased the AUC(0-Infinity)/dose ratio for IV and oral ALF 4- and 10-fold, respectively; reduced hepatic extraction (from 0.26 to 0.07) and intestinal extraction (from 0.51 to 0); and increased bioavailability (from 37 to 95%). Acute ritonavir inhibits first-pass CYP3A > 96%. Chronic ritonavir inhibits hepatic CYP3A (> 70%) and first-pass CYP3A (> 90%). Acute and steady-state ritonavir increased the fexofenadine AUC(0-infinity) 2.8- and 1.4-fold, respectively, suggesting P-gp inhibition. Steady-state compared with acute ritonavir caused mild apparent induction of P-gp and hepatic CYP3A, but net inhibition still predominated. Ritonavir inhibited both intestinal and hepatic CYP3A and drug transport. ALF miosis noninvasively determined CYP3A inhibition by ritonavir. PMID:19238656

  16. [Furanocoumarins contents and cytochrome P450 3A (CYP3A) inhibitory activities of various processed fruit peel products: outflow of 6',7'-Dihydroxybergamottin during processing treatment of peel].

    PubMed

    Ishihara, Masaru; Toda, Hikaru; Sunagane, Nobuyoshi; Ohta, Takafumi

    2011-01-01

    Furanocoumarins (FCs) such as bergamottin (BG) and 6',7'-dihydroxybergamottin (DHBG) contained in grapefruits are known to be cytochrome P450 3A4 (CYP3A4) inhibitors. These are contained in larger quantity in peel than in pulp, and therefore, processed peel products possibly have strong CYP3A4 inhibitory activity. The CYP3A4 inhibitory potency of these processed peel products, however, remains to be elucidated. The FC content and CYP3A inhibitory activities of various processed fruit peel products were investigated. CYP3A inhibitory activities of crystallized grapefruit peel, grapefruit marmalade, lemon peel and bitter orange slice were close to that of 100% grapefruit juice, while the activities of yuzu slice, pomelo (buntan) marmalade and crystallized iyokan peel were very weak, 1/8-1/20 of 100% grapefruit juice. The maximum BG content was 5.6 µg/g in lemon peel. The maximum DHBG content was 7.2 µg/g in crystallized grapefruit peel, about 1/30 that of raw peel. Grapefruit marmalade and crystallized grapefruit peel contained similar amounts of FCs to 100% grapefruit juice, but FCs were not detected in pomelo (buntan) marmalade or crystallized iyokan peel. Good correlation (r=0.78) was observed between the FC contents of these peel products and those CYP3A inhibitory activities. Preparation of homemade grapefruit marmalade and crystallized peel revealed that considerably lower DHBG content in these products and lower CYP3A inhibitory activity than anticipated were attributable to outflow of DHBG to broth during boiling of the raw peel. PMID:21532264

  17. 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

  18. 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

  19. 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

  20. 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

  1. BDE47 induces rat CYP3A1 by targeting the transcriptional regulation of miR-23b.

    PubMed

    Sun, Zhenzhen; Zhang, Zhan; Ji, Minghui; Yang, Hongbao; Cromie, Meghan; Gu, Jun; Wang, Chao; Yang, Lu; Yu, Yongquan; Gao, Weimin; Wang, Shou-Lin

    2016-01-01

    Cytochrome P450 3A (CYP3A) is the most abundant CYP450 enzyme in the liver and is involved in the metabolism of over 50% of xenobiotics. Our previous studies revealed that 2,2',4,4'-tetrabromodiphenyl ether (BDE47) could induce rat CYP3A1 expression, but the molecular basis remains unclear. Using in silico analysis, we identified a potential miR-23b recognition element (MRE23b) in the 3'-UTR region of CYP3A1 mRNA, which was verified by the luciferase assay. The miR-23b mimic and inhibitor significantly down- and up-regulated the expression of CYP3A1, respectively. Additionally, BDE47 significantly down-regulated the expression of miR-23b in rats and in hepatic H4IIE cells. Induction or blockage of CYP3A1 by a miR-23b inhibitor or mimic could correspondingly alter BDE47-induced expression of CYP3A1 and cytotoxicity in H4IIE cells. Furthermore, LV-anti-miR-23b significantly decreased endogenous levels of miR-23b and increased the expression and activity of CYP3A1 in rat liver. LV-anti-miR-23b also significantly increased the hydroxylated metabolites of BDE47 (3-OH-BDE47, 4-OH-BDE42, and 4'-OH-BDE49) in rat serum. In conclusion, we first found that BDE47 induced rat CYP3A1 expression by targeting the transcriptional regulation of miR-23b. This study helps provide a better understanding of CYP3A regulation and offers novel clues for the role of miRNAs in the metabolism and distribution of environmental pollutants. PMID:27546062

  2. BDE47 induces rat CYP3A1 by targeting the transcriptional regulation of miR-23b

    PubMed Central

    Sun, Zhenzhen; Zhang, Zhan; Ji, Minghui; Yang, Hongbao; Cromie, Meghan; Gu, Jun; Wang, Chao; Yang, Lu; Yu, Yongquan; Gao, Weimin; Wang, Shou-Lin

    2016-01-01

    Cytochrome P450 3A (CYP3A) is the most abundant CYP450 enzyme in the liver and is involved in the metabolism of over 50% of xenobiotics. Our previous studies revealed that 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) could induce rat CYP3A1 expression, but the molecular basis remains unclear. Using in silico analysis, we identified a potential miR-23b recognition element (MRE23b) in the 3′-UTR region of CYP3A1 mRNA, which was verified by the luciferase assay. The miR-23b mimic and inhibitor significantly down- and up-regulated the expression of CYP3A1, respectively. Additionally, BDE47 significantly down-regulated the expression of miR-23b in rats and in hepatic H4IIE cells. Induction or blockage of CYP3A1 by a miR-23b inhibitor or mimic could correspondingly alter BDE47-induced expression of CYP3A1 and cytotoxicity in H4IIE cells. Furthermore, LV-anti-miR-23b significantly decreased endogenous levels of miR-23b and increased the expression and activity of CYP3A1 in rat liver. LV-anti-miR-23b also significantly increased the hydroxylated metabolites of BDE47 (3-OH-BDE47, 4-OH-BDE42, and 4′-OH-BDE49) in rat serum. In conclusion, we first found that BDE47 induced rat CYP3A1 expression by targeting the transcriptional regulation of miR-23b. This study helps provide a better understanding of CYP3A regulation and offers novel clues for the role of miRNAs in the metabolism and distribution of environmental pollutants. PMID:27546062

  3. 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

  4. Interactions between xenoestrogens and ketoconazole on hepatic CYP1A and CYP3A, in juvenile Atlantic cod (Gadus morhua)

    PubMed Central

    Hasselberg, Linda; Grøsvik, Bjørn E; Goksøyr, Anders; Celander, Malin C

    2005-01-01

    Background Xenoestrogens and antifungal azoles probably share a common route of metabolism, through hepatic cytochrome P450 (CYP) enzymes. Chemical interactions with metabolic pathways may affect clearance of both xenobiotics and endobiotics. This study was carried out to identify possible chemical interactions by those substances on CYP1A and CYP3A, in Atlantic cod liver. We investigated effects of two xenoestrogens (nonylphenol and ethynylestradiol) and of the model imidazole ketoconazole, alone and in combination. Results Treatment with ketoconazole resulted in 60% increase in CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity. Treatment with nonylphenol resulted in 40% reduction of CYP1A activity. Combined exposure to ketoconazole and nonylphenol resulted in 70% induction of CYP1A activities and 93% increase in CYP1A protein levels. Ketoconazole and nonylphenol alone or in combination had no effect on CYP3A expression, as analyzed by western blots. However, 2-dimensional (2D) gel electrophoresis revealed the presence of two CYP3A-immunoreactive proteins, with a more basic isoform induced by ketoconazole. Treatment with ketoconazole and nonylphenol alone resulted in 54% and 35% reduction of the CYP3A-mediated benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BFCOD) activity. Combined exposure of ketoconazole and nonylphenol resulted in 98% decrease in CYP3A activity. This decrease was greater than the additive effect of each compound alone. In vitro studies revealed that ketoconazole was a potent non-competitive inhibitor of both CYP1A and CYP3A activities and that nonylphenol selectively non-competitively inhibited CYP1A activity. Treatment with ethynylestradiol resulted in 46% decrease in CYP3A activity and 22% decrease in protein expression in vivo. In vitro inhibition studies in liver microsomes showed that ethynylestradiol acted as a non-competitive inhibitor of CYP1A activity and as an uncompetitive inhibitor of CYP3A activity. Conclusions

  5. In Vitro Approaches to Study Regulation of Hepatic Cytochrome P450 (CYP) 3A Expression by Paclitaxel and Rifampicin.

    PubMed

    Ghose, Romi; Mallick, Pankajini; Taneja, Guncha; Chu, Chun; Moorthy, Bhagavatula

    2016-01-01

    Cancer is the second leading cause of mortality worldwide; however the response rate to chemotherapy treatment remains slow, mainly due to narrow therapeutic index and multidrug resistance. Paclitaxel (taxol) has a superior outcome in terms of response rates and progression-free survival. However, numerous cancer patients are resistant to this drug. In this investigation, we tested the hypothesis that induction of cytochrome P450 (Cyp)3a11 gene by paclitaxel is downregulated by the inflammatory mediator, lipopolysaccharide (LPS), and that the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α, attenuates human CYP3A4 gene induction by rifampicin. Primary mouse hepatocytes were pretreated with LPS (1 μg/ml) for 10 min, followed by paclitaxel (20 μM) or vehicle for 24 h. RNA was extracted from the cells by trizol method followed by cDNA synthesis and analysis by real-time PCR. Paclitaxel significantly induced gene expression of Cyp3a11 (~30-fold) and this induction was attenuated in LPS-treated samples. Induction and subsequent downregulation of CYP3A enzyme can impact paclitaxel treatment in cancer patients where inflammatory mediators are activated. It has been shown that the nuclear receptor, pregnane X receptor (PXR), plays a role in the induction of CYP enzymes. In order to understand the mechanisms of regulation of human CYP3A4 gene, we co-transfected HepG2 cells (human liver cell line) with CYP3A4-luciferase construct and a PXR expression plasmid. The cells were then treated with the pro-inflammatory cytokine, TNFα, followed by the prototype CYP3A inducer rifampicin. It is well established that rifampicin activates PXR, leading to CYP3A4 induction. We found that induction of CYP3A4-luciferase activity by rifampicin was significantly attenuated by TNFα. In conclusion, we describe herein several in vitro approaches entailing primary and cultured hepatocytes, real-time PCR, and transcriptional activation (transfection) assays to investigate the

  6. 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

  7. A mechanism-based pharmacokinetic/pharmacodynamic model for CYP3A1/2 induction by dexamethasone in rats

    PubMed Central

    Li, Liang; Li, Zai-quan; Deng, Chen-hui; Ning, Miao-ran; Li, Han-qing; Bi, Shan-shan; Zhou, Tian-yan; Lu, Wei

    2012-01-01

    Aim: To develop a pharmacokinetic/pharmacodynamic (PK/PD) model describing the receptor/gene-mediated induction of CYP3A1/2 by dexamethasone (DEX) in rats. Methods: A group of male Sprague-Dawley rats receiving DEX (100 mg/kg, ip) were sacrificed at various time points up to 60 h post-treatment. Their blood sample and liver were collected. The plasma concentration of DEX was determined with a reverse phase HPLC method. CYP3A1/2 mRNA, protein levels and enzyme activity were measured using RT-PCR, ELISA and the testosterone substrate assay, respectively. Data analyses were performed using a first-order conditional estimate (FOCE) with INTERACTION method in NONMEM version 7.1.2. Results: A two-compartment model with zero-order absorption was applied to describe the pharmacokinetic characteristics of DEX. Systemic clearance, the apparent volume of distribution and the duration of zero-order absorption were calculated to be 172.7 mL·kg−1·h−1, 657.4 mL/kg and 10.47 h, respectively. An indirect response model with a series of transit compartments was developed to describe the induction of CYP3A1/2 via PXR transactivation by DEX. The maximum induction of CYP3A1 and CYP3A2 mRNA levels was achieved, showing nearly 21.29- and 8.67-fold increases relative to the basal levels, respectively. The CYP3A1 and CYP3A2 protein levels were increased by 8.02-fold and 2.49-fold, respectively. The total enzyme activities of CYP3A1/2 were shown to increase by up to 2.79-fold, with a lag time of 40 h from the Tmax of the DEX plasma concentration. The final PK/PD model was able to recapitulate the delayed induction of CYP3A1/2 mRNA, protein and enzyme activity by DEX. Conclusion: A mechanism-based PK/PD model was developed to characterize the complex concentration-induction response relationship between DEX and CYP3A1/2 and to resolve the drug- and system-specific PK/PD parameters for the course of induction. PMID:22212433

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Identification of CYP3A7 for Glyburide Metabolism in Human Fetal Livers

    PubMed Central

    Shuster, Diana L.; Risler, Linda J.; Prasad, Bhagwat; Calamia, Justina C.; Voellinger, Jenna L.; Kelly, Edward J.; Unadkat, Jashvant D.; Hebert, Mary F.; Shen, Danny D.; Thummel, Kenneth E.; Mao, Qingcheng

    2014-01-01

    Glyburide is commonly prescribed for the treatment of gestational diabetes mellitus; however, fetal exposure to glyburide is not well understood and may have short- and long-term consequences for the health of the child. Glyburide can cross the placenta; fetal concentrations at term are nearly comparable to maternal levels. Whether or not glyburide is metabolized in the fetus and by what mechanisms has yet to be determined. In this study, we determined the kinetic parameters for glyburide depletion by CYP3A isoenzymes; characterized glyburide metabolism by human fetal liver tissues collected during the first or early second trimester of pregnancy; and identified the major enzyme responsible for glyburide metabolism in human fetal livers. CYP3A4 had the highest metabolic capacity towards glyburide, followed by CYP3A7 and CYP3A5 (Clint,u = 37.1, 13.0, and 8.7 ml/min/nmol P450, respectively). M5 was the predominant metabolite generated by CYP3A7 and human fetal liver microsomes (HFLMs) with approximately 96% relative abundance. M5 was also the dominant metabolite generated by CYP3A4, CYP3A5, and adult liver microsomes; however, M1-M4 were also present, with up to 15% relative abundance. CYP3A7 protein levels in HFLMs were highly correlated with glyburide Clint, 16α-OH DHEA formation, and 4′-OH midazolam formation. Likewise, glyburide Clint was highly correlated with 16α-OH DHEA formation. Fetal demographics as well as CYP3A5 and CYP3A7 genotype did not alter CYP3A7 protein levels or glyburide Clint. These results indicate that human fetal livers metabolize glyburide predominantly to M5 and that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers. PMID:25450675

  14. Food-drug interactions via human cytochrome P450 3A (CYP3A).

    PubMed

    Fujita, Ken-ichi

    2004-01-01

    Food-drug interactions have been reported to occur in various systems in the body. The causes of these interactions are mainly divided into pharmacodynamic and pharmacokinetic processes. Among these processes, drug metabolism plays a crucial role in drug interactions. Metabolic food-drug interactions occur when a certain food alters the activity of a drug-metabolizing enzyme, leading to a modulation of the pharmacokinetics of drugs metabolized by the enzyme. A variety of interactions have been documented so far. Foods consisting of complex chemical mixtures, such as fruits, alcoholic beverages, teas, and herbs, possess the ability to inhibit or induce the activity of drug-metabolizing enzymes. According to results obtained thus far, cytochrome P450 3A4 (CYP3A4) appears to be a key enzyme in food-drug interactions. For example, interactions of grapefruit juice with felodipine and cyclosporine, red wine with cyclosporine, and St John's wort with various medicines including cyclosporine, have been demonstrated. The results indicate the requirement of dosage adjustment to maintain drug concentrations within their therapeutic windows. The CYP3A4-related interaction by food components may be related to the high level of expression of CYP3A4 in the small intestine, as well as its broad substrate specificity, as CYP3A4 is responsible for the metabolism of more than 50% of clinical pharmaceuticals. This review article summarizes the findings obtained to date concerning food-drug interactions and their clinical implications. It seems likely that more information regarding such interactions will accumulate in the future, and awareness is necessary for achieving optimal drug therapy. PMID:15663291

  15. Acetaminophen induces accumulation of functional rat CYP3A via polyubiquitination dysfunction.

    PubMed

    Santoh, Masataka; Sanoh, Seigo; Takagi, Masashi; Ejiri, Yoko; Kotake, Yaichiro; Ohta, Shigeru

    2016-01-01

    Acetaminophen (APAP) is extensively used as an analgesic and antipyretic drug. APAP is partly metabolized to N-acetyl-p-benzoquinone imine, a reactive metabolite, by cytochrome P450 (CYP) 1A2, 2E1 and 3A4. Some reports have indicated that CYP3A protein production and its metabolic activity are induced by APAP in rats in vivo. The CYP3A subfamily is believed to be transcriptionally regulated by chemical compounds. However, the mechanism underlying these responses is not completely understood. To clarify these mechanisms, we assessed the effects of APAP on CYP3A1/23 protein levels according to mRNA synthesis and protein degradation in rat hepatocyte spheroids, a model of liver tissue, in vivo. APAP induced CYP3A1/23 protein levels and metabolic activity. However, no change in CYP3A1/23 mRNA levels was observed. Moreover, APAP prolonged the half-life of CYP3A1/23 protein. CYP3A is known to be degraded via the ubiquitin-proteasome system. APAP significantly was found to decrease levels of polyubiquitinated CYP3A1/23 and glycoprotein 78, an E3 ligase of CYP3A1/23. These findings demonstrate that APAP induces accumulation of functional CYP3A protein via inhibition of protein degradation. Our findings may lead to the determination of novel drug-drug interactions with APAP. PMID:26900149

  16. Acetaminophen induces accumulation of functional rat CYP3A via polyubiquitination dysfunction

    PubMed Central

    Santoh, Masataka; Sanoh, Seigo; Takagi, Masashi; Ejiri, Yoko; Kotake, Yaichiro; Ohta, Shigeru

    2016-01-01

    Acetaminophen (APAP) is extensively used as an analgesic and antipyretic drug. APAP is partly metabolized to N-acetyl-p-benzoquinone imine, a reactive metabolite, by cytochrome P450 (CYP) 1A2, 2E1 and 3A4. Some reports have indicated that CYP3A protein production and its metabolic activity are induced by APAP in rats in vivo. The CYP3A subfamily is believed to be transcriptionally regulated by chemical compounds. However, the mechanism underlying these responses is not completely understood. To clarify these mechanisms, we assessed the effects of APAP on CYP3A1/23 protein levels according to mRNA synthesis and protein degradation in rat hepatocyte spheroids, a model of liver tissue, in vivo. APAP induced CYP3A1/23 protein levels and metabolic activity. However, no change in CYP3A1/23 mRNA levels was observed. Moreover, APAP prolonged the half-life of CYP3A1/23 protein. CYP3A is known to be degraded via the ubiquitin-proteasome system. APAP significantly was found to decrease levels of polyubiquitinated CYP3A1/23 and glycoprotein 78, an E3 ligase of CYP3A1/23. These findings demonstrate that APAP induces accumulation of functional CYP3A protein via inhibition of protein degradation. Our findings may lead to the determination of novel drug–drug interactions with APAP. PMID:26900149

  17. Loss of CYP3A7 gene induction by 1,25-dihydroxyvitamin D3 is caused by less binding of VDR to the proximal ER6 in CYP3A7 gene.

    PubMed

    Hara, Hirokazu; Yasunami, Yoko; Adachi, Tetsuo

    2004-09-01

    Cytochrome P450 3A4 and 3A7 (CYP3A4 and CYP3A7, respectively) are predominant forms in the human adult and fetal liver, respectively. 1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to be a potent inducer of CYP3A4 in human colon carcinoma Caco-2 via vitamin D receptor (VDR). However, whether CYP3A7 is inducible by 1,25(OH)(2)D(3) has not yet been elucidated. In the present study, we examined the effect of 1,25(OH)(2)D(3) on CYP3A7 gene expression in Caco-2 cells, which express CYP3A4 and CYP3A7 mRNAs. 1,25(OH)(2)D(3) hardly induced the expression of CYP3A7 mRNA in contrast to the marked induction of CYP3A4 mRNA. Reporter assay using 5'-franking region CYP3A4 and CYP3A7 genes also revealed that 1,25(OH)(2)D(3) activates CYP3A4 promoter, but not CYP3A7 promoter, which has two mutations in the proximal ER6 site compared with CYP3A4 promoter. In addition, we found that the binding of VDR to the proximal ER6 in CYP3A7 gene was markedly less than that to the proximal ER6 in CYP3A4 gene using gel shift assay. Taken together, the decrease of VDR binding to the proximal ER6 caused by the mutation results in the loss of CYP3A7 gene activation by 1,25(OH)(2)D(3). PMID:15358113

  18. Cytochrome P450 CYP3A in human renal cell cancer

    PubMed Central

    Murray, G I; McFadyen, M C E; Mitchell, R T; Cheung, Y-L; Kerr, A C; Melvin, W T

    1999-01-01

    Renal cell cancer is the main malignant tumour of the kidney and has an increasing incidence. This type of tumour has a poor prognosis and shows intrinsic resistance to several anti-cancer drugs. The CYP3A P450 family, which consists of three closely related forms, is involved in the oxidative activation and deactivation of a variety of carcinogens and several anti-cancer drugs. In this study the presence and cellular localization of CYP3A has been investigated using a combination of immunohistochemistry, immunoblotting and reverse transcriptase polymerase chain reaction (RT-PCR) in renal cell cancer and corresponding normal kidney. CYP3A was consistently expressed in both renal call cancer and in normal kidney. In renal cell cancer, CYP3A was localized to tumour cells and in normal kidney the predominant cellular localization of CYP3A was to proximal tubular epithelial cells. RT-PCR showed that both CYP3A5 mRNA and CYP3A7 mRNA were consistently present in both tumour and normal samples, while CYP3A4 mRNA was present in 65% of tumours and 90% of normal samples. This study indicates that individual members of the CYP3A family are expressed in renal cell cancer. The presence of CYP3A in renal cell cancer might be important in the metabolic potentiation as well as the detoxification of chemotherapeutic agents used to renal cancer. © 1999 Cancer Research Campaign PMID:10206301

  19. 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

  20. Predicting the "First dose in children" of CYP3A-metabolized drugs: Evaluation of scaling approaches and insights into the CYP3A7-CYP3A4 switch at young ages.

    PubMed

    Strougo, Ashley; Yassen, Ashraf; Monnereau, Claire; Danhof, Meindert; Freijer, Jan

    2014-09-01

    First-dose-in-children relies on the prediction of clearance from adults for which little information is available on the accuracy of the scaling-approaches applied. For CYP3A-metabolized compounds, scaling of clearance is further challenged by different isoforms and by the CYP3A7 to CYP3A4 switch at young ages. This investigation aimed to evaluate the accuracy of two frequently used scaling approaches and to gain insights into the ontogeny of CYP3A. Hence, a literature database was compiled containing 203 clearance values from term-neonates to adults for 18 CYP3A-metabolized compounds. The clearances in adults were scaled to children using (i) allometric scaling plus maturation function and (ii) a mechanistic approach based on the well-stirred model. Three maturation functions were separately evaluated. In children >3 months, all approaches were interchangeable heeding the maturation function applied and biases were mostly observed in children <3 months. The results from a sensitivity analysis indicate that these biases are possibly caused by disregarding the CYP3A7 activity which could account for up to 86% of the metabolism in term-neonates. Only the mechanistic approach using an overall-CYP3A maturation function led to unbiased predictions of clearances across all ages. The current investigation adds to the predictions of the first-dose-in-children of compounds (partially) metabolized by CYP3A. PMID:24676942

  1. 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

  2. Unexpected contribution of cytochrome P450 enzymes CYP11B2 and CYP21, as well as CYP3A4 in xenobiotic androgen elimination - insights from metandienone metabolism.

    PubMed

    Parr, Maria Kristina; Zöllner, Andy; Fusshöller, Gregor; Opfermann, Georg; Schlörer, Nils; Zorio, Mirela; Bureik, Matthias; Schänzer, Wilhelm

    2012-09-18

    The metabolism of a variety of anabolic steroids frequently misused for doping purposes has been investigated in the last years. This research mainly focused on main and long-term metabolites suitable for detection, but detailed clearance mechanisms have rarely been elucidated. Recent studies on metandienone focused on the identification of 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20βOH-NorMD) as long-term metabolite, however, the metabolic pathway of its generation remained unclear. Metandienone and its Wagner-Meerwein rearrangement product 17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one (NorMD) were hydroxylated by different human cytochrome P450 enzymes (CYPs). Some of their hydroxylation products were chemically synthesized and characterized by mass spectrometry to allow for their trace detection in urine samples. Following oral administration of metandienone or NorMD in one human volunteer each the post administration urines were checked for the presence of those hydroxylated metabolites using GC-MS/MS analysis. The human mitochondrial steroid hydroxylating enzymes CYP11B1 and CYP11B2 were capable to metabolize metandienone leading to the formation of 11β-hydroxymetandienone and 18-hydroxymetandienone. Following Wagner-Meerwein rearrangement, the resulting products could be assigned to 20βOH-NorMD and 11βOH-NorMD. The contribution of CYP11B1 and CYP11B2 in human metabolism of metandienone was confirmed by analysis of post-administration samples of metandienone and NorMD. Combined with the results from a previous study, enzymatic pathways were identified that involve CYP21 and CYP3A4 in the hydroxylation of NorMD, while CYP21, CYP3A4 and CYP11B2 take part in 20βOH-NorMD generation from MD. The current study represents a valuable contribution to the elucidation of clearance mechanisms of anabolic steroids and also indicates that mainly non-liver CYPs seem to be involved in these processes. PMID:22885098

  3. Rate of onset of inhibition of gut-wall and hepatic CYP3A by clarithromycin

    PubMed Central

    Quinney, Sara K.; Malireddy, Srikar R.; Vuppalanchi, Raj; Hamman, Mitchell A.; Chalasani, Naga; Gorski, J. Christopher; Hall, Stephen D.

    2013-01-01

    Aims To determine the extent and time-course of hepatic and intestinal cytochrome P450 3A (CYP3A) inactivation due to the mechanism-based inhibitor clarithromycin. Methods Intestinal and hepatic CYP3A inhibition was examined in 12 healthy volunteers following the administration of single and multiple doses of oral clarithromycin (500 mg). Intestinal biopsies were obtained under intravenous midazolam sedation at baseline and after the first dose, on days 2–4, and on days 6–8 of the clarithromycin treatment. The formation of 1′-hydroxymidazolam in biopsy tissue and the serum 1′-hydroxymidazolam:midazolam ratio were indicators of intestinal and hepatic CYP3A activity, respectively. Results Intestinal CYP3A activity decreased by 64 % (p=0.0029) following the first dose of clarithromycin, but hepatic CYP3A activity did not significantly decrease. Repeated dosing of clarithromycin caused a significant decrease in hepatic CYP3A activity (p=0.005), while intestinal activity showed little further decline. The CYP3A5 or CYP3A4*1B genotype were unable to account for inter-individual variability in CYP3A activity. Conclusions Following the administration of clarithromycin, the onset of hepatic CYP3A inactivation is delayed compared to that of intestinal CYP3A. The time-course of drug–drug interactions due to clarithromycin will vary with the relative contribution of intestinal and hepatic CYP3A to the clearance and bioavailability of a victim substrate. PMID:22777148

  4. 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

  5. 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.

  6. 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

  7. 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

  8. The Effect of Ritonavir on Human CYP2B6 Catalytic Activity: Heme Modification Contributes to the Mechanism-Based Inactivation of CYP2B6 and CYP3A4 by Ritonavir

    PubMed Central

    Lin, Hsia-lien; D’Agostino, Jaime; Kenaan, Cesar; Calinski, Diane

    2013-01-01

    The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a KI of 0.9 μM, a kinact of 0.05 min−1, and a partition ratio of approximately 3. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis showed that the protonated molecular ion of RTV exhibits an m/z at 721 and its two major metabolites are an oxidation product with MH+ at m/z 737 and a deacylated product with MH+ at m/z 580. Inactivation of CYP2B6 by incubation with 10 μM RTV for 10 min resulted in an approximately 50% loss of catalytic activity and native heme, but no modification of the apoprotein was observed. RTV was found to be a potent mixed-type reversible inhibitor (Ki = 0.33 μM) and a type II ligand (spectral dissociation constant-Ks = 0.85 μM) of CYP2B6. Although previous studies have demonstrated that RTV is a potent mechanism-based inactivator of CYP3A4, the molecular mechanism responsible for the inactivation has not been determined. Here, we provide evidence that RTV inactivation of CYP3A4 is due to heme destruction with the formation of a heme-protein adduct. Similar to CYP2B6, there is no significant modification of the apoprotein. Furthermore, LC-MS/MS analysis revealed that both CYP3A4 and human liver microsomes form an RTV-glutathione conjugate having a MH+ at m/z 858 during metabolism of RTV, suggesting the formation of an isocyanate intermediate leading to formation of the conjugate. PMID:23886699

  9. Arsenite decreases CYP3A23 induction in cultured rat hepatocytes by transcriptional and translational mechanisms

    SciTech Connect

    Noreault, Trisha L.; Nichols, Ralph C.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Peter R.; Evans, Ronald M.; Sinclair, Jacqueline F. . E-mail: JSINC@dartmouth.edu

    2005-12-01

    Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 {mu}M arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.

  10. CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation

    PubMed Central

    Zia, Hamid; Murray, Graeme I; Vyhlidal, Carrie A; Leeder, J Steven; Anwar, Ahmed E; Bui, Marilyn M; Ahmed, Atif A

    2015-01-01

    Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti-cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra-pelvic). None of the biomarkers showed any correlation with overall or disease-free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours. PMID:25670065

  11. CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation.

    PubMed

    Zia, Hamid; Murray, Graeme I; Vyhlidal, Carrie A; Leeder, J Steven; Anwar, Ahmed E; Bui, Marilyn M; Ahmed, Atif A

    2015-04-01

    Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti-cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra-pelvic). None of the biomarkers showed any correlation with overall or disease-free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours. PMID:25670065

  12. PXR variants and artemisinin use in Vietnamese subjects: frequency distribution and impact on the interindividual variability of CYP3A induction by artemisinin.

    PubMed

    Piedade, Rita; Schaeffeler, Elke; Winter, Stefan; Asimus, Sara; Schwab, Matthias; Ashton, Michael; Burk, Oliver; Gil, José P

    2012-04-01

    Artemisinins induce drug metabolism through the activation of the pregnane X receptor (PXR) in vitro. Here, we report the resequencing and genotyping of PXR variants in 75 Vietnamese individuals previously characterized for CYP3A enzyme activity after artemisinin exposure. We identified a total of 31 PXR variants, including 5 novel single nucleotide polymorphisms (SNPs), and we identified significantly different allele frequencies relative to other ethnic groups. A trend of significance was observed between the level of CYP3A4 induction by artemisinin and two PXR variants, the 8118C→T (Y328Y) and 10719A→G variants. PMID:22252826

  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. High-dose green tea polyphenol intake decreases CYP3A expression in a liver-specific manner with increases in blood substrate drug concentrations.

    PubMed

    Ikarashi, Nobutomo; Ogawa, Sosuke; Hirobe, Ryuta; Kusunoki, Yoshiki; Kon, Risako; Ochiai, Wataru; Sugiyama, Kiyoshi

    2016-06-30

    In recent years, the intake of functional foods containing high-doses of green tea polyphenols (GP) has been increasing. In this study, the long-term safety of high-dose GP was assessed from a pharmacokinetic point of view by focusing on the drug-metabolizing enzyme, cytochrome P450 (CYP). Mice were fed a diet containing 3% GP for 4weeks, and the CYP expression levels and activity were determined. The GP-treated group showed a significant decrease in the hepatic CYP3A and an increase in the hepatic CYP2C expression compared with the control group. CYP1A, CYP2D, and CYP2E expression were not different between the GP-treated and the control groups. In the small intestine, there were no differences in the CYP3A protein levels between the groups. The increase in the plasma triazolam concentration in the GP-treated group was observed. Although no changes were found in the hepatic CYP3A levels in mice receiving a diet containing 0.1% GP for 4weeks, a significant decrease was seen in the hepatic CYP3A level in mice receiving a diet containing 3% GP for only 1week. This study revealed that the intake of a high-dose GP results in a liver-specific decrease in the CYP3A expression level. The results also indicated that the effects of GP on CYP3A were not observed following the intake of a low-dose GP. In the future, caution should be taken in cases when functional foods containing a high-dose GP are concomitantly consumed with a CYP3A substrate drug. PMID:27130545

  15. Population pharmacokinetic modelling to assess the impact of CYP2D6 and CYP3A metabolic phenotypes on the pharmacokinetics of tamoxifen and endoxifen

    PubMed Central

    ter Heine, Rob; Binkhorst, Lisette; de Graan, Anne Joy M; de Bruijn, Peter; Beijnen, Jos H; Mathijssen, Ron H J; Huitema, Alwin D R

    2014-01-01

    Aims Tamoxifen is considered a pro-drug of its active metabolite endoxifen. The major metabolic enzymes involved in endoxifen formation are CYP2D6 and CYP3A. There is considerable evidence that variability in activity of these enzymes influences endoxifen exposure and thereby may influence the clinical outcome of tamoxifen treatment. We aimed to quantify the impact of metabolic phenotype on the pharmacokinetics of tamoxifen and endoxifen. Methods We assessed the CYP2D6 and CYP3A metabolic phenotypes in 40 breast cancer patients on tamoxifen treatment with a single dose of dextromethorphan as a dual phenotypic probe for CYP2D6 and CYP3A. The pharmacokinetics of dextromethorphan, tamoxifen and their relevant metabolites were analyzed using non-linear mixed effects modelling. Results Population pharmacokinetic models were developed for dextromethorphan, tamoxifen and their metabolites. In the final model for tamoxifen, the dextromethorphan derived metabolic phenotypes for CYP2D6 as well as CYP3A significantly (P < 0.0001) explained 54% of the observed variability in endoxifen formation (inter-individual variability reduced from 55% to 25%). Conclusions We have shown that not only CYP2D6, but also CYP3A enzyme activity influences the tamoxifen to endoxifen conversion in breast cancer patients. Our developed model may be used to assess separately the impact of CYP2D6 and CYP3A mediated drug–drug interactions with tamoxifen without the necessity of administering this anti-oestrogenic drug and to support Bayesian guided therapeutic drug monitoring of tamoxifen in routine clinical practice. PMID:24697814

  16. The influence of standardized Valeriana officinalis extract on the CYP3A1 gene expression by nuclear receptors in in vivo model.

    PubMed

    Bogacz, Anna; Mrozikiewicz, Przemyslaw M; Karasiewicz, Monika; Bartkowiak-Wieczorek, Joanna; Majchrzycki, Marian; Mikolajczak, Przemyslaw L; Ozarowski, Marcin; Grzeskowiak, Edmund

    2014-01-01

    Valeriana officinalis is one of the most popular medicinal plants commonly used as a sedative and sleep aid. It is suggested that its pharmacologically active compounds derived from the root may modulate the CYP3A4 gene expression by activation of pregnane X receptor (PXR) or constitutive androstane receptor (CAR) and lead to pharmacokinetic herb-drug interactions. The aim of the study was to determine the influence of valerian on the expression level of CYP3A1 (homologue to human CYP3A4) as well as nuclear receptors PXR, CAR, RXR, GR, and HNF-4α. Male Wistar rats were given standardized valerian extract (300 mg/kg/day, p.o.) for 3 and 10 days. The expression in liver tissue was analyzed by using real-time PCR. Our result showed a decrease of CYP3A1 expression level by 35% (P = 0.248) and 37% (P < 0.001), respectively. Moreover, Valeriana exhibited statistically significant reduction in RXR (approximately 28%) only after 3-day treatment. We also demonstrated a decrease in the amount HNF-4α by 22% (P = 0.005) and 32% (P = 0.012), respectively. In case of CAR, the increase of expression level by 46% (P = 0.023) was noted. These findings suggest that Valeriana officinalis extract can decrease the CYP3A4 expression and therefore may lead to interactions with synthetic drugs metabolized by this enzyme. PMID:25302309

  17. The Influence of Standardized Valeriana officinalis Extract on the CYP3A1 Gene Expression by Nuclear Receptors in In Vivo Model

    PubMed Central

    Mrozikiewicz, Przemyslaw M.; Karasiewicz, Monika; Mikolajczak, Przemyslaw L.; Ozarowski, Marcin; Grzeskowiak, Edmund

    2014-01-01

    Valeriana officinalis is one of the most popular medicinal plants commonly used as a sedative and sleep aid. It is suggested that its pharmacologically active compounds derived from the root may modulate the CYP3A4 gene expression by activation of pregnane X receptor (PXR) or constitutive androstane receptor (CAR) and lead to pharmacokinetic herb-drug interactions. The aim of the study was to determine the influence of valerian on the expression level of CYP3A1 (homologue to human CYP3A4) as well as nuclear receptors PXR, CAR, RXR, GR, and HNF-4α. Male Wistar rats were given standardized valerian extract (300 mg/kg/day, p.o.) for 3 and 10 days. The expression in liver tissue was analyzed by using real-time PCR. Our result showed a decrease of CYP3A1 expression level by 35% (P = 0.248) and 37% (P < 0.001), respectively. Moreover, Valeriana exhibited statistically significant reduction in RXR (approximately 28%) only after 3-day treatment. We also demonstrated a decrease in the amount HNF-4α by 22% (P = 0.005) and 32% (P = 0.012), respectively. In case of CAR, the increase of expression level by 46% (P = 0.023) was noted. These findings suggest that Valeriana officinalis extract can decrease the CYP3A4 expression and therefore may lead to interactions with synthetic drugs metabolized by this enzyme. PMID:25302309

  18. 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

  19. Knockout of mouse Cyp3a gene enhances synthesis of cholesterol and bile acid in the liver[S

    PubMed Central

    Hashimoto, Mari; Kobayashi, Kaoru; Watanabe, Mio; Kazuki, Yasuhiro; Takehara, Shoko; Inaba, Asumi; Nitta, Shin-ichiro; Senda, Naoto; Oshimura, Mitsuo; Chiba, Kan

    2013-01-01

    Here, we studied the effects of cytochrome P450 (CYP)3A deficiency on the mRNA expression of genes encoding regulators of hepatic cholesterol levels using Cyp3a-knockout (Cyp3a−/−) mice. The mRNA expression levels of genes encoding enzymes involved in cholesterol biosynthesis in the livers of Cyp3a−/− mice were higher than those of wild-type (WT) mice. Nuclear levels of sterol regulatory element-binding protein-2 (SREBP-2), which enhances cholesterol biosynthesis, were also higher in the livers of Cyp3a−/− mice. Binding of SREBP-2 to the Hmgcs1 gene promoter was more abundant in the livers of Cyp3a−/− mice. These results suggest that deficiency of CYP3A enzymes enhances transcription of genes encoding enzymes involved in cholesterol biosynthesis via activation of SREBP-2. On the other hand, hepatic cholesterol levels in Cyp3a−/− mice were 20% lower than those in WT mice. The mRNA expression levels of genes encoding enzymes involved in bile acid synthesis, plasma levels of 7α-hydroxy-4-cholesten-3-one and hepatic levels of total bile acid were significantly higher in Cyp3a−/− mice than in WT mice. These findings suggest that reduction of hepatic total cholesterol in Cyp3a−/− mice would be the consequence of enhanced bile acid synthesis. Therefore, CYP3A enzymes appear to play roles in the synthesis of cholesterol and bile acid in vivo. PMID:23709690

  20. A PBPK Model to Predict Disposition of CYP3A-Metabolized Drugs in Pregnant Women: Verification and Discerning the Site of CYP3A Induction.

    PubMed

    Ke, A B; Nallani, S C; Zhao, P; Rostami-Hodjegan, A; Unadkat, J D

    2012-01-01

    Besides logistical and ethical concerns, evaluation of safety and efficacy of medications in pregnant women is complicated by marked changes in pharmacokinetics (PK) of drugs. For example, CYP3A activity is induced during the third trimester (T3). We explored whether a previously published physiologically based pharmacokinetic (PBPK) model could quantitatively predict PK profiles of CYP3A-metabolized drugs during T3, and discern the site of CYP3A induction (i.e., liver, intestine, or both). The model accounted for gestational age-dependent changes in maternal physiological function and hepatic CYP3A activity. For model verification, mean plasma area under the curve (AUC), peak plasma concentration (Cmax), and trough plasma concentration (Cmin) of midazolam (MDZ), nifedipine (NIF), and indinavir (IDV) were predicted and compared with published studies. The PBPK model successfully predicted MDZ, NIF, and IDV disposition during T3. A sensitivity analysis suggested that CYP3A induction in T3 is most likely hepatic and not intestinal. Our PBPK model is a useful tool to evaluate different dosing regimens during T3 for drugs cleared primarily via CYP3A metabolism.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e3; doi:10.1038/psp.2012.2; advance online publication 26 September 2012. PMID:23835883

  1. 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

  2. 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

  3. 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

  4. The effects on metabolic clearance when administering a potent CYP3A autoinducer with the prototypic CYP3A inhibitor, ketoconazole.

    PubMed

    Ayan-Oshodi, Mosun A; Willis, Brian A; Annes, William F; Lowe, Stephen L; Friedrich, Stuart; de la Peña, Amparo; Zhang, Wei; Brown, Thomas; Wise, Stephen D; Hall, Stephen D

    2012-10-01

    Ketoconazole is recognized as the prototypical CYP3A inhibitor and is often used to determine the metabolic CYP3A liabilities of new chemical entities in preclinical and clinical studies. Ketoconazole has been commercially available for approximately 30 years and was marketed before drug-metabolizing enzymes were well characterized; consequently, little is known about its metabolic profile. Semagacestat, a γ-secretase inhibitor investigated as a potential therapy for Alzheimer's disease, was determined to be a potent CYP3A autoinducer in human hepatocytes. Two human studies were conducted to assess the induction potential of semagacestat. In the first study (study 1, n = 20), semagacestat increased the mean apparent clearance (CL/F) of oral midazolam (76-324 l/h) and nifedipine (63-229 l/h) as predicted from hepatocytes. In a second (steady-state) study (study 2, n = 20), semagacestat CL/F increased from 22 after a single dose to 31 l/h. Ketoconazole decreased semagacestat CL/F by 32% after a single dose of semagacestat [geometric means ratio estimate, 0.68; 90% confidence interval (CI). 0.64, 0.73] and 46% at steady state (geometric means ratio estimate. 0.54; 90% CI, 0.51, 0.58). Ketoconazole area under the concentration-time curve over 8 h decreased 49% from first to last day of semagacestat dosing. Semagacestat significantly increases the oral clearance of CYP3A substrates, confirming its inducer designation. More importantly, when administered with a potent CYP3A inducer at steady state, ketoconazole's plasma exposure decreased, indicating that it may also be cleared by CYP3A, other inducible enzymes or transporters, or both. PMID:22789530

  5. Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A.

    PubMed

    Jonsson-Schmunk, Kristina; Wonganan, Piynauch; Choi, Jin Huk; Callahan, Shellie M; Croyle, Maria A

    2016-05-01

    Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrinβ-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states. PMID:26868618

  6. 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

  7. Transient inhibition of cyp3a in rats by star fruit juice.

    PubMed

    Hidaka, Muneaki; Okumura, Manabu; Ogikubo, Tetsuya; Kai, Hirofumi; Fujita, Ken-ichi; Iwakiri, Tomomi; Yamasaki, Keishi; Setoguchi, Nao; Matsunaga, Naoya; Arimori, Kazuhiko

    2006-03-01

    Star fruit juice is a potent in vitro inhibitor of CYP3A; however, few reports are available on the inhibition of CYP3A activities by star fruit juice in vivo. Therefore, in this study, we investigated the CYP3A-mediated star fruit-drug interaction in vivo. The effect of star fruit juice on carbamazepine pharmacokinetics was examined in rats. In comparison with water, the area under the concentration-time curve (AUC) of carbamazepine was approximately 1.3-fold greater when star fruit juice (2 ml) was orally administered 1 h before the oral administration of carbamazepine (50 mg/kg). In contrast, the elimination half-life of carbamazepine and the AUC ratio of carbamazepine 10,11-epoxide to carbamazepine were not altered by the administration of star fruit juice. These results suggest that star fruit juice impairs the function of enteric CYP3A, but not of hepatic CYP3A. In addition, we evaluated the time course of recovery of CYP3A activity that was reduced after the treatment with star fruit juice. The inhibition by star fruit juice was recovered within approximately 24 h. These data suggest that the effect of star fruit juice is mainly reversible and transient. Thus, we discovered that star fruit juice alters the carbamazepine pharmacokinetics in rats. PMID:16326816

  8. 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

  9. 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

  10. 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

  11. PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C from Peucedanum praeruptorum Dunn

    PubMed Central

    Huang, Ling; Wu, Qian; Li, Yu-Hua; Wang, Yi-Tao; Bi, Hui-Chang

    2013-01-01

    We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on the CYP3A gene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level of CYP3a11 gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs. PMID:24379885

  12. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

    SciTech Connect

    Noreault-Conti, Trisha L.; Jacobs, Judith M.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Jacqueline F.; Nichols, Ralph C. . E-mail: ralph.c.nichols@dartmouth.edu

    2006-12-15

    Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

  13. 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

  14. 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

  15. DDPH, a novel antihypertensive agent, is a potential dual inhibitor of hepatic CYP2D and CYP3A.

    PubMed

    Zhu, Yinsong; Hu, Jiong; He, Wenjuan; Gao, Xiujuan; Ren, Ping; Chen, Hui

    2016-03-01

    DDPH (1-(2, 6-dimethylphenoxy)-2-(3, 4-dimethoxyphenylethylamino) propane hydrochloride) is a promising novel antihypertensive agent, with potent antihypertensive, neuroprotective and cardioprotective effects. This study aimed to investigate the effects of DDPH on the expression and activity of hepatic cytochrome P450 (CYP) isoforms and evaluate the metabolic drug-drug interactions of DDPH with propafenone. Our results showed that orally administered DDPH (12.5-50 mg/kg/d) for 7 days significantly inhibited CYP2D1 and CYP3A1 activity and mRNA and protein expression but weakly increased CYP1A2 activity and expression in rats. Enzyme kinetics studies showed that DDPH was a competitive inhibitor of CYP2D1 and mixed inhibitor of CYP3A1 in rat liver microsomes with Ki values of 3.70 ± 0.42 μM and 4.79 ± 1.10 μM respectively. With human liver microsomes, DDPH was a noncompetitive inhibitor of CYP2D6 (Ki = 0.85 ± 0.06 μM) and mixed inhibitor of CYP3A (Ki = 2.15 ± 0.41 μM). Further in vivo study showed that oral administration of DDPH (12.5-50 mg/kg/d) for 7 days in rats significantly increased the area under the plasma concentration-time curve (AUC) of propafenone by 25.4%-63.9%, with a concomitant decrease in the plasma clearance. In conclusion, the results indicated that DDPH inhibited CYP2D and CYP3A activities and down-regulated their protein expression and mRNA transcription. DDPH might cause metabolic drug-drug interactions through modulation of the activity and expression of CYP2D and CYP3A. This information could be important in the prediction of possible drug-drug interactions as well as for the effective therapy and the limitation of toxicity of DDPH in clinical practice. PMID:26827781

  16. 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

  17. 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

  18. 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

  19. 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

  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. Identification of genomic polymorphisms in upstream elements of the bovine CYP3A28 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cytochrome P450 (CYP) enzyme family consists of a group of heme-containing monooxygenases that participate in metabolism and phase I detoxification. Previously, our group reported that the coding sequence for the CYP3A28 gene in cattle was polymorphic and related to cattle performance on toxic t...

  2. 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

  3. 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

  4. 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

  5. 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

  6. Cytochrome P450 Allele CYP3A7*1C Associates with Adverse Outcomes in Chronic Lymphocytic Leukemia, Breast, and Lung Cancer.

    PubMed

    Johnson, Nichola; De Ieso, Paolo; Migliorini, Gabriele; Orr, Nick; Broderick, Peter; Catovsky, Daniel; Matakidou, Athena; Eisen, Timothy; Goldsmith, Christy; Dudbridge, Frank; Peto, Julian; Dos-Santos-Silva, Isabel; Ashworth, Alan; Ross, Gillian; Houlston, Richard S; Fletcher, Olivia

    2016-03-15

    CYP3A enzymes metabolize endogenous hormones and chemotherapeutic agents used to treat cancer, thereby potentially affecting drug effectiveness. Here, we refined the genetic basis underlying the functional effects of a CYP3A haplotype on urinary estrone glucuronide (E1G) levels and tested for an association between CYP3A genotype and outcome in patients with chronic lymphocytic leukemia (CLL), breast, or lung cancers. The most significantly associated SNP was rs45446698, an SNP that tags the CYP3A7*1C allele; this SNP was associated with a 54% decrease in urinary E1G levels. Genotyping this SNP in 1,008 breast cancer, 1,128 lung cancer, and 347 CLL patients, we found that rs45446698 was associated with breast cancer mortality (HR, 1.74; P = 0.03), all-cause mortality in lung cancer patients (HR, 1.43; P = 0.009), and CLL progression (HR, 1.62; P = 0.03). We also found borderline evidence of a statistical interaction between the CYP3A7*1C allele, treatment of patients with a cytotoxic agent that is a CYP3A substrate, and clinical outcome (Pinteraction = 0.06). The CYP3A7*1C allele, which results in adult expression of the fetal CYP3A7 gene, is likely to be the functional allele influencing levels of circulating endogenous sex hormones and outcome in these various malignancies. Further studies confirming these associations and determining the mechanism by which CYP3A7*1C influences outcome are required. One possibility is that standard chemotherapy regimens that include CYP3A substrates may not be optimal for the approximately 8% of cancer patients who are CYP3A7*1C carriers. PMID:26964624

  7. 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

  8. 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

  9. 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

  10. 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

  11. In vitro and in vivo drug interactions involving human CYP3A.

    PubMed

    Thummel, K E; Wilkinson, G R

    1998-01-01

    Cytochrome P4503A (CYP3A) is importantly involved in the metabolism of many chemically diverse drugs administered to humans. Moreover, its localization in high amounts both in the small intestinal epithelium and liver makes it a major contributor to presystemic elimination following oral drug administration. Drug interactions involving enzyme inhibition or induction are common following the coadministration of two or more CYP3A substrates. Studies using in vitro preparations are useful in identifying such potential interactions and possibly permitting extrapolation of in vitro findings to the likely in vivo situation. Even if accurate quantitative predictions cannot be made, several classes of drugs can be expected to result in a drug interaction based on clinical experience. In many instances, the extent of such drug interactions is sufficiently pronounced to contraindicate the therapeutic use of the involved drugs. PMID:9597161

  12. Genomewide Association Study of Tacrolimus Concentrations in African American Kidney Transplant Recipients Identifies Multiple CYP3A5 Alleles

    PubMed Central

    Oetting, W. S.; Schladt, D. P.; Guan, W.; Miller, M. B.; Remmel, R. P.; Dorr, C.; Sanghavi, K.; Mannon, R. B.; Herrera, B.; Matas, A. J.; Salomon, D. R.; Kwok, P.-Y.; Keating, B. J.; Israni, A. K.; Jacobson, P. A.

    2016-01-01

    We previously reported that tacrolimus (TAC) trough blood concentrations for African American (AA) kidney allograft recipients were lower than those observed in white patients. Subtherapeutic TAC troughs may be associated with acute rejection (AR) and AR-associated allograft failure. This variation in TAC troughs is due, in part, to differences in the frequency of the cytochrome P450 CYP3A5*3 allele (rs776746, expresses nonfunctional enzyme) between white and AA recipients; however, even after accounting for this variant, variability in AA-associated troughs is significant. We conducted a genomewide association study of TAC troughs in AA kidney allograft recipients to search for additional genetic variation. We identified two additional CYP3A5 variants in AA recipients independently associated with TAC troughs: CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343). All three variants and clinical factors account for 53.9% of the observed variance in troughs, with 19.8% of the variance coming from demographic and clinical factors including recipient age, glomerular filtration rate, anti-cytomegalovirus drug use, simultaneous pancreas-kidney transplant and antibody induction. There was no evidence of common genetic variants in AA recipients significantly influencing TAC troughs aside from the CYP3A gene. These results reveal that additional and possibly rare functional variants exist that account for the additional variation. PMID:26485092

  13. 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

  14. 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.

  15. 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

  16. Urinary 6β-Hydroxycortisol/Cortisol Ratio Most Highly Correlates With Midazolam Clearance Under Hepatic CYP3A Inhibition and Induction in Females: A Pharmacometabolomics Approach.

    PubMed

    Shin, Kwang-Hee; Ahn, Li Young; Choi, Man Ho; Moon, Ju-Yeon; Lee, Jieon; Jang, In-Jin; Yu, Kyung-Sang; Cho, Joo-Youn

    2016-09-01

    Endogenous metabolites of cytochrome P450 (CYP3A) are useful in predicting drug-drug interactions between in vivo CYP3A inhibitors and inducers for clinical applications of CYP3A substrate drugs. This study aimed to develop predictable markers of the magnitude of hepatic CYP3A induction and inhibition in healthy female subjects using pharmacometabolomics. Twelve female subjects received midazolam during three study phases: 1 mg midazolam (control phase), 1 mg midazolam after pretreatment with 400 mg ketoconazole once daily for 4 days (CYP3A inhibition phase), and 2.5 mg midazolam after pretreatment with 600 mg rifampicin once daily for 10 days (CYP3A induction phase). Throughout the study, blood samples were collected 24 h after midazolam administration and urine samples at 12-h intervals during the 24 h before and after midazolam administration for the analysis of endogenous steroid metabolites. A statistical model was generated to predict midazolam clearance using measurements of endogenous metabolites associated with the inhibition and induction of CYP3A. Mean midazolam clearance decreased to ∼20% of control levels during the inhibition phase and increased more than 2-fold during the induction phase. Of the urine and plasma metabolites measured, the 6β-hydroxycortisol/cortisol ratio was most significantly correlated with midazolam clearance during hepatic CYP3A inhibition and induction. Our results suggest that the urinary 6β-hydroxycortisol/cortisol ratio is the best predictor of hepatic CYP3A activity under both maximal inhibition and maximal induction. Furthermore, the predictive model including 6β-hydroxycortisol/cortisol as a covariate could be applied to predict the magnitude of CYP3A-mediated drug interactions. PMID:27317471

  17. 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

  18. Chemoprotective activity of boldine: modulation of drug-metabolizing enzymes.

    PubMed

    Kubínová, R; Machala, M; Minksová, K; Neca, J; Suchý, V

    2001-03-01

    Possible chemoprotective effects of the naturally occurring alkaloid boldine, a major alkaloid of boldo (Peumus boldus Mol.) leaves and bark, including in vitro modulations of drug-metabolizing enzymes in mouse hepatoma Hepa-1 cell line and mouse hepatic microsomes, were investigated. Boldine manifested inhibition activity on hepatic microsomal CYP1A-dependent 7-ethoxyresorufin O-deethylase and CYP3A-dependent testosterone 6 beta-hydroxylase activities and stimulated glutathione S-transferase activity in Hepa-1 cells. In addition to the known antioxidant activity, boldine could decrease the metabolic activation of other xenobiotics including chemical mutagens. PMID:11265593

  19. 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

  20. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes.

    PubMed

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N; Brantley, Scott J; Paine, Mary F; Oberlies, Nicholas H

    2011-02-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC(50)) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and < 10 µM, respectively, using HIM as the enzyme source and 2.8, 4.3, and < 10 µM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study. PMID:20717876

  1. Isolation and Identification of Intestinal CYP3A Inhibitors from Cranberry (Vaccinium macrocarpon) using Human Intestinal Microsomes

    PubMed Central

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N.; Brantley, Scott J.; Paine, Mary F.; Oberlies, Nicholas H.

    2010-01-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, a cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC50) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and <10 μM, respectively, using HIM as the enzyme source and was 2.8, 4.3, and <10 μM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study. PMID:20717876

  2. 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

  3. Effect of Repeated 1-h Episodes of Immobilization Stress on Activity of Glucocorticoid Metabolism Enzymes in the Liver.

    PubMed

    Tseilikman, V E; Kozochkin, D A; Sinitskii, A I; Tseylikman, O B; Lapshin, M S; Kuzina, O V; Komel'kova, M V; Telesheva, I B

    2016-03-01

    Differences in corticosterone level associated with different activity of glucocorticoid-oxidizing enzymes in the liver were revealed in rats exposed to stress. Pro-inflammatory changes in the liver were associated with enhanced CYP3A-dependent monooxygenation. PMID:27021104

  4. 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

  5. A CAR-responsive enhancer element locating approximately 31 kb upstream in the 5'-flanking region of rat cytochrome P450 (CYP) 3A1 gene.

    PubMed

    Gamou, Toshie; Habano, Wataru; Terashima, Jun; Ozawa, Shogo

    2015-04-01

    Constitutive androstane receptor (CAR) is one of the principal regulators of hepatic cytochrome P450s (CYPs) 3A (CYP3A). cDNA-mediated expression of a mature rat CAR (rCAR) into rat hepatoma cells induced CYP3A1 and CYP2B mRNAs. Aberrant rCAR failed in these inductions. Three important human CYP3A4 regulatory elements (REs), proximal ER6 (proER6), xenobiotic responsive enhancer module (XREM) and constitutive liver enhancer module (CLEM), support constitutive and inducible expression of CYP3As mediated by CAR and pregnane X receptor (PXR). NHR-scan software predicted proER6, XREM and CLEM at -255 b, -8 kb and -11.5 kb, respectively of CYP3A4, but neither XREM nor CLEM was predicted in rat CYP3A. A luciferase reporter construct carrying a 5'-flanking sequence of CYP3A1 (-31,739 to -31,585 from its transcription initiation site) revealed important for the rCAR-dependent transactivation of CYP3A1. This region includes two putative binding motifs of nuclear receptors (DR4 and DR2), a putative hepatocyte nuclear factor-1 binding motif (HNF1), nuclear factor-kappa B binding motif (NFκB), activator protein 1 binding motif (AP-1), and ecotropic viral integration site 1 binding motif (Evi1). We hereby conclude DR4 and/or DR2 motifs being primarily responsible and HNF1 being synergistically functioning elements for the rCAR-mediated transcription of CYP3A1. PMID:25989892

  6. 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

  7. CYP3A5*3 and MDR1 C3435T are influencing factors of inter-subject variability in rupatadine pharmacokinetics in healthy Chinese volunteers.

    PubMed

    Xiong, Yuqing; Yuan, Zhao; Yang, Jingzhi; Xia, Chunhua; Li, Xinhua; Huang, Shibo; Zhang, Hong; Liu, Mingyi

    2016-04-01

    Rupatadine (RUP) is an oral antihistamine and platelet-activating factor antagonist and is shown as the substrate of CYP3A5 and P-gp. The significant interindividual differences of CYP3A5 and P-gp often cause bioavailability differences of some clinical drugs. The present study is aimed to evaluate the effect of genetic polymorphisms of CYP3A5 and MDR1 on RUP pharmacokinetics in healthy male Chinese volunteer subjects. Blood samples were collected from 36 subjects before and after a single, oral RUP 10 mg dose. A PCR-RFLP assay was used to genotype CYP3A5*3 and assess MDR1 C3435T variation. A validated LC-MS/MS method quantified plasma RUP concentration. The relationship between RUP plasma concentration, pharmacokinetic parameters, and polymorphic alleles (CYP3A5 and MDR1) were assessed. Plasma RUP concentrations were lower for CYP3A5*1/*1 carriers than for CYP3A5*3/*3 and CYP3A5*1/*3 carriers. Mean C(max), AUC(0-t) and AUC(0-∞) were significantly lower, and the CLz and Vd were significantly higher in the CYP3A5 wild-type group, than in the CYP3A5 mutated group. MDR1 CT and MDR1 TT carriers had lower plasma RUP concentrations than MDR1 CC carriers. The mean C(max), AUC(0-t), AUC(0-∞) and T max were significantly lower in the TT group than in the CC and CT groups. The mean CLz was higher in the TT group than in the CC and CT groups, but not significantly. These results suggest that CYP3A5 and MDR1 may play a key role in the variability of RUP metabolism and transport, respectively. CYP3A5 and MDR1 polymorphisms may be the main explanation for the differences observed in RUP pharmacokinetics, and therefore may provide a rationale for safe and effective clinical use of RUP. Our research lays down a solid theory foundation to guide the safe and effective clinical use of RUP and a route to achieve individualized therapy. PMID:25427746

  8. 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

  9. 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

    reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase.

  10. 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

  11. 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

  12. Rhubarb decreased the systemic exposure of cyclosporine, a probe substrate of P-glycoprotein and CYP 3A.

    PubMed

    Yu, Chung-Ping; Lin, Hui-Ju; Lin, Shiuan-Pey; Shia, Chi-Sheng; Chang, Pei-Hua; Hou, Yu-Chi; Hsieh, Yo-Wen

    2016-08-01

    1. Rhubarb, rhizome of Rheum palmatum L. (RP), is an important herb in clinical Chinese medicine. 2. Cyclosporine (CSP) is an immunosuppressant with narrow therapeutic window. The oral bioavailability of CSP was associated with P-glycoprotein (P-gp) and CYP 3A4. CSP was used as a probe substrate to investigate the in vivo modulation effects of RP on P-gp and CYP 3A. 3. Rats were orally administered 2.5 mg/kg of CSP with and without 0.25 and 1.0 g/kg of RP. The blood CSP concentration was determined by a specific monoclonal fluorescence polarization immunoassay. 4. Both dosages of RP significantly decreased the Cmax and AUC0-t of CSP in rats. Mechanism studies indicated that RP activated the functions of P-gp and CYP 3A. 5. RP ingestion reduced the systemic exposure of CSP through activating P-gp and CYP 3A. PMID:26634287

  13. 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

  14. Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva.

    PubMed

    Tseng, Hua-Pin; Hseu, Tzong-Hsiung; Buhler, Donald R; Wang, Wen-Der; Hu, Chin-Hwa

    2005-06-15

    In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription. PMID:15922010

  15. Constitutive and xenobiotics-induced expression of a novel CYP3A gene from zebrafish larva

    SciTech Connect

    Tseng, H.-P.; Hseu, Tzong-Hsiung; Buhler, Donald R.; Wang, W.-D.; Hu, C.-H. . E-mail: chhu@mail.ntou.edu.tw

    2005-06-15

    In mammals, CYP3A isozymes collectively comprise the largest portion of the liver and small intestinal CYP protein. They are involved in the metabolism of an extensive range of endogenous substrates and xenobiotics and make a significant contribution to the termination of the action of steroid hormones. A full-length cDNA of CYP3A gene, named CYP3A65, was cloned from zebrafish by RT-PCR. The CYP3A65 mRNA was initially transcribed only in the liver and intestine upon hatching of the zebrafish embryos. Like the human CYP3A genes, CYP3A65 transcription in the foregut region was enhanced by treatment of the zebrafish larvae with the steroid dexamethasone and the macrocyclic antibiotic rifampicin. Differing from mammalian CYP3A genes, CYP3A65 transcription was also elicited by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) during early larval stages. Repression of AHR2 translation by antisense morpholino oligonucleotides abrogated both of constitutive and TCDD-stimulated CYP3A65 transcription in larval intestine. These findings suggested that the AHR2 signaling pathway plays an essential role in CYP3A65 transcription.

  16. 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

  17. 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

  18. Identification of a Cranberry Juice Product that Inhibits Enteric CYP3A-Mediated First-Pass Metabolism in Humans

    PubMed Central

    Ngo, Ngoc; Yan, Zhixia; Graf, Tyler N.; Carrizosa, Daniel R.; Kashuba, Angela D. M.; Dees, E. Claire; Oberlies, Nicholas H.; Paine, Mary F.

    2009-01-01

    An in vivo study in rats showed a cranberry juice product to inhibit the intestinal first-pass metabolism of the CYP3A substrate nifedipine. However, a clinical study involving the CYP3A probe substrate midazolam and a different cranberry juice product showed no interaction. Because the composition of bioactive components in natural products can vary substantially, a systematic in vitro-in vivo approach was taken to identify a cranberry juice capable of inhibiting enteric CYP3A in humans. First, the effects of five cranberry juices, coded A through E, were evaluated on midazolam 1′-hydroxylation activity in human intestinal microsomes. Juice E was the most potent, ablating activity at 0.5% juice (v/v) relative to control. Second, juice E was fractionated to generate hexane-, chloroform-, butanol-, and aqueous-soluble fractions. The hexane- and chloroform-soluble fractions at 50 μg/ml were the most potent, inhibiting by 77 and 63%, respectively, suggesting that the CYP3A inhibitors reside largely in these more lipophilic fractions. Finally, juice E was evaluated on the oral pharmacokinetics of midazolam in 16 healthy volunteers. Relative to water, juice E significantly increased the geometric mean area under the curve (AUC)0-∞ of midazolam by ∼30% (p = 0.001), decreased the geometric mean 1′-hydroxymidazolam/midazolam AUC0-∞ ratio by ∼40% (p < 0.001), and had no effect on geometric mean terminal half-life, indicating inhibition of enteric, but not hepatic, CYP3A-mediated first-pass metabolism of midazolam. This approach both showed a potential drug interaction liability with cranberry juice and substantiated that rigorous in vitro characterization of dietary substances is required before initiation of clinical drug-diet interaction studies. PMID:19114462

  19. Regulation of zebrafish CYP3A65 transcription by AHR2

    SciTech Connect

    Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin; Tzou, Wen-Shyong; Hu, Chin-Hwa

    2013-07-15

    CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′ flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. - Highlights: • Tg(CYP3A65:EGFP) and CYP3A65 exhibits identical expression pattern. • CYP3A65 can be significantly induced by TCDD or kynurenine. • The AHRE elements are required to mediate CYP3A65 transcription. • The AHR2 DNA and ligand-binding domains are required for CYP3A65 transcription. • AHRE elements are present in many teleost CYP3 genes, but not in

  20. Global Pharmacogenomics: Distribution of CYP3A5 Polymorphisms and Phenotypes in the Brazilian Population

    PubMed Central

    Suarez-Kurtz, Guilherme; Vargens, Daniela D.; Santoro, Ana Beatriz; Hutz, Mara H.; de Moraes, Maria Elisabete; Pena, Sérgio D. J.; Ribeiro-dos-Santos, Ândrea; Romano-Silva, Marco A.; Struchiner, Claudio José

    2014-01-01

    The influence of self-reported “race/color”, geographical origin and genetic ancestry on the distribution of three functional CYP3A5 polymorphisms, their imputed haplotypes and inferred phenotypes was examined in 909 healthy, adult Brazilians, self-identified as White, Brown or Black (“race/color” categories of the Brazilian census). The cohort was genotyped for CYP3A5*3 (rs776746), CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343), CYP3A5 haplotypes were imputed and CYP3A5 metabolizer phenotypes were inferred according to the number of defective CYP3A5 alleles. Estimates of the individual proportions of Amerindian, African and European ancestry were available for the entire cohort. Multinomial log-linear regression models were applied to infer the statistical association between the distribution of CYP3A5 alleles, haplotypes and phenotypes (response variables), and self-reported Color, geographical region and ancestry (explanatory variables). We found that Color per se or in combination with geographical region associates significantly with the distribution of CYP3A5 variant alleles and CYP3A5 metabolizer phenotypes, whereas geographical region per se influences the frequency distribution of CYP3A5 variant alleles. The odds of having the default CYP3A5*3 allele and the poor metabolizer phenotype increases continuously with the increase of European ancestry and decrease of African ancestry. The opposite trend is observed in relation to CYP3A5*6, CYP3A5*7, the default CYP3A5*1 allele, and both the extensive and intermediate phenotypes. No significant effect of Amerindian ancestry on the distribution of CYP3A5 alleles or phenotypes was observed. In conclusion, this study strongly supports the notion that the intrinsic heterogeneity of the Brazilian population must be acknowledged in the design and interpretation of pharmacogenomic studies, and dealt with as a continuous variable, rather than proportioned in arbitrary categories that do not capture the diversity

  1. Global pharmacogenomics: distribution of CYP3A5 polymorphisms and phenotypes in the Brazilian population.

    PubMed

    Suarez-Kurtz, Guilherme; Vargens, Daniela D; Santoro, Ana Beatriz; Hutz, Mara H; de Moraes, Maria Elisabete; Pena, Sérgio D J; Ribeiro-dos-Santos, Ândrea; Romano-Silva, Marco A; Struchiner, Claudio José

    2014-01-01

    The influence of self-reported "race/color", geographical origin and genetic ancestry on the distribution of three functional CYP3A5 polymorphisms, their imputed haplotypes and inferred phenotypes was examined in 909 healthy, adult Brazilians, self-identified as White, Brown or Black ("race/color" categories of the Brazilian census). The cohort was genotyped for CYP3A5*3 (rs776746), CYP3A5*6 (rs10264272) and CYP3A5*7 (rs41303343), CYP3A5 haplotypes were imputed and CYP3A5 metabolizer phenotypes were inferred according to the number of defective CYP3A5 alleles. Estimates of the individual proportions of Amerindian, African and European ancestry were available for the entire cohort. Multinomial log-linear regression models were applied to infer the statistical association between the distribution of CYP3A5 alleles, haplotypes and phenotypes (response variables), and self-reported Color, geographical region and ancestry (explanatory variables). We found that Color per se or in combination with geographical region associates significantly with the distribution of CYP3A5 variant alleles and CYP3A5 metabolizer phenotypes, whereas geographical region per se influences the frequency distribution of CYP3A5 variant alleles. The odds of having the default CYP3A5*3 allele and the poor metabolizer phenotype increases continuously with the increase of European ancestry and decrease of African ancestry. The opposite trend is observed in relation to CYP3A5*6, CYP3A5*7, the default CYP3A5*1 allele, and both the extensive and intermediate phenotypes. No significant effect of Amerindian ancestry on the distribution of CYP3A5 alleles or phenotypes was observed. In conclusion, this study strongly supports the notion that the intrinsic heterogeneity of the Brazilian population must be acknowledged in the design and interpretation of pharmacogenomic studies, and dealt with as a continuous variable, rather than proportioned in arbitrary categories that do not capture the diversity of the

  2. Effects of triclosan on the detoxification system in the yellow catfish (Pelteobagrus fulvidraco): expressions of CYP and GST genes and corresponding enzyme activity in phase I, II and antioxidant system.

    PubMed

    Ku, Peijia; Wu, Xiaoyan; Nie, Xiangping; Ou, Ruikang; Wang, Lan; Su, Tian; Li, Yigang

    2014-11-01

    Triclosan (TCS), a broad-spectrum antibacterial agent widely used in pharmaceuticals and personal case products (PPCPs), has been universally detected in aquatic ecosystem in recent years. Unfortunately, there is limited information about its potential impacts on responses of genes and enzymes related to fish detoxification. In the present work, we cloned CYP3A and alpha-GST of yellow catfish (Pelteobagrus fulvidraco) and tested the transcriptional expression of CYP1A, CYP3A and GST as well as the alterations of their corresponding enzymes, including ethoxyresorufin-O-deethylase (EROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (ERND), glutathione S-transferase (GST) and catalase (CAT), and also the oxidative product malondialdehyde (MDA) content in the liver of P. fulvidraco exposed to TCS. Amino acids of CYP3A and GST were deduced and phylogenetic tree was constructed respectively. High identity percent was exhibited between P. fulvidraco and other species, such as other fish, birds and mammals. Results indicated that TCS significantly elevated CYP1A and GST but decreased CYP3A expression, EROD activity and MDA content at lower concentrations of TCS at 24h. Moreover, CYP3A and GST were significantly inhibited at 72 h but induced at 168 h at lower concentrations. However, CYP3A was always induced at the highest concentration during the exposure period. Furthermore, CYP3A, GST, GST enzyme and MDA content exhibited a dose-effect relationship to some extent, but no significant responses were observed in ERND, APND and CAT except for individual treatments. Taken together, EROD was the most sensitive to TCS exposure as compared to other enzymes. Meanwhile, mRNA responses were more sensitive in yellow catfish. PMID:25064140

  3. 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

  4. The effect of piperine on midazolam plasma concentration in healthy volunteers, a research on the CYP3A-involving metabolism

    PubMed Central

    2014-01-01

    Some studies showed that piperine (the alkaloid of piper nigrum) can change the activities of microsomal enzymes. Midazolam concentration is applied as a probe to determine the CYP3A enzyme activity. This study was done to determine piperine pretreatment role on midazolam plasma concentration. Twenty healthy volunteers (14 men and 6 women) received oral dose of piperine (15 mg) or placebo for three days as pretreatment and midazolam (10 mg) on fourth day of study and the blood samples were taken at 0.5, 2.5 and 5 h after midazolam administration. The midazolam plasma levels were assayed using HPLC method (C18 analytical column, 75:25 methanol:water as mobile phase, UV detector at 242 nm wavelength and diazepam as internal standard). Data were fit in a “one-compartment PK model” using P-Pharm 1.5 software and analyzed under statistical tests. The mean ±SD of the age and body mass index were 24.3 ± 1.83 years (range: 21–28 years) and 23.46± 2.85, respectively. The duration of sedation in piperine receiving group was greater that the placebo group (188±59 vs. 102±43 min, p<0.0001). Half-life and clearance of midazolam were higher in piperine pretreatment group compared to placebo [1.88±0.03 vs. 1.71± 0.04 h (p<0.0001) and 33.62 ± 0.4 vs. 37.09 ± 1.07 ml/min (p<0.0001), respectively]. According to the results, piperine can significantly increases half-life and decreases clearance of midazolam compared to placebo. It is suggested that piperine can demonstrate those effects by inhibition CYP3A4 enzyme activity in liver microsomal system. PMID:24398010

  5. 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

  6. 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

  7. Regulation of zebrafish CYP3A65 transcription by AHR2.

    PubMed

    Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin; Tzou, Wen-Shyong; Hu, Chin-Hwa

    2013-07-15

    CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5' flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. PMID:23624173

  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. Pregnane X receptor mediated-transcription regulation of CYP3A by glycyrrhizin: a possible mechanism for its hepatoprotective property against lithocholic acid-induced injury.

    PubMed

    Wang, Yu-Guang; Zhou, Jian-Ming; Ma, Zeng-Chun; Li, Hua; Liang, Qian-De; Tan, Hong-Ling; Xiao, Cheng-Rong; Zhang, Bo-Li; Gao, Yue

    2012-10-25

    Licorice (LE) has been commonly used in traditional Chinese medicine (TCM) for over 4000 years to reconcile various drugs and for hepatic disorders. Glycyrrhizin is the main bioactive component isolated from LE herbs. In the present study we examined the effects of glycyrrhizin on pregnane X receptor (PXR)-mediated CYP3A expression and its hepatoprotective activity. Treatment of HepG2 cells with glycyrrhizin resulted in marked increase in both CYP3A4 mRNA and protein levels. The transcriptional activation of the CYP3A4 gene through glycyrrhizin is PXR-dependent, as shown in transient transfection experiments. Glycyrrhizin activates the DNA-binding capacity of the PXR for the CYP3A4 element responding to xenobiotic signals, as measured by the electrophoretic-mobility shift assay (EMSA). These results indicate that the induction of the hepatic CYP3A4 by glycyrrhizin is mediated through the activation of PXR. The next aim of the current study was to determine whether the activation of PXR and induction of CYP3A by glycyrrhizin prevents hepatotoxicity during cholestasis as a mechanism of hepatoprotection. Mice were pretreated with glycyrrhizin prior to induction of intrahepatic cholestasis using lithocholic acid (LCA). Pre-treatment with glycyrrhizin, as well as the PXR activator pregnenolone 16α-carbontrile (PCN), prevents the increase in plasma ALT and AST activity, multifocal necrosis and prevents an increase in a level of serum LCA level in mice, as compared with the results in the mice treated with LCA alone. Activation of the PXR by glycyrrhizin results in induction of CYP3A11 (CYP3A4 for human) expression and inhibition of CYP7A1 through an increase in small heterodimer partner (SHP) expression. Glycyrrhizin regulates the expression of the gene mentioned above to prevent toxic accumulation of bile acids in the liver and it also protects mouse livers from the harmful effects of LCA. In conclusion, PXR-mediated effects on CYP3A and CYP7A may contribute to the

  10. 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

  11. 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

  12. 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.

  13. The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1.

    PubMed

    Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

    2016-01-01

    Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation. PMID:27624558

  14. 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

  15. Decreased exposure of atorvastatin in diabetic rats partly due to induction of hepatic Cyp3a and Oatp2.

    PubMed

    Shu, Nan; Hu, Mengyue; Liu, Can; Zhang, Mian; Ling, Zhaoli; Zhang, Ji; Xu, Ping; Zhong, Zeyu; Chen, Yang; Liu, Li; Liu, Xiaodong

    2016-10-01

    1. Atorvastatin is frequently prescribed for lowering blood cholesterol and for prevention of events associated with cardiovascular disease. The aim of this study was to investigate the pharmacokinetics of atorvastatin in diabetic rats. 2. Diabetes was induced in rats by combination of high-fat diet and low-dose streptozotocin (35 mg/kg). Plasma concentrations of atorvastatin following oral (10 mg/kg) and intravenous (2 mg/kg) administrations to rats were measured by LC-MS. Metabolism and uptake of atorvastatin in primary hepatocytes of experimental rats were assessed. Protein expressions and activities of hepatic Cyp3a and Oatp2 were further investigated. 3. Clearances of atorvastatin in diabetic rats following oral and intravenous administrations were remarkably increased, leading to marked decreases in area-under-the-plasma concentration-time curve (AUC). The estimated oral and systematic clearances of atorvastatin in diabetic rats were 4.5-fold and 2.0-fold of control rats, respectively. Metabolism and uptake of atorvastatin in primary hepatocytes isolated from diabetic rats were significantly increased, which were consistent with the up-regulated protein expressions and activities of hepatic Cyp3a and Oatp2. 4. All these results demonstrated that the plasma exposure of atorvastatin was significantly decreased in diabetic rats, which was partly due to the up-regulated activities and expressions of both hepatic Cyp3a and Oatp2. PMID:26864241

  16. The Psychostimulant Khat (Catha edulis) Inhibits CYP2D6 Enzyme Activity in Humans.

    PubMed

    Bedada, Worku; de Andrés, Fernando; Engidawork, Ephrem; Pohanka, Anton; Beck, Olof; Bertilsson, Leif; Llerena, Adrián; Aklillu, Eleni

    2015-12-01

    The use of khat (Catha edulis) while on medication may alter treatment outcome. In particular, the influence of khat on the metabolic activities of drug-metabolizing enzymes is not known. We performed a comparative 1-way crossover study to evaluate the effect of khat on cytochrome P450 (CYP)2D6 and CYP3A4 enzyme activity. After 1 week of khat abstinence, baseline CYP2D6 and CYP3A4 metabolic activities were determined in 40 Ethiopian male volunteers using 30 mg dextromethorphan (DM) as a probe drug and then repeated after 1 week of daily use of 400 g fresh khat leaves. Urinary concentrations of cathinone and cathine were determined to monitor the subjects' compliance to the study protocol. Genotyping for CYP2D6*3 and CYP2D6*4 was done. Plasma DM, dextrorphan and 3-methoxymorphinan concentrations were quantified. CYP2D6 and CYP3A4 enzyme activities were assessed by comparing plasma log DM/dextrorphan and log DM/methoxymorphinan metabolic ratio (MR) respectively in the presence and absence of khat. Cytochrome 2D6 MR was significantly increased from baseline by concurrent khat use (paired t test, P = 0.003; geometric mean ratio, 1.38; 95% confidence interval [95% CI], 1.12-1.53). Moreover, the inhibition of CYP2D6 activity by khat was more pronounced in CYP2D6*1/*1 compared with CYP2D6*1/*4 genotypes (P = 0.01). A marginal inhibition of CYP3A4 activity in the presence of khat was observed (P = 0.24). The mean percentage increase of CYP2D6 and CYP3A4 MR from baseline by khat use was 46% (95% CI, 20-72) and 31% (95% CI, 8-54), respectively. This is the first report linking khat use with significant inhibition of CYP2D6 metabolic activity in humans. PMID:26444948

  17. Effects of cytochrome P450 3A (CYP3A) and the drug transporters P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) on the pharmacokinetics of lopinavir

    PubMed Central

    van Waterschoot, RAB; ter Heine, R; Wagenaar, E; van der Kruijssen, CMM; Rooswinkel, RW; Huitema, ADR; Beijnen, JH; Schinkel, AH

    2010-01-01

    Background and purpose: Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2. Experimental approach: Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/− ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine. Key results: Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration – time curve (AUC)oral was increased in Abcb1a/b−/− mice (approximately ninefold vs. wild-type) but not in Abcc2−/− mice. Increased lopinavir AUCoral (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a−/−) mice compared with wild-type mice. No difference in AUCoral between Cyp3a−/− and Cyp3a/Abcb1a/b/Abcc2−/− mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUCoral (>100-fold), compared with Cyp3a−/− mice. Ritonavir markedly increased lopinavir AUCoral in all CYP3A-containing mouse strains. Conclusions and implications: CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity. PMID:20590614

  18. 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

  19. 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

  20. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guidelines for CYP3A5 Genotype and Tacrolimus Dosing

    PubMed Central

    Birdwell, Kelly A.; Decker, Brian; Barbarino, Julia M.; Peterson, Josh F.; Stein, C. Michael; Sadee, Wolfgang; Wang, Danxin; Vinks, Alexander A.; He, Yijing; Swen, Jesse J.; Leeder, J. Steven; van Schaik, RHN; Thummel, Kenneth E.; Klein, Teri E.; Caudle, Kelly E.; MacPhee, Iain A.M.

    2015-01-01

    Tacrolimus is the mainstay immunosuppressant drug used after solid organ and hematopoietic stem cell transplantation. Individuals who express CYP3A5 (extensive and intermediate metabolizers) generally have decreased dose-adjusted trough concentrations of tacrolimus as compared to those who are CYP3A5 non-expressers (poor metabolizers), possibly delaying achievement of target blood concentrations. We summarize evidence from the published literature supporting this association and provide dosing recommendations for tacrolimus based on CYP3A5 genotype when known (updates at www.pharmgkb.org). PMID:25801146

  1. Drug Metabolism Enzyme Expression and Activity in Primary Cultures of Human Proximal Tubular Cells

    PubMed Central

    Lash, Lawrence H.; Putt, David A.; Cai, Hongliang

    2008-01-01

    We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Toxicology 228, 200–218 (2006)]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism. PMID:18055091

  2. 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

  3. Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities

    PubMed Central

    Hanapi, N. A.; Ismail, S.; Mansor, S. M.

    2013-01-01

    Context: To date, many findings reveal that most of the modern drugs have the ability to interact with herbal drugs. Aims: This study was conducted to determine the inhibitory effects of mitragynine on cytochrome P450 2C9, 2D6 and 3A4 activities. Methods and Material: The in vitro study was conducted using a high-throughput luminescence assay. Statistical Analysis: Statistical analysis was conducted using one-way ANOVA and Dunnett's test with P < 0.05 vs. control. The IC50 values were calculated using the GraphPad Prism® 5 (Version 5.01, GraphPad Software, Inc., USA). Results: Assessment using recombinant enzymes showed that mitragynine gave the strongest inhibitory effect on CYP2D6 with an IC50 value of 0.45±0.33 mM, followed by CYP2C9 and CYP3A4 with IC50 values of 9.70±4.80 and 41.32±6.74 μM respectively. Positive inhibitors appropriate for CYP2C9, CYP2D6, and CYP3A4 which are sulfaphenazole, quinidine and ketoconazole were used respectively. Vmax values of CYP2C9, CYP2D6 and CYP3A4 were 0.0005, 0.01155 and 0.0137 μM luciferin formed/pmol/min respectively. Km values of CYP2C9, CYP2D6, and CYP3A4 were 32.65, 56.01, and 103.30 μM respectively. Mitragynine noncompetitively inhibits CYP2C9 and CYP2D6 activities with the Ki values of 61.48 and 12.86 μM respectively. On the other hand, mitragynine inhibits CYP3A4 competitively with a Ki value of 379.18 μM. Conclusions: The findings of this study reveal that mitragynine might inhibit cytochrome P450 enzyme activities, specifically CYP2D6. Therefore, administration of mitragynine together with herbal or modern drugs which follow the same metabolic pathway may contribute to herb-drug interactions. PMID:24174816

  4. Identification of rifampin-inducible P450IIIA4 (CYP3A4) in human small bowel enterocytes.

    PubMed Central

    Kolars, J C; Schmiedlin-Ren, P; Schuetz, J D; Fang, C; Watkins, P B

    1992-01-01

    Enzymes within the P450IIIA (CYP3A) subfamily appear to account for significant "first pass" metabolism of some drugs in the intestine. To identify which of the known P450IIIA genes are expressed in intestine, enterocyte RNA was hybridized on Northern blots with synthetic oligonucleotides complementary to hypervariable regions of hepatic P450IIIA4, P450IIIA5, and P450IIIA7 cDNAs. Hybridization was detected only with the P450IIIA4-specific oligonucleotide. The identity of the hybridizing mRNA was confirmed to be P450IIIA4 by direct sequencing of a DNA fragment amplified from enterocyte cDNA by the polymerase chain reaction. To determine if enterocyte P450IIIA4 is inducible, biopsies of small bowel mucosa were obtained from five volunteers before and after they received 7d of treatment with rifampin, a known inducer of P450IIIA4 in liver. Rifampin treatment resulted in a five- or eightfold mean increase (P < 0.05) in the biopsy concentration of P450IIIA4 mRNA when normalized for content of sucrase isomaltase or intestinal fatty acid binding protein mRNAs, respectively. Rifampin also induced P450IIIA immunoreactive protein in enterocytes in each of the subjects, as judged by immunohistochemistry, and resulted in a 10-fold increase in P450IIIA4-specific catalytic activity (erythromycin N-demethylation) in the one patient studied. Our identification of inducible P450IIIA4 in enterocytes may in part account for drug interactions characteristic of P450IIIA4 substrates and suggests a strategy for controlling entry into the body of a major class of xenobiotics. Images PMID:1430211

  5. 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

  6. 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

  7. 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

  8. Experimental approaches to evaluate activities of cytochromes P450 3A

    PubMed Central

    Bořek-Dohalská, Lucie; Hodek, Petr; Hudeček, Jiří; Stiborová, Marie

    2008-01-01

    Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro. PMID:21218106

  9. 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

  10. 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

  11. 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

  12. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    PubMed Central

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

    2013-01-01

    Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

  13. Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11) in the Liver of Mouse Induced by Microcystin-LR

    PubMed Central

    Zhang, Bangjun; Liu, Yang; Li, Xiaoyu

    2015-01-01

    Microcystins (MCs) are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs) play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR) on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11) at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD) (CYP1A1) and erythromycin N-demthylase (ERND) (CYP3A11) activities and increased aniline hydroxylase (ANH) activity (CYP2E1) in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice. PMID:25831226

  14. CYP3A5 genotyping for assessing the efficacy of treatment with simvastatin and atorvastatin

    PubMed Central

    Kolovou, Genovefa; Kolovou, Vana; Ragia, Georgia; Mihas, Constantinos; Diakoumakou, Olga; Vasiliadis, Ioannis; Mavrogeni, Sophie; Vartela, Vassiliki; Manolopoulos, Vangelis G

    2015-01-01

    In this work, we examined the impact of polymorphism in the cytochrome P450 (CYP) 3A5 gene, CYP3A5*1 (6986A > G, rs 776746), on the reduction in the lipid levels caused by simvastatin and atorvastatin. We studied 350 hyperlipidemic patients who received 10-40 mg of atorvastatin (n = 175) or simvastatin (n = 175) daily. Genotyping for CYP3A5 was done by PCR-RFLP analysis. Differences in the lipid profile before and after treatment were expressed as the % difference. The frequency of CYP3A5polymorphism was 13.4% for heterozygotes and 86.6% for homozygotes. Comparison of the responses to same dose of each drug showed that the highest % difference was associated with total cholesterol (TC) in subjects receiving atorvastatin 40 mg compared with simvastatin 40 mg (p = 0.048). However, comparison of the responses to equivalent doses of atorvastatin vs. simvastatin revealed no difference in the % change in any of the lipid parameters examined. In individuals with the same CYP3A5 genotype, a head to head comparison of the efficacy of the same dose of simvastatin vs. atorvastatin revealed an advantage for atorvastatin. For equivalent doses of atorvastatin vs. simvastatin there was no difference in the % change in any of the lipid parameters examined. Within the same genotype there was a significant difference in the % change related to the drug treatment. PMID:26273214

  15. Cytotoxicity of luteolin in primary rat hepatocytes: the role of CYP3A-mediated ortho-benzoquinone metabolite formation and glutathione depletion.

    PubMed

    Shi, Fuguo; Zhao, Peng; Li, Xiaobing; Pan, Hong; Ma, Shiping; Ding, Li

    2015-11-01

    Luteolin (LUT), an active ingredient in traditional Chinese medicines and an integral part of the human diet, has shown promising pharmacological activities with a great potential for clinical use. The purpose of this study was to evaluate the role of cytochrome P450 (CYP450)-mediated reactive ortho-benzoquinone metabolites formation and glutathione (GSH) depletion in LUT-induced cytotoxicity in primary rat hepatocytes. A reactive ortho-benzoquinone metabolite was identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in rat liver microsomes (RLMs) and rat hepatocytes. Using a specific chemical inhibitor method, the CYP3A subfamily was found to be responsible for the reactive metabolite formation in RLMs. Induction of CYP3A by dexamethasone enhanced LUT-induced cytotoxicity, whereas inhibition of CYP3A by ketoconazole (Keto) decreased the cytotoxicity. The cytotoxicity and cell apoptosis induced by LUT were related to the amount of reactive metabolite formation. Furthermore, Keto inhibited the LUT-induced GSH exhaustion. The cytotoxicity was significantly enhanced by pretreatment with L-buthionine sulfoximine to deplete the intracellular GSH. A time course experiment showed that GSH depletion by LUT was not via oxidation of GSH and occurred prior to the increase in 2', 7'-dichlorofluorescein in hepatocytes. Collectively, these data suggest that CYP3A-mediated reactive metabolite formation plays a critical role in LUT-induced hepatotoxicity, and the direct GSH depletion is an initiating event in LUT-mediated cytotoxicity in primary rat hepatocytes. PMID:25612170

  16. Semiphysiologically based pharmacokinetic model for midazolam and CYP3A mediated metabolite 1-OH-midazolam in morbidly obese and weight loss surgery patients.

    PubMed

    Brill, M J E; Välitalo, P A J; Darwich, A S; van Ramshorst, B; van Dongen, H P A; Rostami-Hodjegan, A; Danhof, M; Knibbe, C A J

    2016-01-01

    This study aimed to describe the pharmacokinetics of midazolam and its cytochrome P450 3A (CYP3A) mediated metabolite 1-OH-midazolam in morbidly obese patients receiving oral and i.v. midazolam before (n = 20) and one year after weight loss surgery (n = 18), thereby providing insight into the influence of weight loss surgery on CYP3A activity in the gut wall and liver. In a semiphysiologically based pharmacokinetic (semi-PBPK) model in which different blood flow scenarios were evaluated, intrinsic hepatic clearance of midazolam (CLint,H) was 2 (95% CI 1.40-1.64) times higher compared to morbidly obese patients before surgery (P < 0.01). Midazolam gut wall clearance (CLint,G) was slightly lower in patients after surgery (P > 0.05), with low values for both groups. The results of the semi-PBPK model suggest that, in patients after weight loss surgery, CYP3A hepatic metabolizing capacity seems to recover compared to morbidly obese patients, whereas CYP3A mediated CLint,G was low for both populations and showed large interindividual variability. PMID:26844012

  17. Carbamazepine-Induced Liver Injury Requires CYP3A-Mediated Metabolism and Glutathione Depletion in Rats.

    PubMed

    Iida, Azumi; Sasaki, Eita; Yano, Azusa; Tsuneyama, Koichi; Fukami, Tatsuki; Nakajima, Miki; Yokoi, Tsuyoshi

    2015-07-01

    Carbamazepine (CBZ) is widely used as an antiepileptic agent and causes rare but severe liver injury in humans. It has been generally recognized that reactive metabolites formed via the metabolic activation reaction contribute to the onset of liver injuries by several drugs. However, the role of CBZ metabolism in the development of liver injury is not fully understood. In this study, we developed a novel rat model of CBZ-induced liver injury and attempted to elucidate the associated mechanisms by focusing on the metabolism of CBZ. The repeated administration of CBZ for 5 days in combination with l-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor, resulted in increases in the plasma alanine aminotransferase (ALT) levels and centrilobular necrosis in the liver that were observed in various degrees. The CBZ and 2-hydroxy-CBZ concentrations in the plasma after the last CBZ administration were lower in the rats with high plasma ALT levels compared with those with normal plasma ALT levels, showing the possibility that the further metabolism of CBZ and/or 2-hydroxy-CBZ is associated with the liver injury. Although a single administration of CBZ did not affect the plasma ALT levels, even when cotreated with BSO, pretreatment with dexamethasone, a CYP3A inducer, increased the plasma ALT levels. In addition, the rats cotreated with troleandomycin or ketoconazole, CYP3A inhibitors, suppressed the increased plasma ALT levels. In conclusion, reactive metabolite(s) of CBZ produced by CYP3A under the GSH-depleted condition might be involved in the development of liver injury in rats. PMID:25870103

  18. A Systematic Approach to Evaluate Herb-Drug Interaction Mechanisms: Investigation of Milk Thistle Extracts and Eight Isolated Constituents as CYP3A Inhibitors

    PubMed Central

    Brantley, Scott J.; Graf, Tyler N.; Oberlies, Nicholas H.

    2013-01-01

    Despite increasing recognition of potential untoward interactions between herbal products and conventional medications, a standard system for prospective assessment of these interactions remains elusive. This information gap was addressed by evaluating the drug interaction liability of the model herbal product milk thistle (Silybum marianum) with the CYP3A probe substrate midazolam. The inhibitory effects of commercially available milk thistle extracts and isolated constituents on midazolam 1′-hydroxylation were screened using human liver and intestinal microsomes. Relative to vehicle, the extract silymarin and constituents silybin A, isosilybin A, isosilybin B, and silychristin at 100 μM demonstrated >50% inhibition of CYP3A activity with at least one microsomal preparation, prompting IC50 determination. The IC50s for isosilybin B and silychristin were ∼60 and 90 μM, respectively, whereas those for the remaining constituents were >100 μM. Extracts and constituents that contained the 1,4-dioxane moiety demonstrated a >1.5-fold shift in IC50 when tested as potential mechanism-based inhibitors. The semipurified extract, silibinin, and the two associated constituents (silybin A and silybin B) demonstrated mechanism-based inhibition of recombinant CYP3A4 (KI, ∼100 μM; kinact, ∼0.20 min−1) but not microsomal CYP3A activity. The maximum predicted increases in midazolam area under the curve using the static mechanistic equation and recombinant CYP3A4 data were 1.75-fold, which may necessitate clinical assessment. Evaluation of the interaction liability of single herbal product constituents, in addition to commercially available extracts, will enable elucidation of mechanisms underlying potential clinically significant herb-drug interactions. Application of this framework to other herbal products would permit predictions of herb-drug interactions and assist in prioritizing clinical evaluation. PMID:23801821

  19. Structure-Activity Relationship and Substrate-Dependent Phenomena in Effects of Ginsenosides on Activities of Drug-Metabolizing P450 Enzymes

    PubMed Central

    Hao, Miao; Zhao, Yuqing; Chen, Peizhan; Huang, He; Liu, Hong; Jiang, Hualiang; Zhang, Ruiwen; Wang, Hui

    2008-01-01

    Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs). Results from previous studies on ginsenosides' effects on human drug-metabolizing P450 enzymes are inconsistent and confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4. Moreover, substrate-dependent phenomena were found in ginsenosides' effects on CYP3A4 when another fluorescent probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in previous GDI reports. PMID:18628990

  20. Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

    PubMed

    Bozcaarmutlu, Azra; Turna, Sema; Sapmaz, Canan; Arinc, Emel; Yenisoy-Karakaş, Serpil

    2014-06-01

    The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey. PMID:24756956

  1. 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).

  2. 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

  3. Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

    PubMed Central

    Andersson, T; Miners, J O; Veronese, M E; Birkett, D J

    1994-01-01

    1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase. PMID:7981013

  4. Effect of CYP3A perpetrators on ibrutinib exposure in healthy participants

    PubMed Central

    de Jong, Jan; Skee, Donna; Murphy, Joe; Sukbuntherng, Juthamas; Hellemans, Peter; Smit, Johan; de Vries, Ronald; Jiao, Juhui James; Snoeys, Jan; Mannaert, Erik

    2015-01-01

    Ibrutinib (PCI-32765), a potent covalent inhibitor of Bruton’s tyrosine kinase, has shown efficacy against a variety of B-cell malignancies. Given the prominent role of CYP3A in ibrutinib metabolism, effect of coadministration of CYP3A perpetrators with ibrutinib was evaluated in healthy adults. Ibrutinib (120 mg [Study 1, fasted], 560 mg [studies 2 (fasted), and 3 (nonfasted)]) was given alone and with ketoconazole [Study 1; 400 mg q.d.], rifampin [Study 2; 600 mg q.d.], and grapefruit juice [GFJ, Study 3]. Lower doses of ibrutinib were used together with CYP3A inhibitors [Study 1: 40 mg; Study 3: 140 mg], as safety precaution. Under fasted condition, ketoconazole increased ibrutinib dose-normalized (DN) exposure [DN-AUClast: 24-fold; DN-Cmax: 29-fold], rifampin decreased ibrutinib exposure [Cmax: 13-fold; AUClast: 10-fold]. Under nonfasted condition, GFJ caused a moderate increase [DN-Cmax: 3.5-fold; DN-AUC: 2.2-fold], most likely through inhibition of intestinal CYP3A. Half-life was not affected by CYP perpetrators indicating the interaction was mainly on first-pass extraction. All treatments were well-tolerated. PMID:26171235

  5. 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

  6. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method.

    PubMed

    Midde, Narasimha M; Rahman, Mohammad A; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34 ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  7. Effect of Ethanol on the Metabolic Characteristics of HIV-1 Integrase Inhibitor Elvitegravir and Elvitegravir/Cobicistat with CYP3A: An Analysis Using a Newly Developed LC-MS/MS Method

    PubMed Central

    Midde, Narasimha M.; Rahman, Mohammad A.; Rathi, Chetan; Li, Junhao; Meibohm, Bernd; Li, Weihua; Kumar, Santosh

    2016-01-01

    Elvitegravir (EVG), an integrase inhibitor for the treatment HIV infection, is increasingly becoming the part of first-line antiretroviral therapy (ART) regimen. EVG is mainly metabolized through cytochrome P450 (CYP) 3A4. Previously, we have shown that ethanol alters ART-CYP3A4 interactions with protease inhibitors thereby altering their metabolisms. However, as EVG is a fairly new class of drug, its kinetic characteristics and the effect of ethanol on EVG-CYPP3A4 interaction is poorly understood. In this study, we characterized EVG and cobicistat (COBI)-boosted EVG metabolism in human microsomes followed by ethanol-EVG, ethanol-COBI-EVG interaction with CYP3A. First, we developed and validated a simple, sensitive, and robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of EVG in the human liver microsomes. The lower limit of quantification for the drug was at 0.003 μM (1.34ng/ml). Extraction yield, matrix effects, drug stability, and calibration curves for the proposed method were validated according to the FDA guidelines. Time dependent kinetics data showed that 20mM ethanol decreases the apparent half-life of EVG degradation by ~50% compared to EVG alone. Our substrate kinetic results revealed that ethanol mildly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that EVG inhibits CYP3A4, and 20 mM ethanol causes a decrease in the IC50 of EVG. However, in the presence of COBI we were unable to determine these parameters effectively because COBI, being a strong inhibitor of CYP3A4, blocked the EVG/ethanol-CYP3A4 interactions. Docking studies predicted a shift of EVG or COBI binding to the active site of CYP3A4 in the presence of ethanol. Taken together, these results suggest that ethanol interacts with microsomal CYP3A and alters EVG-CYP3A4 interaction thereby altering EVG metabolism and inhibition of CYP3A4 by EVG. This finding has clinical significance because alcohol use is

  8. Human variation and CYP enzyme contribution in benfuracarb metabolism in human in vitro hepatic models.

    PubMed

    Abass, Khaled; Reponen, Petri; Mattila, Sampo; Rautio, Arja; Pelkonen, Olavi

    2014-01-13

    Human responses to the toxicological effects of chemicals are often complicated by a substantial interindividual variability in toxicokinetics, of which metabolism is often the most important factor. Therefore, we investigated human variation and the contributions of human-CYP isoforms to in vitro metabolism of benfuracarb. The primary metabolic pathways were the initial sulfur oxidation to benfuracarb-sulfoxide and the nitrogen-sulfur bond cleavage to carbofuran (activation). The Km, Vmax, and CL(int) values of carbofuran production in ten individual hepatic samples varied 7.3-, 3.4-, and 5.4-fold, respectively. CYP2C9 and CYP2C19 catalyzed benfuracarb sulphur oxidation. Carbofuran formation, representing from 79% to 98% of the total metabolism, was catalyzed predominantly by CYP3A4. The calculated relative contribution of CYP3A4 to carbofuran formation was 93%, while it was 4.4% for CYP2C9. The major contribution of CYP3A4 in benfuracarb metabolism was further substantiated by showing a strong correlation with CYP3A4-selective markers midazolam-1'-hydroxylation and omeprazole-sulfoxidation (r=0.885 and 0.772, respectively). Carbofuran formation was highly inhibited by the CYP3A inhibitor ketoconazole. Moreover, CYP3A4 marker activities were relatively inhibited by benfuracarb. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro activation of benfuracarb and that CYP3A4-catalyzed metabolism is the primary source of interindividual differences. PMID:24016712

  9. 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

  10. Genetic variation of CYP3A5 influences paclitaxel/carboplatin-induced toxicity in Chinese epithelial ovarian cancer patients.

    PubMed

    Hu, Lei; Lv, Qiao-Li; Guo, Ying; Cheng, Lin; Wu, Na-Yiyuan; Qin, Chong-Zhen; Zhou, Hong-Hao

    2016-03-01

    Combination chemotherapy with platinum and taxane is the first-line treatment for ovarian cancer. The dose-limiting toxicities of these drugs include neuropathy, leukopenia, and neutropenia, but they exhibit substantial interindividual variability. This study investigated the relationship between CYP3A5 polymorphisms and paclitaxel/carboplatin-induced toxicity in Chinese epithelial ovarian cancer patients. Seventy-five patients with epithelial ovarian cancer were recruited. After combination chemotherapy, genotype analysis was conducted, and toxic effects were evaluated according to the Common Toxicity Criteria. A significant association was found between myelosuppression and the CYP3A5*3 genotype. CYP3A5*3/*1 patients showed a significantly higher risk of developing leukopenia (P < .001; Pearson's χ(2) test) and neutropenia (P < .001; Pearson's χ(2) test) than CYP3A5*3*3 patients. CYP3A5*3/*3 patients had significantly higher median leukocyte and neutrophil nadir counts than CYP3A5*3*1 patients (P < .001, Mann-Whitney U test). However, we did not observe an association between neuropathy and CYP3A5*3 in this study (P =.64; Pearson's χ(2) test). This is the first study to verify the influence of CYP3A5 polymorphisms on paclitaxel/carboplatin-induced toxicity in Chinese epithelial ovarian cancer patients. Our findings suggest that interindividual variability in paclitaxel/carboplatin-induced myelosuppression can be predicted by CYP3A5*3 genotyping and that incorporation of CYP3A5*3 genetic data in treatment selection could help to reduce myelosuppression events, thereby individualizing paclitaxel/carboplatin pharmacotherapy. PMID:26179145

  11. Itraconazole and clarithromycin as ketoconazole alternatives for clinical CYP3A inhibition studies.

    PubMed

    Ke, A B; Zamek-Gliszczynski, M J; Higgins, J W; Hall, S D

    2014-05-01

    High-dose ketoconazole (400 mg q.d. for ≥5 days) has been the gold-standard strong cytochrome P450 3A (CYP3A) inhibitor in drug development drug-drug interaction (DDI) studies. In 2013, the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) advised against using this ketoconazole regimen following review of clinical safety reports. We systematically evaluated 19 strong CYP3A inhibitors from regulatory guidances and a literature database to identify itraconazole (200 mg b.i.d. on day 1, q.d. on days 2-6) and clarithromycin (500 mg b.i.d. for 7 days) as acceptable ketoconazole alternatives. PMID:24747234

  12. Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver.

    PubMed

    Khan, Ansar A; Chow, Edwin C Y; van Loenen-Weemaes, Anne-miek M A; Porte, Robert J; Pang, K Sandy; Groothuis, Geny M M

    2009-05-12

    In this study, we compared the regulation of CYP3A isozymes by the vitamin D receptor (VDR) ligand 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) against ligands of the pregnane X receptor (PXR), the glucocorticoid receptor (GR) and the farnesoid X receptor (FXR) in precision-cut tissue slices of the rat jejunum, ileum, colon and liver, and human ileum and liver. In the rat, 1,25(OH)(2)D(3) strongly induced CYP3A1 mRNA, quantified by qRT-PCR, along the entire length of the intestine, induced CYP3A2 only in ileum but had no effect on CYP3A9. In contrast, the PXR/GR ligand, dexamethasone (DEX), the PXR ligand, pregnenolone-16 alpha carbonitrile (PCN), and the FXR ligand, chenodeoxycholic acid (CDCA), but not the GR ligand, budesonide (BUD), induced CYP3A1 only in the ileum, none of them influenced CYP3A2 expression, and PCN, DEX and BUD but not CDCA induced CYP3A9 in jejunum, ileum and colon. In rat liver, CYP3A1, CYP3A2 and CYP3A9 mRNA expression was unaffected by 1,25(OH)(2)D(3), whereas CDCA decreased the mRNA of all CYP3A isozymes; PCN induced CYP3A1 and CYP3A9, BUD induced CYP3A9, and DEX induced all three CYP3A isozymes. In human ileum and liver, 1,25(OH)(2)D(3) and DEX induced CYP3A4 expression, whereas CDCA induced CYP3A4 expression in liver only. In conclusion, the regulation of rat CYP3A isozymes by VDR, PXR, FXR and GR ligands differed for different segments of the rat and human intestine and liver, and the changes did not parallel expression levels of the nuclear receptors. PMID:19429418

  13. Characterization of biotransformation enzyme activities in primary rat proximal tubular cells.

    PubMed

    Schaaf, G J; de Groene, E M; Maas, R F; Commandeur, J N; Fink-Gremmels, J

    2001-04-16

    The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application. PMID:11311212

  14. 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

  15. Photoperiodism and Enzyme Activity

    PubMed Central

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  16. 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

  17. 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

  18. Echinacea Purpurea Significantly Induces Cytochrome P450 3A (CYP3A) but does not alter Lopinavir-Ritonavir Exposure in Healthy Subjects

    PubMed Central

    Penzak, Scott R.; Robertson, Sarah M.; Hunt, Jennifer D.; Chairez, Cheryl; Malati, Christine Y.; Alfaro, Raul M.; Stevenson, James M.; Candidate, Pharm.D.; Kovacs, Joseph A.

    2012-01-01

    Study Objective To determine the influence of Echinacea purpurea on the pharmacokinetics of lopinavir-ritonavir, and on CYP3A and P-glycoprotein (P-gp) activity using the probe substrates midazolam, and fexofenadine, respectively. Design Open label, single-sequence pharmacokinetic study. Setting Outpatient clinic in a Federal Government research hospital. Subjects Thirteen (8 males) healthy volunteers (median age: 31 yrs). Measurements and main results Healthy volunteers received lopinavir-ritonavir (400/100 mg) twice daily for 30 days. On study day 16, subjects began taking Echinacea purpurea 500 mg three times daily, which they continued for four weeks, the first two weeks in combination with lopinavir-ritonavir. On days 15 and 30 of lopinavir-ritonavir administration (pre and post-Echinacea, respectively), serial blood samples were collected over 12 hrs to determine lopinavir and ritonavir concentrations and subsequent pharmacokinetic parameters using non-compartmental methods. Study subjects also received single doses of midazolam (8 mg orally) and fexofenadine (120 mg orally) before- and after 28 days of Echinacea purpurea to assess CYP3A and P-glycoprotein (P-gp) activity, respectively. Neither lopinavir nor ritonavir pharmacokinetics were significantly altered by 2 weeks of Echinacea coadministration. The geometric mean ratios (GMR, 90% CI) for lopinavir area under the concentration vs. time curve from zero to 12 hrs (AUC0–12) and maximum concentration (post-Echinacea/pre-Echinacea) were 0.96 (0.83, 1.10) and 1.00 (0.88, 1.12), respectively (P > 0.05). Conversely, GMRs (90% CIs) for midazolam AUC from time zero to infinity (AUC0-∞) and oral clearance were 0.73 (0.61, 0.85) (P = 0.008) and 1.37 (1.10, 1.63) (P = 0.02), respectively. Fexofenadine pharmacokinetics did not significantly differ pre- and post-echinacea administration (P > 0.05). Conclusion Echinacea purpurea induced CYP3A activity but did not alter lopinavir concentrations, most likely due to

  19. VX-509 (Decernotinib)-Mediated CYP3A Time-Dependent Inhibition: An Aldehyde Oxidase Metabolite as a Perpetrator of Drug-Drug Interactions.

    PubMed

    Zetterberg, Craig; Maltais, Francois; Laitinen, Leena; Liao, Shengkai; Tsao, Hong; Chakilam, Ananthsrinivas; Hariparsad, Niresh

    2016-08-01

    (R)-2-((2-(1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methyl-N-(2,2,2-trifluoroethyl)butanamide (VX-509, decernotinib) is an oral Janus kinase 3 inhibitor that has been studied in patients with rheumatoid arthritis. Patients with rheumatoid arthritis often receive multiple medications, such as statins and steroids, to manage the signs and symptoms of comorbidities, which increases the chances of drug-drug interactions (DDIs). Mechanism-based inhibition is a subset of time-dependent inhibition (TDI) and occurs when a molecule forms a reactive metabolite which irreversibly binds and inactivates drug-metabolizing enzymes, potentially increasing the systemic load to toxic concentrations. Traditionally, perpetrating compounds are screened using human liver microsomes (HLMs); however, this system may be inadequate when the precipitant is activated by a non-cytochrome P450 (P450)-mediated pathway. Even though studies assessing competitive inhibition and TDI using HLM suggested a low risk for CYP3A4-mediated DDI in the clinic, VX-509 increased the area under the curve of midazolam, atorvastatin, and methyl-prednisolone by approximately 12.0-, 2.7-, and 4.3-fold, respectively. Metabolite identification studies using human liver cytosol indicated that VX-509 is converted to an oxidative metabolite, which is the perpetrator of the DDIs observed in the clinic. As opposed to HLM, hepatocytes contain the full complement of drug-metabolizing enzymes and transporters and can be used to assess TDI arising from non-P450-mediated metabolic pathways. In the current study, we highlight the role of aldehyde oxidase in the formation of the hydroxyl-metabolite of VX-509, which is involved in clinically significant TDI-based DDIs and represents an additional example in which a system-dependent prediction of TDI would be evident. PMID:27298338

  20. Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

    PubMed

    Bártíková, Hana; Boušová, Iva; Jedličková, Pavla; Lněničková, Kateřina; Skálová, Lenka; Szotáková, Barbora

    2014-01-01

    The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious. PMID:25237750

  1. Dydrogesterone metabolism in human liver by aldo-keto reductases and cytochrome P450 enzymes.

    PubMed

    Olbrich, Matthias; Weigl, Kevin; Kahler, Elke; Mihara, Katsuhiro

    2016-10-01

    1. The metabolism of dydrogesterone was investigated in human liver cytosol (HLC) and human liver microsomes (HLM). Enzymes involved in dydrogesterone metabolism were identified and their relative contributions were estimated. 2. Dydrogesterone clearance was clearly higher in HLC compared to HLM. The major active metabolite 20α-dihydrodydrogesterone (20α-DHD) was only produced in HLC. 3. The formation of 20α-DHD by cytosolic aldo-keto reductase 1C (AKR1C) was confirmed with isoenzyme-specific AKR inhibitors. 4. Using recombinantly expressed human cytochrome P450 (CYP) isoenzymes, dydrogesterone was shown to be metabolically transformed by CYP3A4 and CYP2C19. 5. A clear contribution of CYP3A4 to microsomal metabolism of dydrogesterone was demonstrated with HLM and isoenzyme-specific CYP inhibitors, and confirmed by a significant correlation between dydrogesterone clearance and CYP3A4 activity. 6. Contribution of CYP2C19 was shown to be clearly less than CYP3A4 and restricted to a small group of human individuals with very high CYP2C19 activity. Therefore, it is expected that CYP2C19 genetic variations will not affect dydrogesterone pharmacokinetics in man. 7. In conclusion, dydrogesterone metabolism in the liver is dominated primarily by cytosolic enzymes (particularly AKR1C) and secondarily by CYP3A4, with the former exclusively responsible for 20α-DHD formation. PMID:26796435

  2. Effect of the CYP3A inhibitors, diltiazem and ketoconazole, on ticagrelor pharmacokinetics in healthy volunteers

    PubMed Central

    Teng, Renli; Butler, Kathleen

    2013-01-01

    Objectives Two open-label, two-period, crossover studies in healthy volunteers were designed to determine the pharmacokinetic interactions between ticagrelor, a P2Y12 receptor antagonist, and a moderate (diltiazem) and a strong (ketoconazole) cytochrome P450 (CYP) 3A inhibitor. Methods Seventeen volunteers received diltiazem (240 mg once daily) for 14 days. In the second study, ketoconazole (n = 14) 200 mg twice daily was given for 10 days. A single oral 90-mg ticagrelor dose was administered on day 8 (diltiazem) or day 4 (ketoconazole). In each study, volunteers received a single 90-mg oral dose of ticagrelor before or after washout (≥14 days). Pharmacokinetic parameters for ticagrelor, AR-C124910XX (primary metabolite), diltiazem, and ketoconazole were assessed. Results Compared with ticagrelor alone, diltiazem co-administration significantly increased the mean maximum concentration (Cmax) and mean area under the plasma concentration–time curve (AUC) for ticagrelor by 69% and 174%, respectively. Diltiazem co-administration reduced Cmax by 38% but had no significant effect on AUC for AR-C124910XX. Cmax and AUC for ticagrelor were increased by 135% and 632%, respectively, by ketoconazole co-administration, whereas these parameters were reduced by 89% and 56%, respectively, for AR-C124910XX. Diltiazem and ketoconazole pharmacokinetic parameters were not significantly affected by the presence of ticagrelor. Conclusions These results suggest that ticagrelor can be co-administered with moderate CYP3A inhibitors. However, co-administration of strong CYP3A inhibitors with ticagrelor is not recommended.

  3. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma.

    PubMed

    Noll, Elisa M; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V; Eils, Roland; Giese, Nathalia A; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R

    2016-03-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  4. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

    PubMed Central

    Noll, Elisa M.; Eisen, Christian; Stenzinger, Albrecht; Espinet, Elisa; Muckenhuber, Alexander; Klein, Corinna; Vogel, Vanessa; Klaus, Bernd; Nadler, Wiebke; Rösli, Christoph; Lutz, Christian; Kulke, Michael; Engelhardt, Jan; Zickgraf, Franziska M.; Espinosa, Octavio; Schlesner, Matthias; Jiang, Xiaoqi; Kopp-Schneider, Annette; Neuhaus, Peter; Bahra, Marcus; Sinn, Bruno V.; Eils, Roland; Giese, Nathalia A.; Hackert, Thilo; Strobel, Oliver; Werner, Jens; Büchler, Markus W.; Weichert, Wilko; Trumpp, Andreas; Sprick, Martin R.

    2016-01-01

    Although subtypes of pancreatic ductal adenocarcinoma (PDAC) were described, this malignancy is clinically still treated as a single disease. Here, we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Individuals bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or shRNA-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4 alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. PMID:26855150

  5. Sex differences in constitutive mRNA levels of CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46 in the pig liver: Comparison between Meishan and Landrace pigs.

    PubMed

    Kojima, Misaki; Degawa, Masakuni

    2016-06-01

    Breed and sex differences in hepatic mRNA levels of cytochrome P450 (CYP) isoforms (CYP2B22, CYP2C33, CYP2C49, CYP3A22, CYP3A29 and CYP3A46) were examined in 5-month-old Meishan, Landrace, and their crossbred F1 (LM and ML) pigs. Serum testosterone levels in male Meishan, LM, and ML pigs were 2.5-3.5-fold higher than in Landrace pigs. CYP3A46 mRNA was breed-specifically detected only in Landrace, LM, and ML pigs. In Meishan, LM, and ML pigs only, male-predominant expressions of CYP2B22, CYP2C33, CYP2C49 and CYP3A29 mRNAs were observed; CYP3A22 mRNA expression showed the opposite pattern. Male-dominant mRNA expression was also observed in LM and ML pigs for CYP3A46. The sex differences in CYP mRNA levels in Meishan pigs disappeared when males were castrated and were restored by testosterone propionate (TP) administration to the castrated males. In Landrace pigs, TP administration to castrated males and intact females significantly increased the levels of CYP2B22, CYP2C33, and CYP3A46 mRNAs. Immature (1-month-old) pigs showed no breed or sex differences in CYP mRNA expressions. The results demonstrated that androgen is an important determinant of sex-associated expression of several CYPs and suggested that breed differences in sex-associated expression could be caused by differences in serum androgen level and by other genetic traits. PMID:27080814

  6. 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

  7. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro

    PubMed Central

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-01-01

    In this study, we investigated the enzymes catalyzing the phaseⅠmetabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern. PMID:26404338

  8. 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

  9. 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...

  10. Differential roles of P-glycoprotein, multidrug resistance-associated protein 2, and CYP3A on saquinavir oral absorption in Sprague-Dawley rats.

    PubMed

    Usansky, Helen H; Hu, Peidi; Sinko, Patrick J

    2008-05-01

    The objective of this investigation was to differentiate the roles of P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (Mrp2), and CYP3A on saquinavir (SQV) oral absorption. With use of single-pass jejunal perfusion (in situ) and portal vein-cannulated rats (in vivo), SQV absorption was studied under chemical inhibition of Pgp [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2 isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)], Mrp2 [(3-(((3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl) ((3-(dimethylamino-3-oxopropyl)thio)methyl)-thio) propanoic acid (MK571)], and/or CYP3A (midazolam). Plasma concentrations of SQV and related metabolites were analyzed by liquid chromatography-tandem mass spectrometry. When given alone, SQV absorption was extremely low both in situ (F(a) = 0.07%) and in vivo [C(max) = 0.068 microg/ml; area under the curve (AUC) = 6.8 microg x min/ml]. Coadministration of GF120918 boosted SQV absorption by more than 20-fold with decreased variation in AUCs (percent coefficient of variation = 30% versus 100%). In contrast, coadministration of MK571 or midazolam increased SQV absorption only 2- to 3-fold without improving the variation in AUCs. SQV oral absorption was not further improved when it was given with GF120918 and midazolam or with GF120918 and MK571. The current results provide, for the first time, direct and explicit evidence that the low oral absorption of SQV is controlled by a secretory transporter, Pgp, and not by limited passive diffusion owing to its poor physicochemical properties. Pgp-mediated transport is also responsible for the highly variable oral bioavailability of SQV. In contrast, intestinal Mrp2 and intestinal CYP3A appear to play minor roles in SQV oral bioavailability. Given the differential and complex roles of Pgp and CYP3A in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration to avoid therapy-limiting drug-drug transporter

  11. Characterization of hepatic enzyme activity in older adults with dementia: potential impact on personalizing pharmacotherapy

    PubMed Central

    Campbell, Noll L; Skaar, Todd C; Perkins, Anthony J; Gao, Sujuan; Li, Lang; Khan, Babar A; Boustani, Malaz A

    2015-01-01

    Objective To determine the frequency of pharmacogenomic variants and concurrent medications that may alter the efficacy and tolerability of acetylcholinesterase inhibitors (AChEIs). Materials and methods A multisite cross-sectional study was carried out across four memory care practices in the greater Indianapolis area. Participants were adults aged 65 years and older with a diagnosis of probable or possible Alzheimer’s disease (AD) (n=105). Blood samples and self-reported medication data were collected. Since two of the three AChEIs are metabolized by cytochrome P450 (CYP)-2D6, we determined the frequency of functional genetic variants in the CYP2D6 gene and calculated their predicted CYP2D6-activity scores. Concurrent medication data were collected from self-reported medication surveys, and their predicted effect on the pharmacokinetics of AChEIs was determined based on their known effects on CYP2D6 and CYP3A4/5 enzyme activities. Results Among the 105 subjects enrolled, 72% were female and 36% were African American. Subjects had a mean age of 79.6 years. The population used a mean of eight medications per day (prescription and nonprescription). The CYP2D6 activity score frequencies were 0 (3.8%), 0.5 (4.8%), 1.0 (36.2%), 1.5–2.0 (51.4%), and >2.0 (3.8%). Nineteen subjects (18.1%) used a medication considered a strong or moderate inhibitor of CYP2D6, and eight subjects (7.6%) used a medication considered a strong or moderate inhibitor of CYP3A4/5. In total, 28.6% of the study population was predicted to have reduced activity of the CYP2D6 or CYP3A4/5 enzymes due to either genetic variants or concomitant medications. Conclusion Both pharmacogenetic variants and concurrent drug therapies that are predicted to alter the pharmacokinetics of AChEIs should be evaluated in older adults with AD. Pharmacogenetic and drug-interaction data may help personalize AD therapy and increase adherence by improving tolerability. PMID:25609939

  12. 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

  13. 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

  14. 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

  15. 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

  16. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    SciTech Connect

    Jin, Hua; Shen, Shuijie; Chen, Xiaoyan; Zhong, Dafang; Zheng, Jiang

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  17. The Absence of CYP3A5*3 Is a Protective Factor to Anticonvulsants Hypersensitivity Reactions: A Case-Control Study in Brazilian Subjects

    PubMed Central

    dos Santos, Bernardo; Talib, Leda Leme; Yamaguti, Célia; Rodrigues, Helcio; Gattaz, Wagner Farid; Kalil, Jorge

    2015-01-01

    Although aromatic anticonvulsants are usually well tolerated, they can cause cutaneous adverse drug reactions in up to 10% of patients. The clinical manifestations of the antiepileptics-induced hypersensitivity reactions (AHR) vary from mild skin rashes to severe cutaneous drug adverse reactions which are related to high mortality and significant morbidity. Genetic polymorphisms in cytochrome P450 genes are associated with altered enzymatic activity and may contribute to the risk of AHR. Here we present a case-control study in which we genotyped SNPs of CYP2C19, 2C9 and 3A5 of 55 individuals with varying severities of AHR, 83 tolerant, and 366 healthy control subjects from São Paulo, Brazil. Clinical characterization was based on standardized scoring systems and drug patch test. All in vivo investigation followed the ENDA (European Network of Drug Allergy) recommendations. Genotype was determined by real time PCR using peripheral blood DNA as a template. Of all 504 subjects, 65% were females, 45% self-identified as Afro-American, 38% as Caucasian and 17% as having non-African mixed ascendancy. Amongst 55 subjects with AHR, 44 had severe cutaneous drug adverse reactions. Of the 46 drug patch tests performed, 29 (63%) were positive. We found a strong association between the absence of CYP3A5*3 and tolerant subjects when compared to AHR (p = 0.0002, OR = 5.28 [CI95% 2.09–14.84]). None of our groups presented positive association with CYP2C19 and 2C9 polymorphisms, however, both SNPs contributed to separation of cases and tolerants in a Classification and Regression Tree. Our findings indicate that drug metabolism genes can contribute in the tolerability of antiepileptics. CYP3A5*3 is the most prevalent CYP3A5 allele associated with reduced enzymatic function. The current study provides evidence that normal CYP3A5 activity might be a protective factor to aromatic antiepileptics-induced hypersensitivity reactions in Brazilian subjects. PMID:26291084

  18. Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

    PubMed Central

    Vyskočilová, Erika; Szotáková, Barbora; Skálová, Lenka; Bártíková, Hana; Hlaváčová, Jitka

    2013-01-01

    Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates. PMID:23971034

  19. Physiologically based predictions of the impact of inhibition of intestinal and hepatic metabolism on human pharmacokinetics of CYP3A substrates.

    PubMed

    Fenneteau, Frederique; Poulin, Patrick; Nekka, Fahima

    2010-01-01

    The first objective of the present study was to predict the pharmacokinetics of selected CYP3A substrates administered at a single oral dose to human. The second objective was to predict pharmacokinetics of the selected drugs in presence of inhibitors of the intestinal and/or hepatic CYP3A activity. We developed a whole-body physiologically based pharmacokinetics (WB-PBPK) model accounting for presystemic elimination of midazolam (MDZ), alprazolam (APZ), triazolam (TRZ), and simvastatin (SMV). The model also accounted for concomitant administration of the above-mentioned drugs with CYP3A inhibitors, namely ketoconazole (KTZ), itraconazole (ITZ), diltiazem (DTZ), saquinavir (SQV), and a furanocoumarin contained in grape-fruit juice (GFJ), namely 6',7'-dihydroxybergamottin (DHB). Model predictions were compared to published clinical data. An uncertainty analysis was performed to account for the variability and uncertainty of model parameters when predicting the model outcomes. We also briefly report on the results of our efforts to develop a global sensitivity analysis and its application to the current WB-PBPK model. Considering the current criterion for a successful prediction, judged satisfied once the clinical data are captured within the 5th and 95th percentiles of the predicted concentration-time profiles, a successful prediction has been obtained for a single oral administration of MDZ and SMV. For APZ and TRZ, however, a slight deviation toward the 95th percentile was observed especially for C(max) but, overall, the in vivo profiles were well captured by the PBPK model. Moreover, the impact of DHB-mediated inhibition on the extent of intestinal pre-systemic elimination of MDZ and SMV has been accurately predicted by the proposed PBPK model. For concomitant administrations of MDZ and ITZ, APZ and KTZ, as well as SMV and DTZ, the in vivo concentration-time profiles were accurately captured by the model. A slight deviation was observed for SMV when

  20. 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

  1. Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites.

    PubMed

    Rendic, S; Nolteernsting, E; Schänzer, W

    1999-11-26

    Metabolism of steroid hormones with anabolic properties was studied in vitro using human recombinant CYP3A4, CYP2C9 and 2B6 enzymes. The enzyme formats used for CYP3A4 and CYP2C9 were insect cell microsomes expressing human CYP enzymes and purified recombinant human CYP enzymes in a reconstituted system. CYP3A4 enzyme formats incubated with anabolic steroids, testosterone, 17alpha-methyltestosterone, metandienone, boldenone and 4-chloro-1,2-dehydro-17alpha-methyltestosterone, produced 6beta-hydroxyl metabolites identified as trimethylsilyl (TMS)-ethers by a gas chromatography-mass spectrometry (GC-MS) method. When the same formats of CYP2C9 were incubated with the anabolic steroids, no 6beta-hydroxyl metabolites were formed. Human lymphoblast cell microsomes expressing human CYP2B6 incubated with the steroids investigated produced traces of 6beta-hydroxyl metabolites with testosterone and 17alpha-methyltestosterone only. We suggest that the electronic effects of the 3-keto-4-ene structural moiety contribute to the selectivity within the active site of CYP3A4 enzyme resulting in selective 6beta-hydroxylation. PMID:10630892

  2. Genetic polymorphisms of the drug-metabolizing enzyme cytochrome P450 3A5 in a Uyghur Chinese population.

    PubMed

    Chen, Zhengshuai; Li, Jingjie; Chen, Peng; Wang, Fengjiao; Zhang, Ning; Yang, Min; Jin, Tianbo; Chen, Chao

    2016-09-01

    1.  Detection of CYP3A5 variant alleles, and knowledge about their allelic frequency in Uyghur ethnic groups, is important to establish the clinical relevance of screening for these polymorphisms to optimize pharmacotherapy. 2. We used DNA sequencing to investigate the promoter, exons and surrounding introns, and 3'-untranslated region of the CYP3A5 gene in 96 unrelated healthy Uyghur individuals. We also used SIFT and PolyPhen-2 to predict the protein function of the novel non-synonymous mutation in CYP3A5 coding regions. 3. We found 24 different CYP3A5 polymorphisms in the Uyghur population, three of which were novel: the synonymous mutation 43C > T in exon 1, two mutations 32120C > G and 32245T > C in 3'-untranslated region, and we detected the allele frequencies of CYP3A5*1 and *3 as 64.58% and 35.42%, respectively. While no subjects with CYP3A5*6 were identified. Other identified genotypes included the heterozygous genotype 1A/3A (59.38%) and 1A/3E (11.46%), which lead to decreased enzyme activity. In addition, the frequency of haplotype "TTAGGT" was the most prevalent with 0.781. 4. Our data provide new information regarding CYP3A5 genetic polymorphisms in Uyghur individuals, which may help to improve individualization of drug therapy and offer a preliminary basis for more rational use of drugs. PMID:26739429

  3. Exploratory effects of a strong CYP3A inhibitor (ketoconazole), a strong CYP3A inducer (rifampicin), and concomitant ethanol on piragliatin pharmacokinetics and pharmacodynamics in type 2 diabetic patients.

    PubMed

    Zhi, Jianguo; Zhai, Suoping; Georgy, Angela; Liang, Zhenming; Boldrin, Mark

    2016-05-01

    Piragliatin is a CYP3A substrate; its inactive metabolite M4, formed through cytosolic reductase, is reversibly metabolized back to piragliatin through CYP3A. The impact of concomitant CYP3A modifiers thus cannot be predicted. Drinking alcohol under fasting conditions is associated with a recognized glucose-lowering effect, which might be synergistic with piragliatin's hypoglycemic effect. Two exploratory studies were conducted to examine these potential interactions in type 2 diabetes (T2D) patients: 16 completed an open-label, sequential 2-way crossover, 2-arm (randomized to ketoconazole and rifampicin) CYP3A study; another 18 participated in a double-blind, placebo-controlled, randomized 3-way crossover ethanol study. Administration of piragliatin (100-mg single dose) resulted in a 32% Cmax and 44% area under the curve (AUC∞ ) increase in piragliatin exposure without affecting glucose AUC0-6h following ketoconazole (400 mg QD × 5 days); 30% Cmax and 72% AUC∞ decrease in piragliatin exposure with a 13% increase in glucose AUC0-6h following rifampicin (600 mg QD × 5 days); and, unexpectedly, a 32% Cmax and 23% AUC0-6h decrease (no change in AUC∞ ) in piragliatin exposure with a 13% increase in glucose AUC0-6h following alcohol (40-g single dose). In conclusion, a strong CYP3A modifier or concomitant alcohol could lead to a change in exposure to piragliatin with a potential alteration in glucose-lowering effect. PMID:26272330

  4. Two CYP3A-like genes in the marine mussel Mytilus edulis: mRNA expression modulation following short-term exposure to endocrine disruptors.

    PubMed

    Cubero-Leon, Elena; Puinean, A Mirel; Labadie, Pierre; Ciocan, Corina; Itoh, Naoki; Kishida, Mitsuyo; Osada, Makoto; Minier, Christophe; Hill, Elizabeth M; Rotchell, Jeanette M

    2012-03-01

    Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation. PMID:22189070

  5. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

    PubMed

    Zhang, Qing-Hao; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2012-01-01

    Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC₅₀) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC₅₀ > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids. PMID:22375877

  6. 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

  7. Genomic comparisons of bovine CYP3A28 of Angus, Brahman, and reciprocal crosses and milk characteristics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450s are a group of heme-containing monooxygenases necessary for the oxidative metabolism of foreign biological substances. Our goal was to determine the frequency of single nucleotide polymorphism (SNP) 994 in the CYP3A28 sequence of three breed types of cattle. The distribution of geno...

  8. Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model.

    PubMed

    Almond, Lisa M; Mukadam, Sophie; Gardner, Iain; Okialda, Krystle; Wong, Susan; Hatley, Oliver; Tay, Suzanne; Rowland-Yeo, Karen; Jamei, Masoud; Rostami-Hodjegan, Amin; Kenny, Jane R

    2016-06-01

    Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach. PMID:27026679

  9. Prediction of Drug-Drug Interactions Arising from CYP3A induction Using a Physiologically Based Dynamic Model

    PubMed Central

    Mukadam, Sophie; Gardner, Iain; Okialda, Krystle; Wong, Susan; Hatley, Oliver; Tay, Suzanne; Rowland-Yeo, Karen; Jamei, Masoud; Rostami-Hodjegan, Amin; Kenny, Jane R.

    2016-01-01

    Using physiologically based pharmacokinetic modeling, we predicted the magnitude of drug-drug interactions (DDIs) for studies with rifampicin and seven CYP3A4 probe substrates administered i.v. (10 studies) or orally (19 studies). The results showed a tendency to underpredict the DDI magnitude when the victim drug was administered orally. Possible sources of inaccuracy were investigated systematically to determine the most appropriate model refinement. When the maximal fold induction (Indmax) for rifampicin was increased (from 8 to 16) in both the liver and the gut, or when the Indmax was increased in the gut but not in liver, there was a decrease in bias and increased precision compared with the base model (Indmax = 8) [geometric mean fold error (GMFE) 2.12 vs. 1.48 and 1.77, respectively]. Induction parameters (mRNA and activity), determined for rifampicin, carbamazepine, phenytoin, and phenobarbital in hepatocytes from four donors, were then used to evaluate use of the refined rifampicin model for calibration. Calibration of mRNA and activity data for other inducers using the refined rifampicin model led to more accurate DDI predictions compared with the initial model (activity GMFE 1.49 vs. 1.68; mRNA GMFE 1.35 vs. 1.46), suggesting that robust in vivo reference values can be used to overcome interdonor and laboratory-to-laboratory variability. Use of uncalibrated data also performed well (GMFE 1.39 and 1.44 for activity and mRNA). As a result of experimental variability (i.e., in donors and protocols), it is prudent to fully characterize in vitro induction with prototypical inducers to give an understanding of how that particular system extrapolates to the in vivo situation when using an uncalibrated approach. PMID:27026679

  10. Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes.

    PubMed

    Kudo, Toshiyuki; Ozaki, Yuya; Kusano, Tomomi; Hotta, Eri; Oya, Yuka; Komatsu, Seina; Goda, Hitomi; Ito, Kiyomi

    2016-01-01

    1. Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions. 2. The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10-200 mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates. 3. The Km (or S50) and Vmax values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis-Menten or Hill equation, greatly depended on the buffer components and their concentration. 4. The CLint values in phosphate buffer were 1.2-3.0-fold (paclitaxel) or 3.1-6.4-fold (triazolam) higher than in Tris-HCl buffer at 50-100 mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100 mM which are both commonly used in drug metabolism studies. 5. These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro-in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism. PMID:26290405

  11. Pharmacokinetic interaction studies of fenugreek with CYP3A substrates cyclosporine and carbamazepine.

    PubMed

    Al-Jenoobi, Fahad I; Alam, Mohd Aftab; Alkharfy, Khalid M; Al-Suwayeh, Saleh A; Korashy, Hesham M; Al-Mohizea, Abdullah M; Iqbal, Muzaffar; Ahad, Abdul; Raish, Mohammad

    2014-06-01

    The present study investigated the effect of fenugreek seed powder on disposition of CYP3A substrates, cyclosporine and carbamazepine. Rabbits were treated with fenugreek seed powder (300 mg/kg p.o.) for 8 days and on 8th day the single dose of cyclosporine (30 mg/kg, p.o.) and carbamazepine (40 mg/kg, p.o.) were administered to the corresponding group after 1 h of fenugreek administration. Blood samples were drawn at several time points and analyzed by using UPLC-MS (cyclosporine) and HPLC (carbamazepine). Pharmacokinetic parameters were calculated by using PK Solver. The present investigation reveals that there was no statistically significant difference between pre- and post-treated pharmacokinetic parameters such as AUC(o-t), AUC(o-∞), C(max), T(max), T(1/2), K(el), MRT(o-∞) , V(z/F), and Cl/F for cyclosporine and carbamazepine. Two tailed "P" values for all these pharmacokinetic parameters were more than 0.05, indicating insignificant impact of fenugreek treatment on the disposition of cyclosporine and carbamazepine. Further, fenugreek may also not have any significant effect on the functionality of P-glycoprotein as cyclosporine is a substrate to P-glycoprotein. The outcomes of present study suggested that fenugreek may not likely to interfere cyclosporine and carbamazepine pharmacokinetics, when co-administered with these drugs. PMID:24022709

  12. Enzyme activity determination using ultrasound

    NASA Astrophysics Data System (ADS)

    Holmes, M. J.; Southworth, T.; Watson, N. G.; Povey, M. J. W.

    2014-04-01

    Here are presented the results of a novel approach to the measurement of enzyme reaction rates in which ultrasound velocity measurement is used. Our results show enzyme activity is observable, in the acoustic context, and that furthermore this offers the potential to estimate the rate of reaction over different substrate concentrations and temperatures. Findings are corroborated with optical microscopy and rheological measurements. Ultrasound velocity measurement can be performed without the need for aliquot extraction and offers an efficient, non-invasive and dynamic method to monitor enzyme activity.

  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

    obtained using estimates of enzyme Ki, inhibitor and substrate concentrations and absorption rate for substrate and inhibitor. WHAT THIS STUDY ADDS Using a generic approach for all test compounds, the findings from the current study showed the use of recombinant P450s provide a more robust in vitro measure of P450 contribution (fraction metabolized, fm) than that achieved when using chemical inhibitors in combination with human liver microsomes, for the prediction of potential CYP3A4 drug–drug interactions prior to clinical investigation. The current study supported the use of SIMCYP®, a modelling and simulation software in utilizing the in vitro measures in the prediction of potential drug–drug interactions. PMID:18279465

  14. No Impact of Vitamin D on the CYP3A Biomarker 4β-Hydroxycholesterol in Patients with Abnormal Glucose Regulation

    PubMed Central

    Mannheimer, Buster; Wagner, Henrik; Östenson, Claes-Göran; Diczfalusy, Ulf

    2015-01-01

    Purpose To investigate the effect of vitamin D3 on hepatic Cytochrome P450 enzyme (CYP) 3A4 in patients with abnormal glucose regulation using the endogenous marker 4β-hydroxycholesterol (4β-OHC):cholesterol ratio. Methods The present study took advantage of a trial primarily aiming to investigate the effect of vitamin D3 on beta cell function and insulin sensitivity in patients with abnormal glucose regulation. 44 subjects were randomized to receive vitamin D3, 30000 IU given orally once weekly or placebo for 8 weeks. The two sample t-test was used to test the means of the intra-individual differences of 4β-OHC:cholesterol ratio between the two groups. Results Mean (SD) 4β-OHC in the whole group of patients before and after the intervention was 26 (11) ng/ml and 26 (12). Mean (SD) 4β-OHC:cholesterol ratio in the whole group of patients before and after the intervention was 0.12 (0.046) and 0.13 (0.047). In the Vitamin D group mean (SD) serum 25-OH-vitamin D3 increased from 46 (16) to 85nM (13) during the corresponding time period. To investigate the impact of vitamin D3 on hepatic CYP3A4 we calculated the mean intra-individual differences in 4β-OHC:cholesterol ratio (delta 4β-OHC:cholesterol ratio) before versus after the intervention in the two treatment groups. The difference (95% CI) between delta 4β-OHC:cholesterol ratio in the control group and intervention group was -0.0010 (-0.0093, 0.0072), a difference being not statistically significant (p = 0.80). Conclusions We provide further evidence that vitamin D3 may not substantially affect hepatic CYP3A4. This does not exclude the possibility of an impact of intestinal first-pass metabolism of orally administered drugs which should be investigated. Trial Registration ClinicalTrials.gov NCT01497132 PMID:25835492

  15. 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.

  16. Pharmacokinetics of Lidocaine Hydrochloride Metabolized by CYP3A4 in Chinese Han Volunteers Living at Low Altitude and in Native Han and Tibetan Chinese Volunteers Living at High Altitude.

    PubMed

    Zhang, Juanling; Zhu, Junbo; Yao, Xingchen; Duan, Yabin; Zhou, Xuejiao; Yang, Meng; Li, Xiangyang

    2016-01-01

    To investigate the pharmacokinetics of lidocaine hydrochloride metabolized by cytochrome P450 3A4 (CYP3A4) in Chinese Han volunteers living at low altitude (LA) and in native Han and Tibetan Chinese volunteers living at high altitude, lidocaine hydrochloride 10 mg was given by intramuscular injection to 3 groups: Han volunteers living at LA, and native Han and Tibetan volunteers living at a high altitude. Blood samples were collected before the (baseline) study drug was given and at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0 h after study drug administration. Lidocaine hydrochloride in plasma was determined by RP-HPLC. Pharmacokinetics parameters of lidocaine hydrochloride showed that there were no significant difference between the native Han and Tibetan volunteers, but the t1/2 was 29.8 and 29.8% higher in 2 groups, respectively, than in the LA group. To study related mechanism, the effects of exposure to chronic high-altitude hypoxia (CHH) on the activity and expression of CYP3A1 were examined in rats. Rats were divided into LA, chronic moderate altitude hypoxia, and CHH groups. CHH caused significant decreases in the activity and protein and mRNA expression of rat CYP3A1 in vivo. This study found significant changes in the disposition of lidocaine hydrochloride in native healthy Tibetan and Han Chinese subjects living at a high altitude in comparison to healthy Han Chinese subjects living at LA, it might be due to significant decreases in the activity and protein and mRNA expression of CYP3A4 under CHH condition. PMID:26730802

  17. Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

    PubMed Central

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

  18. Differential expression of cytochrome P450 enzymes in normal and tumor tissues from childhood rhabdomyosarcoma.

    PubMed

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

  19. Reduced Methylprednisolone Clearance Causing Prolonged Pharmacodynamics in a Healthy Subject Was Not Associated With CYP3A5*3 Allele or a Change in Diet Composition

    PubMed Central

    Lee, Su-Jun; Jusko, William J.; Salaita, Christine G.; Calis, Karim A.; Jann, Michael W.; Spratlin, Vicky E.; Goldstein, Joyce A.; Hon, Yuen Yi

    2014-01-01

    The influence of diet and genetics was investigated in a healthy white person who had distinctly low methylprednisolone clearance. Pharmacokinetic and pharmacodynamic parameter values were similar on 2 occasions during the consumption of a low-carbohydrate diet and a Weight Watchers diet, indicating that the decreased clearance was unlikely attributable to a change in diet composition. Although the subject was found to be homozygous for CYP3A5*3, genetic findings were not significant for a number of other CYP3A4 and CYP3A5 allelic variants. Because of the high prevalence of CYP3A5*3/*3 in whites and because 5 of 7 white control subjects are also homozygous for CYP3A5*3, this genotype cannot fully explain the reduced metabolism of the drug. Other genetic or contributing factors might have been involved. New polymerase chain reaction–based genotyping methods for functionally defective CYP3A5*6, *8, *9, and *10 alleles were developed in this study. These assays will be useful for CYP3A5 genotype analysis in future clinical studies. PMID:16638735

  20. Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5'-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kwon, Soon-Sang; Kim, Ju-Hyun; Jeong, Hyeon-Uk; Cho, Yong Yeon; Oh, Sei-Ryang; Lee, Hye Suk

    2016-01-01

    Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca(2+)-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin-enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4'-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4'-hydroxylation, and CYP3A4-mediated midazolam 1'-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1'-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4. PMID:27128896

  1. 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

  2. Plasma levels of 25-hydroxyvitamin D3 and in vivo markers of cytochrome P450 3A activity in Swedes and Koreans: effects of a genetic polymorphism and oral contraceptives.

    PubMed

    Nylén, Hanna; Björkhem-Bergman, Linda; Ekström, Lena; Roh, Hyung-Keun; Bertilsson, Leif; Eliasson, Erik; Lindh, Jonatan D; Diczfalusy, Ulf

    2014-10-01

    In vitro studies have shown that vitamin D may induce several cytochrome P450 (CYP) enzymes in general and CYP3A4 in particular. The primary aim of this study was to investigate the relationship between plasma levels of 25-hydroxyvitamin D3 and suggested in vivo markers of CYP3A activity in healthy volunteers from Sweden and Korea. Plasma concentrations of 25-hydroxyvitamin D3 were analysed in samples from three previously performed studies, and the correlation between these levels and suggested in vivo markers of CYP3A activity was investigated by means of nonparametric correlation. In addition, we studied the modulating effects of three vitamin D receptor promoter polymorphisms on the association between 25-hydroxyvitamin D3 and CYP3A enzyme activity in Swedish subjects. The plasma levels of 25-hydroxyvitamin D3 were not significantly associated with CYP3A phenotypes in any of the three studies, but after accounting for the vitamin D receptor polymorphism rs4516035, there was a significant positive association between 25-hydroxyvitamin D3 and CYP3A activity (p = 0.004). Swedes (n = 65) had significantly higher 25-hydroxyvitamin D3 levels than Koreans (n = 67), 75 nM compared with 31 nM (p < 0.001). Swedish women taking oral contraceptives (OC) (n = 19) had somewhat higher plasma levels of 25-hydroxyvitamin D3 compared with Swedish women not taking oral contraceptives (n = 21), 89 and 72 nM, respectively (p = 0.02). In conclusion, our results suggest that the overall influence on the CYP3A activity by 25-hydroxyvitamin D3 is of marginal importance. PMID:24655660

  3. Prevalence of CYP2D6*2, CYP2D6*4, CYP2D6*10, and CYP3A5*3 in Thai breast cancer patients undergoing tamoxifen treatment

    PubMed Central

    Charoenchokthavee, Wanaporn; Panomvana, Duangchit; Sriuranpong, Virote; Areepium, Nutthada

    2016-01-01

    Background Tamoxifen (TAM) is used in breast cancer treatment, but interindividual variabilities in TAM-metabolizing enzymes exist and have been linked to single nucleotide polymorphisms in the respective encoding genes. The different alleles and genotypes of these genes have been presented for Caucasians and Asians. This study aimed to explore the prevalence of the incomplete functional alleles and genotypes of the CYP2D6 and CYP3A5 genes in Thai breast cancer patients undergoing TAM treatment. Patients and methods In total, 134 Thai breast cancer patients were randomly invited to join the Thai Tamoxifen Project. Their blood samples were collected and extracted for individual DNA. The alleles and genotypes were determined by real-time polymerase chain reaction with TaqMan® Drug Metabolism Genotyping Assays. Results The patients were aged from 27.0 years to 82.0 years with a body mass index range from 15.4 to 40.0, with the majority (103/134) in the early stage (stages 0–II) of breast cancer. The median duration of TAM administration was 17.2 months (interquartile range 16.1 months). Most (53%) of the patients were premenopausal with an estrogen receptor (ER) and progesterone receptor (PR) status of ER+/PR+ (71.7%), ER+/PR− (26.9%), ER−/PR+ (0.7%), and ER−/PR− (0.7%). The allele frequencies of CYP2D6*1, CYP2D6*2, CYP2D6*4, CYP2D6*10, CYP3A5*1, and CYP3A5*3 were 72.9%, 3.2%, 1.1%, 22.8%, 37.3%, and 62.7%, respectively, while the genotype frequencies of CYP2D6*1/*1, CYP2D6*1/*2, CYP2D6*2/*2, CYP2D6*4/*4, CYP2D6*1/*10, CYP2D6*2/*10, CYP2D6*4/*10, CYP2D6*10/*10, CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3 were 9.7%, 2.2%, 3.7%, 1.5%, 15.7%, 9.7%, 3.7%, 53.7%, 13.4%, 47.8%, and 38.8%, respectively. Conclusion The majority (97.8%) of Thai breast cancer patients undergoing TAM treatment carry at least one incomplete functional allele, including 20.9% of the patients who carry only incomplete functional alleles for both the CYP2D6 and CYP3A5 genes. This research

  4. Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

    PubMed

    Ghosal, Anima; Yuan, Yuan; Tong, Wei; Su, Ai-Duen; Gu, Chunyan; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-03-01

    Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 μM flufenamic acid (80.3%), 200 μM mefenamic acid (83.7%), and 100 μM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 μM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 μM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways. PMID:21123164

  5. Inhibitory effect of single and repeated doses of nilotinib on the pharmacokinetics of CYP3A substrate midazolam.

    PubMed

    Zhang, Hefei; Sheng, Jennifer; Ko, Jin H; Zheng, Cheng; Zhou, Wei; Priess, Petra; Lin, Wen; Novick, Steven

    2015-04-01

    Effects of single and repeated doses of nilotinib on the pharmacokinetics of midazolam, a cytochrome P450 3A (CYP3A) substrate, were assessed in 2 separate studies. In the single-dose nilotinib study, 18 healthy subjects were randomized to 6 treatment sequences to receive single dose of nilotinib 600 mg, midazolam 4 mg, and coadministration of both in a crossover manner. In the repeated-dose nilotinib study, 19 chronic myeloid leukemia patients took a single dose of midazolam 2 mg on days 1 and 13, and nilotinib 400 mg twice daily from days 2-13. In the single-dose study, the geometric mean ratio of the area under the plasma concentration time curve extrapolated to infinity (AUC(inf)) of midazolam plus nilotinib vs. midazolam was 1.3 (90%CI, 1.2-1.5) and the maximum observed serum concentration (C(max)) was 1.2 (90%CI, 1.0-1.4). In the repeated-dose study, the values for AUC(inf) and C(max) were 2.6 (90%CI, 2.1-3.3) and 2.0 (90%CI, 1.7-2.4), respectively. These results indicate that single-dose and repeated-dose administration of nilotinib results in weak and moderate inhibition of CYP3A, respectively. Therefore, appropriate monitoring and dose adjustment may be needed for drugs that are mainly metabolized by CYP3A, and have narrow therapeutic index, when coadministered with nilotinib. PMID:25418605

  6. 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

  7. 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.

  8. Activation status of the Pregnane X Receptor (PXR) influences Vemurafenib availability in humanized mouse models

    PubMed Central

    MacLeod, A. Kenneth; McLaughlin, Lesley A.; Henderson, Colin J.; Wolf, C. Roland

    2015-01-01

    Vemurafenib is a revolutionary treatment for melanoma, but the magnitude of therapeutic response is highly variable and the rapid acquisition of resistance is frequent. Here, we examined how vemurafenib disposition, particularly through cytochrome P450-mediated oxidation pathways, could potentially influence these outcomes using a panel of knockout and transgenic humanized mouse models. We identified CYP3A4 as the major enzyme involved in the metabolism of vemurafenib in in vitro assays with human liver microsomes. However, mice expressing human CYP3A4 did not process vemurafenib to a greater extent than CYP3A4 null animals, suggesting that other pregnane X receptor (PXR)-regulated pathways may contribute more significantly to vemurafenib metabolism in vivo. Activation of PXR, but not of the closely related constitutive androstane receptor (CAR), profoundly reduced circulating levels of vemurafenib in humanized mice. This effect was independent of CYP3A4 and was negated by co-treatment with the drug efflux transporter inhibitor, elacridar. Finally, vemurafenib strongly induced PXR activity in vitro, but only weakly induced PXR in vivo. Taken together, our findings demonstrate that vemurafenib is unlikely to exhibit a clinically significant interaction with CYP3A4, but that modulation of bioavailability through PXR-mediated regulation of drug transporters (for example by other drugs) has the potential to markedly influence systemic exposure and thereby therapeutic outcomes. Activation status of the Pregnane X Receptor (PXR) influences Vemurafenib availability in humanized mouse models. PMID:26363009

  9. 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

  10. Long-Term Clinical Impact of Adaptation of Initial Tacrolimus Dosing to CYP3A5 Genotype.

    PubMed

    Pallet, N; Etienne, I; Buchler, M; Bailly, E; Hurault de Ligny, B; Choukroun, G; Colosio, C; Thierry, A; Vigneau, C; Moulin, B; Le Meur, Y; Heng, A-E; Legendre, C; Beaune, P; Loriot, M A; Thervet, E

    2016-09-01

    Pretransplantation adaptation of the daily dose of tacrolimus to CYP3A5 genotype is associated with improved achievement of target trough concentration (C0 ), but whether this improvement affects clinical outcomes is unknown. In the present study, we have evaluated the long-term clinical impact of the adaptation of initial tacrolimus dosing according to CYP3A5 genotype: The transplantation outcomes of the 236 kidney transplant recipients included in the Tactique study were retrospectively investigated over a period of more than 5 years. In the Tactique study, patients were randomly assigned to receive tacrolimus at either a fixed dosage or a dosage determined by their genotype, and the primary efficacy end point was the proportion of patients for whom tacrolimus C0 was within target range (10-15 ng/mL) at day 10. Our results indicate that the incidence of biopsy-proven acute rejection and graft survival were similar between the control and the adapted tacrolimus dose groups, as well as between the patients who achieve the tacrolimus C0 target ranges earlier. Patients' death, cancer, cardiovascular events, and infections were also similar, and renal function did not change. We conclude that optimization of initial tacrolimus dose using pharmacogenetic testing does not improve clinical outcomes. PMID:26990694

  11. Capsaicin pretreatment increased the bioavailability of cyclosporin in rats: involvement of P-glycoprotein and CYP 3A inhibition.

    PubMed

    Zhai, Xue-jia; Shi, Fang; Chen, Fen; Lu, Yong-ning

    2013-12-01

    Capsaicin (CAP), the main ingredient responsible for the hot pungent taste of chilli peppers. This study investigated the effect of CAP on the pharmacokinetics of Cyclosporin A (CyA) in rats and the mechanism of this food-drug interaction. The results indicated that after 7 days of low or middle dose of CAP (0.3 or 1.0 mg/kg), the blood concentration of CyA was not significantly changed compared with that of vehicle-treated rats, whereas the blood concentration of CyA in high dose group (3.0 mg/kg) was significantly increased. The total clearance (CL/F) of CyA was decreased, and the bioavailability was significantly increased to about 1.44-fold of that in vehicle-treated rats after 7 days of high dose CAP treatment. At this time, the P-gp and CYP3A1/2 in the liver and intestine were decreased at both the mRNA and protein levels. These results demonstrated that chronic ingestion of high doses of CAP will increase the bioavailability of CyA to a significant extent in rats and the food-drug interaction between CAP and CyA appears to be due to modulation of P-gp and CYP3A gene expression by CAP, with differential dose-dependence. PMID:24013073

  12. Quercetin, an in vitro inhibitor of CYP3A, does not contribute to the interaction between nifedipine and grapefruit juice.

    PubMed Central

    Rashid, J; McKinstry, C; Renwick, A G; Dirnhuber, M; Waller, D G; George, C F

    1993-01-01

    Quercetin, a flavonoid present in various fruits, is a potent in vitro inhibitor of CYP3A. Its role in the reported interaction between grapefruit juice and nifedipine has been determined in vivo in humans. Eight healthy volunteers were given in random order 10 mg nifedipine orally, either alone or with 200 ml double strength grapefruit juice, or with 400 mg quercetin. The area under the plasma concentration-time curve (AUC) for nifedipine with grapefruit juice (mean 320 ng ml(-1) h) was increased significantly (P < 0.01) compared with the AUC when nifedipine was given alone (mean 218 ng ml(-1) h). The time to peak plasma concentration for nifedipine with grapefruit juice (1.5 h) was also increased (P < 0.05) compared with control (0.5 h) suggesting delayed absorption. Although quercetin delayed the time to peak nifedipine concentration (1.3 h) it did not alter the AUC of either the parent drug (mean 209 ng ml(-1) h) or its first-pass metabolite. The results suggest that quercetin does not contribute to the effects of grapefruit juice (which contains <10 mg of quercetin 200 ml(-1)) on the metabolism of nifedipine. Oral doses of quercetin, similar to those possible from the ingestion of other fruits such as strawberries, do not produce in vivo inhibition of CYP3A mediated metabolism of nifedipine. PMID:12959295

  13. 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

  14. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein.

    PubMed

    Evans, David C; O'Connor, Desmond; Lake, Brian G; Evers, Raymond; Allen, Christopher; Hargreaves, Richard

    2003-07-01

    "Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity. PMID:12814962

  15. Enzyme- and transporter-mediated beverage-drug interactions: An update on fruit juices and green tea.

    PubMed

    An, Guohua; Mukker, Jatinder Kaur; Derendorf, Hartmut; Frye, Reginald F

    2015-12-01

    Beverage-drug interactions have remained an active area of research and have been the subject of extensive investigations in the past 2 decades. The known mechanisms of clinically relevant beverage-drug interactions include modulation of the activity of cytochrome P450 (CYP) 3A and organic anion-transporting polypeptide (OATP). For CYP3A-mediated beverage-drug interaction, the in vivo CYP3A inhibitory effect is limited to grapefruit juice (GFJ), which increases the bioavailability of several orally administered drugs that undergo extensive first-pass metabolism via enteric CYP3A. In contrast, clinically significant OATP-mediated beverage-drug interactions have been observed with not only GFJ but also orange juice, apple juice, and, most recently, green tea. Fruit juices and green tea are all a mixture of a large number of constituents. The investigation of specific constituent(s) responsible for the enzyme and/or transporter inhibition remains an active area of research, and many new findings have been obtained on this subject in the past several years. This review highlights the multiple mechanisms through which beverages can alter drug disposition and provides an update on the new findings of beverage-drug interactions, with a focus on fruit juices and green tea. PMID:26095990

  16. Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes.

    PubMed

    Dixit, Vaishali; Moore, Amanda; Tsao, Hong; Hariparsad, Niresh

    2016-02-01

    Long-term coculture models of hepatocytes are promising tools to study drug transport, clearance, and hepatoxicity. In this report we compare the basal expression of drug disposition genes and the inductive response of prototypical inducers (rifampin, phenobarbital, phenytoin) in hepatocyte two-dimensional monocultures and the long-term coculture model (HepatoPac). All the inducers used in the study increased the expression and activity of CYP3A4, CYP2B6 and CYP2C enzymes in the HepatoPac cultures. The coculture model showed a consistent and higher induction of CYP2C enzymes compared with the monocultures. The EC50 of rifampin for CYP3A4 and CYP2C9 was up to 10-fold lower in HepatoPac than the monocultures. The EC50 of rifampin calculated from the clinical drug interaction studies correlated well with the EC50 observed in the HepatoPac cultures. Owing to the long-term stability of the HepatoPac cultures, we were able to directly measure a half-life (t1/2) for both CYP3A4 and CYP2B6 using the depletion kinetics of mRNA and functional activity. The t1/2 for CYP3A4 mRNA was 26 hours and that for the functional protein was 49 hours. The t1/2 of CYP2B6 was 38 hours (mRNA) and 68 hours (activity), which is longer than CYP3A4 and shows the differential turnover of these two proteins. This is the first study to our knowledge to report the turnover rate of CYP2B6 in human hepatocytes. The data presented here demonstrate that the HepatoPac cultures have the potential to be used in long-term culture to mimic complex clinical scenarios. PMID:26658225

  17. 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

  18. Associations of CYP3A4, NR1I2, CYP2C19 and P2RY12 polymorphisms with clopidogrel resistance in Chinese patients with ischemic stroke

    PubMed Central

    Liu, Rui; Zhou, Zi-yi; Chen, Yi-bei; Li, Jia-li; Yu, Wei-bang; Chen, Xin-meng; Zhao, Min; Zhao, Yuan-qi; Cai, Ye-feng; Jin, Jing; Huang, Min

    2016-01-01

    Aim: There is a high incidence of the antiplatelet drug clopidogrel resistance (CR) in Asian populations. Because clopidogrel is a prodrug, polymorphisms of genes encoding the enzymes involved in its biotransformation may be the primary influential factors. The goal of this study was to investigate the associations of polymorphisms of CYP3A4, NR1I2, CYP2C19 and P2RY12 genes with CR in Chinese patients with ischemic stroke. Methods: A total of 191 patients with ischemic stroke were enrolled. The patients were treated with clopidogrel for at least 5 days. Platelet function was measured by light transmission aggregometry. The SNPs NR1I2 (rs13059232), CYP3A4*1G (rs2242480), CYP2C19*2 (rs4244285) and P2RY12 (rs2046934) were genotyped. Results: The CR rate in this population was 36%. The CYP2C19*2 variant was a risk factor for CR (*2/*2+wt/*2 vs wt/wt, OR: 2.366, 95% CI: 1.180–4.741, P=0.014), whereas the CYP3A4*1G variant had a protective effect on CR (*1/*1 vs *1G/*1G+*1/*1G, OR: 2.360, 95% CI: 1.247–4.468, P=0.008). The NR1I2 (rs13059232) polymorphism was moderately associated with CR (CC vs TT+TC, OR: 0.533, 95% CI: 0.286–0.991, P=0.046). The C allele in P2RY12 (rs2046934) was predicted to be a protective factor for CR (CC+TC vs TT, OR: 0.407, 95% CI: 0.191–0.867, P=0.018). In addition, an association was found between hypertension and CR (P=0.022). Conclusion: The individuals with both the CYP2C19*2 allele and hypertension are at high risk of CR during anti-thrombosis therapy. The CYP3A4*1G allele, P2RY12 (rs2046934) C allele and NR1I2 (rs13059232) CC genotype may be protective factors for CR. The associated SNPs studied may be useful to predict clopidogrel resistance in Chinese patients with ischemic stroke. PMID:27133299

  19. Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

    PubMed Central

    Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

    1994-01-01

    1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

  20. Phenotyping studies to assess the effects of phytopharmaceuticals on in vivo activity of main human cytochrome p450 enzymes.

    PubMed

    Zadoyan, Gregor; Fuhr, Uwe

    2012-09-01

    The extensive use of herbal drugs and their multiple components and modes of action suggests that they may also cause drug interactions by changing the activity of human cytochrome P450 enzymes. The purpose of the present review is to present the available data for the top 14 herbal drug sales in the U. S. Studies describing the effects of herbal drugs on phenotyping substrates for individual CYPs were identified by a comprehensive MEDLINE search. Drugs included Allium sativum (Liliaceae), Echinacea purpurea (Asteraceae), Serenoa repens (Arecaceae), Ginkgo biloba (Ginkgoaceae), Vaccinium macrocarpon (Ericaceae), Glycine max (Fabaceae), Panax ginseng (Araliaceae), Actea racemosa (Ranunculaceae), Hypericum perforatum (Hypericaceae), Silybum marianum (Asteraceae), Camellia sinensis (Theaceae), Valeriana officinalis (Valerianaceae), Piper methysticum (Piperaceae), and Hydrastis canadensis (Ranunculaceae) preparations. We identified 70 clinical studies in 69 publications. The majority of the herbal drugs appeared to have no clear effects on most of the CYPs examined. If there was an effect, there was mild inhibition in almost all cases, as seen with garlic or kava effects on CYP2E1 and with soybean components on CYP1A2. The most pronounced effects were induction of CYP3A and other CYPs by St. John's wort and the inhibitory effect of goldenseal on CYP3A and CYP2D6, both being borderline between mild and moderate in magnitude. With the exceptions of St.John's wort and goldenseal, the information currently available suggests that concomitant intake of the herbal drugs addressed here is not a major risk for drugs that are metabolized by CYPs. PMID:22588833

  1. [Effect of diet overloaded by fats on the enzyme systems of metabolism in rats].

    PubMed

    Herych, O Kh; Pentiuk, O O

    2008-01-01

    The feeding of rats with high-fat diet (a part of fats was 50% of energy value of ration against 20% in control) during 4 weeks increased the level of cytochrome P-450 in the liver and enhanced aniline- and p-nitrophenol hydroxylase activity of CYP2E1 as well as erythromycin N-demethylase activity of CYP3A; the activity of aldehyde dehydrogenase class 3, UDP-glucuronosyl transferase, glutathione-S-transferase and N-acetyl transferase. At the same time, the moderate decrease of indomethacin-O-demethylase of CYP2C both phenolsulfotransferase activity were fixed. The changes of enzymatic activity correlated with activating of processes of gluconeogenesis, glycogenolysis and, especially, ketogenesis. A high-fat diet enhanced reactions of biotransformation of amidopyrine, acetanilide, toluene, sulfadimezine, were catalyzed with CYP2E1, CYP3A and enzymes of conjugation, simultaneously it increased the hepatotoxicity of paracetamol. PMID:18710030

  2. Effects of orally applied butyrate bolus on histone acetylation and cytochrome P450 enzyme activity in the liver of chicken – a randomized controlled trial

    PubMed Central

    2013-01-01

    Background Butyrate is known as histone deacetylase inhibitor, inducing histone hyperacetylation in vitro and playing a predominant role in the epigenetic regulation of gene expression and cell function. We hypothesized that butyrate, endogenously produced by intestinal microbial fermentation or applied as a nutritional supplement, might cause similar in vivo modifications in the chromatin structure of the hepatocytes, influencing the expression of certain genes and therefore modifying the activity of hepatic microsomal drug-metabolizing cytochrome P450 (CYP) enzymes. Methods An animal study was carried out in chicken as a model to investigate the molecular mechanisms of butyrate’s epigenetic actions in the liver. Broiler chicks in the early post-hatch period were treated once daily with orally administered bolus of butyrate following overnight starvation with two different doses (0.25 or 1.25 g/kg body weight per day) for five days. After slaughtering, cell nucleus and microsomal fractions were separated by differential centrifugation from the livers. Histones were isolated from cell nuclei and acetylation of hepatic core histones was screened by western blotting. The activity of CYP2H and CYP3A37, enzymes involved in biotransformation in chicken, was detected by aminopyrine N-demethylation and aniline-hydroxylation assays from the microsomal suspensions. Results Orally added butyrate, applied in bolus, had a remarkable impact on nucleosome structure of hepatocytes: independently of the dose, butyrate caused hyperacetylation of histone H2A, but no changes were monitored in the acetylation state of H2B. Intensive hyperacetylation of H3 was induced by the higher administered dose, while the lower dose tended to increase acetylation ratio of H4. In spite of the observed modification in histone acetylation, no significant changes were observed in the hepatic microsomal CYP2H and CYP3A37 activity. Conclusion Orally added butyrate in bolus could cause in vivo

  3. The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

    PubMed

    Soraoka, Hiromi; Oniki, Kentaro; Matsuda, Kazuki; Ono, Tatsumasa; Taharazako, Kosuke; Uchiyashiki, Yoshihiro; Kamihashi, Ryoko; Kita, Ayana; Takashima, Ayaka; Nakagawa, Kazuko; Yasui-Furukori, Norio; Kadowaki, Daisuke; Miyata, Keishi; Saruwatari, Junji

    2016-01-01

    The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6β-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2. PMID:27582327

  4. 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

  5. Determining Enzyme Activity by Radial Diffusion

    ERIC Educational Resources Information Center

    Davis, Bill D.

    1977-01-01

    Discusses advantages of radial diffusion assay in determining presence of enzyme and/or rough approximation of amount of enzyme activities. Procedures are included for the preparation of starch-agar plates, and the application and determination of enzyme. Techniques using plant materials (homogenates, tissues, ungerminated embryos, and seedlings)…

  6. Effects of poloxamer 407-induced hyperlipidemia on the pharmacokinetics of carbamazepine and its 10,11-epoxide metabolite in rats: Impact of decreased expression of both CYP3A1/2 and microsomal epoxide hydrolase.

    PubMed

    Lee, Young Sun; Kim, Young Woo; Kim, Sang Geon; Lee, Inchul; Lee, Myung Gull; Kang, Hee Eun

    2012-06-01

    The pharmacokinetics of carbamazepine (CBZ) and its active 10,11-epoxide metabolite (CBZ-E) were evaluated after intravenous and oral administration of 5 mg/kg CBZ to rats with hyperlipidemia induced by poloxamer 407 (HL rats) and controls. The total area under the plasma concentration-time curve (AUC) of CBZ in HL rats after intravenous administration was significantly greater than that in controls due to their slower non-renal clearance (CL(NR)). This was due to slower hepatic CL(int) for metabolism of CBZ to CBZ-E in HL rats via CYP3A1/2. This result was consistent with a previous study indicating reduced hepatic CYP3A1/2 expression in HL rats. Interestingly, the AUC of CBZ-E was also increased in HL rats, while AUC(CBZ-E)/AUC(CBZ) ratios remained unchanged. These results suggested that further metabolism of CBZ-E to the inactive metabolite trans-10,11-dihydoxyl-10,11-dihydro-CBZ (CBZ-D) via microsomal epoxide hydrolase (mEH) was also slowed in HL rats. The significantly reduced hepatic mRNA level and expression of mEH protein in HL rats compared to controls confirmed the above hypothesis. Similar pharmacokinetic changes were observed in HL rats after oral administration of CBZ. These findings have potential therapeutic implications assuming that the HL rat model qualitatively reflects similar changes in patients with hyperlipidemia. Caution is required regarding pharmacotherapy in the hyperlipidemic state in cases where drugs that are metabolized principally by CYP3A1/2 or mEH and have a narrow therapeutic range are in use. PMID:22137858

  7. Evacetrapib: in vitro and clinical disposition, metabolism, excretion, and assessment of drug interaction potential with strong CYP3A and CYP2C8 inhibitors.

    PubMed

    Cannady, Ellen A; Wang, Ming-Dauh; Friedrich, Stuart; Rehmel, Jessica L F; Yi, Ping; Small, David S; Zhang, Wei; Suico, Jeffrey G

    2015-10-01

    Evacetrapib is an investigational cholesteryl ester transfer protein inhibitor (CETPi) for reduction of risk of major adverse cardiovascular events in patients with high-risk vascular disease. Understanding evacetrapib disposition, metabolism, and the potential for drug-drug interactions (DDI) may help guide prescribing recommendations. In vitro, evacetrapib metabolism was investigated with a panel of human recombinant cytochromes P450 (CYP). The disposition, metabolism, and excretion of evacetrapib following a single 100-mg oral dose of (14)C-evacetrapib were determined in healthy subjects, and the pharmacokinetics of evacetrapib were evaluated in the presence of strong CYP3A or CYP2C8 inhibitors. In vitro, CYP3A was responsible for about 90% of evacetrapib's CYP-associated clearance, while CYP2C8 accounted for about 10%. In the clinical disposition study, only evacetrapib and two minor metabolites circulated in plasma. Evacetrapib metabolism was extensive. A mean of 93.1% and 2.30% of the dose was excreted in feces and urine, respectively. In clinical DDI studies, the ratios of geometric least squares means for evacetrapib with/without the CYP3A inhibitor ketoconazole were 2.37 for area under the curve (AUC)(0-∞) and 1.94 for C max. There was no significant difference in evacetrapib AUC(0-τ) or C max with/without the CYP2C8 inhibitor gemfibrozil, with ratios of 0.996 and 1.02, respectively. Although in vitro results indicated that both CYP3A and CYP2C8 metabolized evacetrapib, clinical studies confirmed that evacetrapib is primarily metabolized by CYP3A. However, given the modest increase in evacetrapib exposure and robust clinical safety profile to date, there is a low likelihood of clinically relevant DDI with concomitant use of strong CYP3A or CYP2C8 inhibitors. PMID:26516590

  8. Evacetrapib: in vitro and clinical disposition, metabolism, excretion, and assessment of drug interaction potential with strong CYP3A and CYP2C8 inhibitors

    PubMed Central

    Cannady, Ellen A; Wang, Ming-Dauh; Friedrich, Stuart; Rehmel, Jessica L F; Yi, Ping; Small, David S; Zhang, Wei; Suico, Jeffrey G

    2015-01-01

    Evacetrapib is an investigational cholesteryl ester transfer protein inhibitor (CETPi) for reduction of risk of major adverse cardiovascular events in patients with high-risk vascular disease. Understanding evacetrapib disposition, metabolism, and the potential for drug–drug interactions (DDI) may help guide prescribing recommendations. In vitro, evacetrapib metabolism was investigated with a panel of human recombinant cytochromes P450 (CYP). The disposition, metabolism, and excretion of evacetrapib following a single 100-mg oral dose of 14C-evacetrapib were determined in healthy subjects, and the pharmacokinetics of evacetrapib were evaluated in the presence of strong CYP3A or CYP2C8 inhibitors. In vitro, CYP3A was responsible for about 90% of evacetrapib's CYP-associated clearance, while CYP2C8 accounted for about 10%. In the clinical disposition study, only evacetrapib and two minor metabolites circulated in plasma. Evacetrapib metabolism was extensive. A mean of 93.1% and 2.30% of the dose was excreted in feces and urine, respectively. In clinical DDI studies, the ratios of geometric least squares means for evacetrapib with/without the CYP3A inhibitor ketoconazole were 2.37 for area under the curve (AUC)(0–∞) and 1.94 for Cmax. There was no significant difference in evacetrapib AUC(0–τ) or Cmax with/without the CYP2C8 inhibitor gemfibrozil, with ratios of 0.996 and 1.02, respectively. Although in vitro results indicated that both CYP3A and CYP2C8 metabolized evacetrapib, clinical studies confirmed that evacetrapib is primarily metabolized by CYP3A. However, given the modest increase in evacetrapib exposure and robust clinical safety profile to date, there is a low likelihood of clinically relevant DDI with concomitant use of strong CYP3A or CYP2C8 inhibitors. PMID:26516590

  9. Pharmacokinetics of a Once-Daily Dose of Tacrolimus Early After Liver Transplantation: With Special Reference to CYP3A5 and ABCB1 Single Nucleotide Polymorphisms.

    PubMed

    Miyata, Yoichi; Akamatsu, Nobuhisa; Sugawara, Yasuhiko; Kaneko, Junichi; Yamamoto, Takehito; Suzuki, Hiroshi; Arita, Junichi; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Tamura, Sumihito; Kokudo, Norihiro

    2016-01-01

    BACKGROUND The aim of the present study was to investigate the pharmacokinetics of the once-daily tacrolimus formulation (QD form) in relation to polymorphisms of the donor cytochrome P450 family 3 sub-family A polypeptide 5 (CYP3A5) gene and recipient adenosine triphosphate-binding cassette sub-family B member 1 (ABCB1) gene. MATERIAL AND METHODS A total of 80 consecutive living-donor liver transplant (LDLT) recipients were started on the QD form of tacrolimus (day 1), and 60 patients were completely followed for 7 days early after liver transplantation in order to evaluate the pharmacokinetics. RESULTS The concentration/dose (C/D) ratio in recipients with the donor CYP3A5 *1 allele was significantly lower throughout the observation period compared with those with the CYP3A5 genotype *3/*3 (p<0.001), while no effect of single-nucleotide polymorphisms (SNPs) of ABCB1 was observed. The administered doses required to achieve the target trough level were significantly higher on day 7 than on day 1 among all groups, regardless of the differences in the SNPs, especially among those with donor CYP3A5 *1 allele. The tacrolimus concentration was kept within the targeted level all through the study regardless of SNPs. CONCLUSIONS The donor CYP3A5 *1 allele correlated with the lower C/D ratio after administration of the QD form, and higher doses of QD-form tacrolimus and careful monitoring for the trough level should be considered, especially in recipients with the donor CYP3A5 *1 allele. PMID:27503662

  10. 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

  11. Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

    PubMed Central

    Lin, Xiu-Xian; Peng, Shi-Fang; Xiao, Mei-Fang; Huang, Wei-Hua; Wang, Yi-Cheng; Peng, Jing-Bo; Zhang, Wei; Ouyang, Dong-Sheng; Chen, Yao

    2016-01-01

    Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The Km and Vmax values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC50 values were 16.00 μM and 9.83 μM, and Ki values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors. PMID:26845774

  12. Enzyme Activity Experiments Using a Simple Spectrophotometer

    ERIC Educational Resources Information Center

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  13. Effects of methoxychlor and 2,2-bis ( p -hydroxyphenyl)-1,1,1-trichloroethane on cytochrome P450 enzyme activities in human and rat livers.

    PubMed

    Chen, Bingbing; Pan, Peipei; Wang, Li; Chen, Menchun; Dong, Yaoyao; Ge, Ren-Shan; Hu, Guo-Xin

    2015-01-01

    Cytochrome P450 (CYP) enzymes are involved in the metabolism of endogenous and exogenous compounds. Human and rat liver microsomes were used to investigate the inhibitory effects of methoxychlor (MXC) and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on the activities of corresponding human and rat CYPs. Probe drugs were used to test the inhibitory effects of MXC and HPTE on human and rat CYPs. The results showed that MXC and HPTE inhibited both human CYP2C9 and rat liver CYP2C11 activity, with half-maximal inhibitory concentration (IC50) values of 15.47 ± 0.36 (MXC) and 8.87 ± 0.53 μmol/l (HPTE) for human CYP2C9, and of 22.45 ± 1.48 (MXC) and 24.63 ± 1.35 μmol/l (HPTE) for rat CYP2C11. MXC and HPTE had no effects on human CYP2C19 activity but inhibited rat CYP2C6 activity with IC50 values of 14.84 ± 0.04 (MXC) and 8.72 ± 0.25 μmol/l (HPTE). With regard to human CYP2D6 and rat CYP2D2 activity, only HPTE potently inhibited human CYP2D6 activity, with an IC50 value of 16.56 ± 0.69 μmol/l. Both chemicals had no effect on human CYP3A4 and rat CYP3A1 activity. In summary, MXC and HPTE are potent inhibitors of some human and rat CYPs. PMID:25833162

  14. Effects of CYP3A5 genetic polymorphism and smoking on the prognosis of non-small-cell lung cancer

    PubMed Central

    Jiang, Li-Peng; Zhu, Zhi-Tu; He, Chun-Yan

    2016-01-01

    Objective We aimed to explore the impacts of the rs776746 polymorphism in the CYP3A5 gene and smoking on the prognosis of non-small-cell lung cancer (NSCLC). Materials and methods Our study enrolled 104 early NSCLC patients undergoing surgery and 107 advanced NSCLC patients undergoing chemotherapy, hospitalized between December 2009 and December 2012 at the First Affiliated Hospital of Liaoning Medical University. All subjects with complete follow-up data were pathologically diagnosed. The rs776746 polymorphism and different genotypes (*1/*1, *1/*3, and *3/*3) were identified by polymerase chain-reaction restriction fragment-length polymorphism. Results Clinical response to chemotherapy in NSCLC patients with *1/*1 + *1/*3 genotypes were significantly worse than in those with the *3/*3 genotype (17.78% vs 56.45%, P<0.001), and after Bonferroni adjustment, the differences still showed significance (Pc<0.01). The mortality risk of NSCLC patients undergoing chemotherapy with the *3/*3 genotype was 0.617 times those with *1/*1 + *1/*3 genotypes (relative risk [RR] 0.617, 95% confidence interval [CI] 0.402–0.948; P=0.028), while the mortality risk of smoking patients was 1.743 times greater than that of nonsmoker patients (RR 1.743, 95% CI 1.133–2.679; P=0.042). Furthermore, a 3.087-fold mortality risk was found in NSCLC patients undergoing surgery with the *3/*3 genotype compared with those with *1/*1 + *1/*3 genotypes (RR 3.087, 95% CI 1.197–7.961; P=0.020). In NSCLC patients undergoing surgery, the mortality risk of smokers was 1.896 times greater than nonsmokers (RR 1.896, 95% CI 1.040–3.455; P=0.037). Conclusion Our study demonstrated that the CYP3A5 rs776746 polymorphism and smoking may influence the prognosis of NSCLC patients undergoing chemotherapy and surgery. PMID:27042114

  15. Effect of the ethinylestradiol/levonorgestrel combined oral contraceptive on the activity of cytochrome P4503A in obese women

    PubMed Central

    Edelman, Alison; Munar, Myrna; Elman, Miriam R; Koop, Dennis; Cherala, Ganesh

    2012-01-01

    AIM(S) While it is known that CYP3A4/5 activity is decreased with combined oral contraceptive (COC) use and obesity suppresses CYP expression, the combined effects of obesity and COC use on CYP3A4/5 activity are unclear. Therefore, our aim was to examine the effect of COC usage on CYP3A4/5 activity in obese women. METHODS Thirty-four, obese (body mass index, BMI > 30 kg m−2) women of reproductive age (18–35 years old) were placed on a COC pill containing 20 µg ethinylestradiol/100 µg levonorgestrel for 21 days starting at the onset of menses. A midazolam pharmacokinetic study was conducted prior to initiation and after 21 days of COC treatment. Serial blood samples were collected and plasma concentrations of midazolam were measured using liquid chromatography tandem mass spectrometry. Pharmacokinetic parameters were estimated using a non-compartmental method. RESULTS Midazolam clearance, a surrogate measure of CYP3A4/5 activity, was significantly decreased upon COC use (63.3 l h−1vs. 53.9 l h−1, P < 0.05). A median decrease of 5.6 l h−1 (95% CI −4.1, 13.3 l h−1) was observed. However, the magnitude of change was similar to that reported in women with normal BMI. CONCLUSIONS Although we hypothesized that obesity might amplify the impact on CYP3A4/5 activity in COC users, we found that this was not the case. This finding is reassuring regarding potential additional drug−drug interactions in obese COC users as CYP3A4/5 is a major enzyme in the metabolism of many marketed drugs. PMID:22299599

  16. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    PubMed

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  17. The effect of pomelo mix ethyl acetate extract on CYP3A6 and P-glycoprotein gene transcripts in rabbits.

    PubMed

    Irshaid, Yacoub M; Zihlef, Malek A; Zmeili, Suheil M; Al-Antary, Eman T; Zmaily, Mais G; Al-Embideen, Somya N; Amireh, Abdallah O

    2014-07-01

    Pomelo fruit juice and pomelo ethylacetate extract have been shown to increase the bioavailability of some CYP3A substrates. The purpose of this study was to investigate if this effect might be contributed to by changes in CYP3A and p-glycoprotein mRNAs levels in the liver and proximal small intestine. The ethyl acetate extract of pomelo mix was administered for 7 days to 10 rabbits. Nine rabbits were administered tap water for 7 days. The administration was through oral intubation to the stomach. On the 8(th) day, the rabbits were sacrificed, and the liver and the proximal 15 cm of the small intestine were dissected. Total RNA was extracted from the specimens and cDNA was prepared by quantitative real-time-polymerase chain reaction (RT-PCR) using specific primers. The ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 almost 5-folds in the intestine and 2-folds in the liver. In contrast, a 1-fold increase to the p-glycoprotein mRNA expression was observed under the same experimental conditions. In conclusion, the ethyl acetate extract of pomelo mix reduced the mRNA expression of CYP3A6 in both intestine and liver but to different degrees, while the p-glycoprotein mRNA expression was not reduced. PMID:24856265

  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. Integrated Transcriptional and Proteomic Analysis with In Vitro Biochemical Assay Reveal the Important Role of CYP3A46 in T-2 Toxin Hydroxylation in Porcine Primary Hepatocytes*

    PubMed Central

    Wang, Jianshe; Jiang, Jun; Zhang, Hongxia; Wang, Junping; Cai, Hua; Li, Cheng; Li, Kangbai; Liu, Jing; Guo, Xuejiang; Zou, Guangxun; Wang, Dazhi; Deng, Yiqun; Dai, Jiayin

    2011-01-01

    Both T-2 toxin and its metabolites are highly potent mycotoxins that can cause severe human and animal diseases upon exposure. Understanding the toxic mechanism and biotransformation process of T-2 toxin at a cellular level is essential for the development of counter-measures. We investigated the effect of T-2 toxin in porcine primary hepatocytes using porcine genome array and two-dimensional difference gel electrophoresis with matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. Integrated transcriptional and proteomic analysis demonstrated that T-2 toxin adversely affected porcine hepatocytes by initiating lipid metabolism disorder, oxidative stress response, and apoptosis. In addition, xenobiotic metabolism genes, including cytochrome P450 3As (CYP3A46 and CYP3A39), carboxylesterase 1Cs (CES1C4 and CES1C5), and epoxide hydrolase (EPHX1), increased in T-2 toxin treatment cells. Using HepG2 cells to over-express the recombinant xenobiotic metabolism genes above and rapid resolution liquid chromatography/tandem mass spectrometry to detect metabolites of T-2 toxin, we determined that porcine CYP3A46 mainly catalyzed T-2 to form 3′-hydroxy-T-2, which was further confirmed by purified CYP3A46 protein. However, recombinant porcine CES1C5 and EPHX1 did not enhance hydrolysis and de-epoxidation of T-2 implying that other esterases and epoxide hydrolases may play dominant roles in those reactions. PMID:21685020

  20. Relationship between the plasma fentanyl and serum 4β-hydroxycholesterol based on CYP3A5 genotype and gender in patients with cancer pain.

    PubMed

    Ishida, Takuya; Naito, Takafumi; Sato, Hikaru; Kawakami, Junichi

    2016-06-01

    This study aimed to evaluate the relationship between the concentrations of plasma fentanyl and serum 4β-hydroxycholesterol based on CYP3A5 genotype and gender in cancer patients. Thirty-three Japanese cancer patients treated with transdermal fentanyl were enrolled. The concentrations of plasma fentanyl and norfentanyl, and serum 4β-hydroxycholesterol and total cholesterol were determined at day 8 or later after starting the medication. The plasma fentanyl concentration was significantly higher in the CYP3A5*3/*3 group than in the *1 allele carrier group. The *3/*3 group had a lower metabolic ratio of fentanyl. The serum 4β-hydroxycholesterol concentration and its ratio to total cholesterol were significantly lower in the CYP3A5*3/*3 group than in the *1 allele carrier group. The concentrations of plasma fentanyl and serum 4β-hydroxycholesterol were significantly higher in women than in men. Gender did not affect the metabolic ratio of fentanyl or the concentration ratio of 4β-hydroxycholesterol. The plasma fentanyl concentration was not correlated with the serum 4β-hydroxycholesterol concentration, while the metabolic ratio of fentanyl was slightly correlated with the serum concentration ratio of 4β-hydroxycholesterol. In conclusion, CYP3A5*3 and gender affected the plasma fentanyl and serum 4β-hydroxycholesterol concentrations in cancer patients. However, the plasma disposition of fentanyl was not determined using the serum 4β-hydroxycholesterol concentration. PMID:27236640

  1. 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

  2. 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

  3. The impact of genetic polymorphisms of drug metabolizing enzymes on the pharmacodynamics of clopidogrel under steady state conditions.

    PubMed

    Nakkam, Nontaya; Tiamkao, Somsak; Kanjanawart, Sirimas; Tiamkao, Siriporn; Vannaprasaht, Suda; Tassaneeyakul, Wongwiwat; Tassaneeyakul, Wichittra

    2015-08-01

    Clopidogrel is an antiplatelet drug that requires biotransformation steps to its active metabolite via cytochromes P450 (CYP), particularly CYP2C19 and CYP3A5 as well as paraoxonase-1 (PON1). The impact of CYP3A5 and PON1 genetic polymorphisms on the response of this drug is unclear. This study aimed to elucidate the degree of genetic polymorphisms of key drug metabolizing enzymes on the antiplatelet effect of clopidogrel. Thirty-five healthy subjects were treated with 75 mg/day clopidogrel for 7 days and serial blood samples were collected for measurement of antiplatelet effect using whole blood impedance aggregometry and VerifyNow(®) P2Y12 methods. The areas under the antiplatelet effect-time curves, maximal and minimal antiplatelet effects of clopidogrel obtained from both methods were significantly different among subjects with different CYP2C19 genotypes. In contrast, these pharmacodymamic parameters measured by both methods of subjects with different PON1 or CYP3A5 genotypes were not significantly different. Among the heterozygous CYP2C19*2 subjects, all pharmacodynamic parameters measured by whole blood impedance aggregometry were significantly different between subjects with different CYP3A5*3 genotypes. Our data suggests that CYP2C19 genetic polymorphism play a major role in the clopidogrel response, however, the impact of CYP3A5 genetic polymorphism, may be pronounced in the subjects who carried the loss-functional allele of CYP2C19. PMID:26099919

  4. Characterization of Soil Samples of Enzyme Activity

    ERIC Educational Resources Information Center

    Freeland, P. W.

    1977-01-01

    Described are nine enzyme essays for distinguishing soil samples. Colorimetric methods are used to compare enzyme levels in soils from different sites. Each soil tested had its own spectrum of activity. Attention is drawn to applications of this technique in forensic science and in studies of soil fertility. (Author/AJ)

  5. Enzyme Activities in Polarized Cell Membranes

    PubMed Central

    Bass, L.; McIlroy, D. K.

    1968-01-01

    The theoretical pH dependence of enzyme activities in membranes of low dielectric constant is estimated. It is shown that in biological membranes some types of enzymes may attain a limiting pH sensitivity such that an increment of only 0.2 pH unit (sufficient to induce action potentials in squid axons) causes a relative activity change of over 25%. The transients of enzyme activity generated by membrane depolarization and by pH increments in the bathing solution are discussed in relation to the transients of nervous excitation. PMID:5641405

  6. [Muscle enzyme activity and exercise].

    PubMed

    Gojanovic, B; Feihl, F; Gremion, G; Waeber, B

    2009-02-01

    Exercise is classically associated with muscular soreness, presenting one to two days later, delayed onset muscular soreness. Blood muscle enzymes and protein elevations are characteristic, and may cause renal failure. Creatin phosphokinase peak appears on the fourth day and depends on exercise type and individual parameters. This effect is attenuated with repeated bouts, by habituation. Metabolic complications are rare. The knowledge of this reaction, even with common exercises, allows to postpone investigations for a complex metabolic disorder, or to avoid stopping a medication for fear of a side effect, as with statins. Indeed, it is necessary to wait for seven days without any exercise before interpreting an elevated CK result. PMID:19180440

  7. Activity of xenobiotic-metabolizing enzymes in the liver of rats with multi-vitamin deficiency.

    PubMed

    Tutelyan, Victor A; Kravchenko, Lidia V; Aksenov, Ilya V; Trusov, Nikita V; Guseva, Galina V; Kodentsova, Vera M; Vrzhesinskaya, Oksana A; Beketova, Nina A

    2013-01-01

    The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection. PMID:24220160

  8. Role of Enzyme Flexibility in Ligand Access and Egress to Active Site: Bias-Exchange Metadynamics Study of 1,3,7-Trimethyluric Acid in Cytochrome P450 3A4.

    PubMed

    Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Laio, Alessandro; Otyepka, Michal

    2016-04-12

    Although the majority of enzymes have buried active sites, very little is known about the energetics and mechanisms associated with substrate and product channeling in and out. Gaining direct information about these processes is a challenging task both for experimental and theoretical techniques. Here, we present a methodology that enables following of a ligand during its passage to the active site of cytochrome P450 (CYP) 3A4 and mapping of the free energy associated with this process. The technique is based on a combination of a bioinformatics tool for identifying access channels and bias-exchange metadynamics and provides converged free energies in good agreement with experimental data. In addition, it identifies the energetically preferred escape routes, limiting steps, and amino acids residues lining the channel. The approach was applied to mapping of a complex channel network in a complex environment, i.e., CYP3A4 attached to a lipid bilayer mimicking an endoplasmic reticulum membrane. The results provided direct information about the energetics and conformational changes associated with the ligand channeling. The methodology can easily be adapted to study channeling through other flexible biomacromolecular channels. PMID:26967371

  9. Visualization of enzyme activities inside earthworm pores

    NASA Astrophysics Data System (ADS)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  10. 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

  11. Interaction between ALOX5AP and CYP3A5 gene variants significantly increases the risk for cerebral infarctions in Chinese.

    PubMed

    Chi, Li-Fen; Yi, Xing-Yang; Shao, Min-Jie; Lin, Jing; Zhou, Qiang

    2014-05-01

    In this study, we investigated associations between susceptibility genes and cerebral infarctions in a Chinese population, and whether gene-gene interactions increase the risk of cerebral infarctions. Overall, 292 patients with cerebral infarctions and 259 healthy control individuals were included. Eight variants in five candidate genes were examined for the risk of stroke, including the SG13S32 (rs9551963), SG13S42 (rs4769060), SG13S89 (rs4769874), and SG13S114 (rs10507391) variants of the 5-lipoxygenase activating protein (ALOX5AP) gene, the G860A (rs751141) variant of the soluble epoxide hydrolase (EPHX2) gene, the A1075C (rs1057910) variant of the CYP2C9*2 gene, the C430T (rs1799853) variant of the CYP2C9*3 gene, and the A6986G (rs776746) variant of the CYP3A5 gene. Gene-gene interactions were explored using generalized multifactor dimensionality reduction methods. There were no statistically significant differences in the frequencies of the genotypes of the eight candidate genes. The generalized multifactor dimensionality reduction analysis showed a significant gene-gene interaction between SG13S114 and A6986G, with scores of 10 for cross-validation consistency and 9 for the sign test (P=0.0107). These gene-gene interactions predicted a significantly higher risk of cerebral infarction (adjusted for age, hypertension, and diabetes mellitus; odds ratio=1.80495%, confidence interval: 1.180-2.759, P=0.006). A two-loci gene interaction confers a significantly higher risk for cerebral infarction. The combinational analysis used in this study may be helpful in the elucidation of genetic risk factors for common and complex diseases. PMID:24368493

  12. The effect of CYP3A inhibitors and inducers on the pharmacokinetics of telaprevir in healthy volunteers

    PubMed Central

    Garg, Varun; Chandorkar, Gurudatt; Yang, Yijun; Adda, Nathalie; McNair, Lindsay; Alves, Katia; Smith, Frances; Heeswijk, Rolf P G

    2013-01-01

    AIM To evaluate the effects of ketoconazole, rifampicin and efavirenz on the pharmacokinetics of telaprevir in healthy volunteers. METHOD Results from three clinical studies are described. (1) Volunteers received a single 750 mg dose telaprevir with and without a single 400 mg dose ketoconazole. (2) Volunteers received (a) 1250 mg telaprevir followed by three 750 mg doses given every 8 h and (b) four 1250 mg telaprevir doses given every 8 h, with a single 400 mg dose ketoconazole given with the fourth dose of telaprevir. (3) Volunteers received either a single 750 mg dose telaprevir with or without 600 mg once daily rifampicin, or 750 mg every 8 h telaprevir with and without 600 mg once daily efavirenz. RESULTS A single 400 mg dose of ketoconazole increased single dose telaprevir exposure: the geometric least-squares mean ratio (GLSMR, with 90% confidence limits) was 1.24 (1.10, 1.41) for Cmax and 1.62 (1.45, 1.81) for AUC(0,∞). However, after multiple doses of telaprevir, there was no discernible effect of ketoconazole on telaprevir exposure. Co-administration of rifampicin at steady-state markedly reduced single dose telaprevir exposure with GLSMRs of 0.14 (0.11, 0.18) for Cmax and 0.08 (0.07, 0.11) for AUC(0,∞), whereas efavirenz had a smaller effect on telaprevir exposure when both drugs were co-administered at steady-state, with GLSMRs of 0.91 (0.81, 1.02) for Cmax, 0.53 (0.44, 0.65) for Cmin, and 0.74 (0.65, 0.84) for AUC(0,8 h). CONCLUSION CYP3A inducers, rifampicin and efavirenz, can reduce telaprevir exposure to varying degrees based on their potency. The effect of ketoconazole as an inhibitor of telaprevir metabolism is more pronounced after a single dose of telaprevir than after repeated administration. PMID:22642697

  13. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  14. Enzyme activity in dialkyl phosphate ionic liquids.

    PubMed

    Thomas, Marie F; Li, Luen-Luen; Handley-Pendleton, Jocelyn M; van der Lelie, Daniel; Dunn, John J; Wishart, James F

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariella volvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v. PMID:22001053

  15. Enzyme activity in dialkyl phosphate ionic liquids

    SciTech Connect

    Thomas, M.F.; Dunn, J.; Li, L.-L.; Handley-Pendleton, J. M.; van der lelie, D.; Wishart, J. F.

    2011-12-01

    The activity of four metagenomic enzymes and an enzyme cloned from the straw mushroom, Volvariellavolvacea were studied in the following ionic liquids, 1,3-dimethylimidazolium dimethyl phosphate, [mmim][dmp], 1-ethyl-3-methylimidazolium dimethyl phosphate, [emim][dmp], 1-ethyl-3-methylimidazolium diethyl phosphate, [emim][dep] and 1-ethyl-3-methylimidazolium acetate, [emim][OAc]. Activity was determined by analyzing the hydrolysis of para-nitrobenzene carbohydrate derivatives. In general, the enzymes were most active in the dimethyl phosphate ionic liquids, followed by acetate. Generally speaking, activity decreased sharply for concentrations of [emim][dep] above 10% v/v, while the other ionic liquids showed less impact on activity up to 20% v/v.

  16. 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

  17. 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

  18. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited. PMID:15778010

  19. A Randomized Controlled Trial Comparing the Efficacy of Cyp3a5 Genotype-Based With Body-Weight-Based Tacrolimus Dosing After Living Donor Kidney Transplantation.

    PubMed

    Shuker, N; Bouamar, R; van Schaik, R H N; Clahsen-van Groningen, M C; Damman, J; Baan, C C; van de Wetering, J; Rowshani, A T; Weimar, W; van Gelder, T; Hesselink, D A

    2016-07-01

    Patients expressing the cytochrome P450 (CYP) 3A5 gene require a higher tacrolimus dose to achieve therapeutic exposure compared with nonexpressers. This randomized-controlled study investigated whether adaptation of the tacrolimus starting dose according to CYP3A5 genotype increases the proportion of kidney transplant recipients being within the target tacrolimus predose concentration range (10-15 ng/mL) at first steady-state. Two hundred forty living-donor, renal transplant recipients were assigned to either receive a standard, body-weight-based or a CYP3A5 genotype-based tacrolimus starting dose. At day 3, no difference in the proportion of patients having a tacrolimus exposure within the target range was observed between the standard-dose and genotype-based groups: 37.4% versus 35.6%, respectively; p = 0.79. The proportion of patients with a subtherapeutic (i.e. <10 ng/mL) or a supratherapeutic (i.e. >15 ng/mL) Tac predose concentration in the two groups was also not significantly different. The incidence of acute rejection was comparable between both groups (p = 0.82). Pharmacogenetic adaptation of the tacrolimus starting dose does not increase the number of patients having therapeutic tacrolimus exposure early after transplantation and does not lead to improved clinical outcome in a low immunological risk population. PMID:26714287

  20. Halophilic enzyme activation induced by salts

    PubMed Central

    Ortega, Gabriel; Laín, Ana; Tadeo, Xavier; López-Méndez, Blanca; Castaño, David; Millet, Oscar

    2011-01-01

    Halophilic archea (halobacteriae) thrive in hypersaline environments, avoiding osmotic shock by increasing the ion concentration of their cytoplasm by up to 3–6 M. To remain folded and active, their constitutive proteins have evolved towards a biased amino acid composition. High salt concentration affects catalytic activity in an enzyme-dependent way and a unified molecular mechanism remains elusive. Here, we have investigated a DNA ligase from Haloferax volcanii (Hv LigN) to show that K+ triggers catalytic activity by preferentially stabilising a specific conformation in the reaction coordinate. Sodium ions, in turn, do not populate such isoform and the enzyme remains inactive in the presence of this co-solute. Our results show that the halophilic amino acid signature enhances the enzyme's thermodynamic stability, with an indirect effect on its catalytic activity. This model has been successfully applied to reengineer Hv LigN into an enzyme that is catalytically active in the presence of NaCl. PMID:22355525

  1. An NMR Study of Enzyme Activity.

    ERIC Educational Resources Information Center

    Peterman, Keith E.; And Others

    1989-01-01

    A laboratory experiment designed as a model for studying enzyme activity with a basic spectrometer is presented. Included are background information, experimental procedures, and a discussion of probable results. Stressed is the value of the use of Nuclear Magnetic Resonance in biochemistry. (CW)

  2. Enzyme Specific Activity in Functionalized Nanoporous Supports

    SciTech Connect

    Lei, Chenghong; Soares, Thereza A.; Shin, Yongsoon; Liu, Jun; Ackerman, Eric J.

    2008-03-26

    Enzyme specific activity can be increased or decreased to a large extent by changing protein loading density in functionalized nanoporous support, where organophosphorus hydrolase can display a constructive orientation and thus leave a completely open entrance for substrate even at higher protein loading density, but glucose oxidase can not.

  3. Multiple plasma enzyme activities in liver disease

    PubMed Central

    Hargreaves, T.; Janota, I.; Smith, M. J. H.

    1961-01-01

    The measurement of the plasma activities of glutamic-oxaloacetic and glutamic-pyruvic transaminases, aldolase, cholinesterase, and isocitric, lactic, and phosphogluconic dehydrogenases in random samples of blood was found to be of no value in the differential diagnosis of hepatitis, obstructive jaundice, hepatic cirrhosis, and neoplastic conditions involving the liver. Serial determinations of the enzyme activities provided useful information about the course of certain hepatic disorders, particularly acute viral hepatitis. PMID:13711559

  4. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  5. Unequal prognostic potentials of p53 gain-of-function mutations in human cancers associate with drug-metabolizing activity.

    PubMed

    Xu, J; Wang, J; Hu, Y; Qian, J; Xu, B; Chen, H; Zou, W; Fang, J-Y

    2014-01-01

    Mutation of p53 is the most common genetic change in human cancer, causing complex effects including not only loss of wild-type function but also gain of novel oncogenic functions (GOF). It is increasingly likely that p53-hotspot mutations may confer different types and magnitudes of GOF, but the evidences are mainly supported by cellular and transgenic animal models. Here we combine large-scale cancer genomic data to characterize the prognostic significance of different p53 mutations in human cancers. Unexpectedly, only mutations on the Arg248 and Arg282 positions displayed significant association with shorter patient survival, but such association was not evident for other hotspot GOF mutations. Gene set enrichment analysis on these mutations revealed higher activity of drug-metabolizing enzymes, including the CYP3A4 cytochrome P450. Ectopic expression of p53 mutant R282W in H1299 and SaOS2 cells significantly upregulated CYP3A4 mRNA and protein levels, and cancer cell lines bearing mortality-associated p53 mutations display higher CYP3A4 expression and resistance to several CYP3A4-metabolized chemotherapeutic drugs. Our results suggest that p53 mutations have unequal GOF activities in human cancers, and future evaluation of p53 as a cancer biomarker should consider which mutation is present in the tumor, rather than having comparison between wild-type and mutant genotypes. PMID:24603336

  6. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  7. 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

  8. Effect of pluronic P123 and F127 block copolymer on P-glycoprotein transport and CYP3A metabolism.

    PubMed

    Guan, Yanbin; Huang, Jiangeng; Zuo, Lan; Xu, Jiaqiang; Si, Luqin; Qiu, Jun; Li, Gao

    2011-10-01

    The aim of the present study was to evaluate the effect of pluronic P123 (P123) and pluronic F127 (F127) on intestinal P-glycoprotein (P-gp) and cytochrome P450 3A using the specific substrates rhodamine-123 (R-123) and midazolam, respectively. Caco-2 cells and everted gut sacs were used as models of intestinal mucosa to assess intestinal absorption of R-123, while rat intestinal microsomes were utilized to examine the effect of P123 and F127 on in vitro midazolam metabolism. P123 and F127 were observed to increase the intracellular accumulation of R-123 in Caco-2 cells in a dose-dependent manner. P123 significantly lowered the efflux ratio of R-123 at two concentrations in Caco-2 monolayers, whereas F127 lowered the efflux ratio only at 1%. Moreover, both pluronics markedly enhanced mucosal to serosal absorption of R-123 in excised ileum of rats. However, no significant difference in relative enzyme activity were observed between P123- or F127-treated and control groups, regardless of the concentrations of P123 and F127 studied. Collectively, these results obtained from the present study demonstrated that P123 and F127 were capable of inhibiting the intestinal P-gp activity, but had little or no effect on intestinal cytochrome P450 3A activity, indicating that P123 and F127 can potentially be used as pharmaceutical ingredients to improve the oral bioavailability of coadministered P-gp substrates via P-gp efflux pump inhibition. PMID:22076772

  9. Characterization and biological properties of NanoCUR formulation and its effect on major human cytochrome P450 enzymes.

    PubMed

    Shamsi, Suhaili; Chen, Yan; Lim, Lee Yong

    2015-11-10

    Curcumin (CUR) has been formulated into a host of nano-sized formulations in a bid to improve its in vivo solubility, stability and bioavailability. The aim of this study was to investigate whether the encapsulation of CUR in nanocarriers would impede its biological interactivity, specifically its potential anti-cancer adjuvant activity via the modulation of CYP enzymes in vitro. NanoCUR, a micellar dispersion prepared via a thin film method using only Pluronic F127 as excipient, was amenable to lyophilization, and retained its nano-sized spherical dimensions (17-33 nm) upon reconstitution with water followed by dilution to 5 μM with HBSS or EMEM. NanoCUR was a weaker cytotoxic agent compared to CUR in solution (sCUR), affecting HepG2 cell viability only when the incubation time was prolonged from 4h to 48 h. Correlation with 2h uptake data suggests this was due to a lower cellular uptake rate of CUR from NanoCUR than from sCUR. The poorer CUR accessibility might also account for NanoCUR being a weaker inhibitor of CYP2C9 and CYP2D6 than sCUR. NanoCUR was, however, 1.76-fold more potent against the CYP3A4 (IC50 5.13 ± 0.91 μM) metabolic function. The higher activity against CYP3A4 might be attributed to the synergistic action of Pluronic F127, since the blank micellar dispersion also inhibited CYP3A4 activity. Both sCUR and NanoCUR had no effect on the CYP3A4 mRNA levels in the HepG2 cells. NanoCUR therefore, maintained most of the biological activities of CUR in vitro, albeit at a lower potency and response rate. PMID:26319630

  10. Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

    PubMed

    Celik, Gurbet; Semiz, Aslı; Karakurt, Serdar; Gencler-Ozkan, Ayse Mine; Arslan, Sevki; Adali, Orhan; Sen, Alaattin

    2016-04-01

    Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20 mg/kg, respectively, for 9 days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21 % in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43 %, and 57 and 57 % in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82 % as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37 % with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content. PMID:25425117

  11. 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

  12. Technical pentabromodiphenyl ether and hexabromocyclododecane as activators of the pregnane-X-receptor (PXR).

    PubMed

    Fery, Yvonne; Buschauer, Inga; Salzig, Christian; Lang, Patrick; Schrenk, Dieter

    2009-10-01

    Technical pentabrominated diphenyl ether (pentaBDE mix) is a mixture of polybrominated diphenyl ethers (PBDEs) which has been widely used as a flame retardant. Since its ban in several countries it has been replaced by other brominated flame retardants such as hexabromocyclododecane (HBCD). Both certain PBDE congeners and HBCD are present in environmental and human samples reflecting their persistent and bioaccumulative properties. PentaBDE mix and HBCD have recently been found to induce cytochrome P450 (CYP) 3 enzymes in rat liver. In this study we tested both technical pentaBDE mix and HBCD for their potency to induce CYP3A enzymes in rat hepatocytes in primary culture, and in rat H4IIE and human HepG2 hepatoma cells. In rat hepatocytes, HBCD was a more effective CYP3A1 inducer than pentaBDE mix, being less effective, however, than the prototype inducer dexamethasone. In human HepG2 cells, both compounds and the prototype inducer rifampicin were about equally effective. In contrast, in HepG2 cells, HBCD failed to induce luciferin-PFBE dealkylase, a common catalytic activity of a number of CYP3A enzymes, possibly reflecting enzyme inhibition. A significant induction of catalytic activity was observed in rat hepatocytes with both compounds. Analysis of a XREM-driven reporter gene activity in transfected cells confirmed that both compounds act as agonists of the human and rat pregnane-X-receptor, which was detectable in all cell types used. PMID:19631710

  13. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    PubMed

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug. PMID:20446912

  14. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

    PubMed

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

    2015-12-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. PMID:26400395

  15. Mechanism-based inhibitory and peroxisome proliferator-activated receptor α-dependent modulating effects of silybin on principal hepatic drug-metabolizing enzymes.

    PubMed

    Wang, Hong; Yan, Tingting; Xie, Yuan; Zhao, Min; Che, Yuan; Zhang, Jun; Liu, Huiying; Cao, Lijuan; Cheng, Xuefang; Xie, Yang; Li, Feiyan; Qi, Qu; Wang, Guangji; Hao, Haiping

    2015-04-01

    Silybin, a major pharmacologically active compound in silymarin, has been widely used in combination with other prescriptions in the clinic to treat hepatitis and a host of other diseases. Previous studies suggested that silybin is a potential inhibitor of multiple drug-metabolizing enzymes (DMEs); however, the in vitro to in vivo translation and the mechanisms involved remain established. The aim of this study was to provide a mechanistic understanding of the regulatory effects of silybin on principal DMEs. Silybin (50 or 150 mg/kg/d) was administered to mice for a consecutive 14 days. The plasma and hepatic exposure of silybin were detected; the mRNA, protein levels, and enzyme activities of principal DMEs were determined. The results demonstrated that the enzyme activities of CYP1A2, CYP2C, CYP3A11, and UGT1A1 were significantly repressed, whereas little alteration of the mRNA and protein levels was observed. Silybin inhibits these DMEs in a mechanism-based and/or substrate-competitive manner. More importantly, silybin was found to be a weak agonist of peroxisome proliferator-activated receptor (PPAR)α, as evidenced from the molecular docking, reporter gene assay, and the targeting gene expression analysis. However, silybin could significantly compromise the activation of PPARα by fenofibrate, characterized with significantly repressed expression of PPARα targeting genes, including L-FABP, ACOX1, and UGT1A6. This study suggests that silybin, despite its low bioavailability, may inhibit enzyme activities of multiple DMEs in a mechanism-based mode, and more importantly, may confer significant drug-drug interaction with PPARα agonists via the repression of PPARα activation in a competitive mode. PMID:25587127

  16. An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

    PubMed

    Bansal, Sumit; Chai, Swee Fen; Lau, Aik Jiang

    2016-06-15

    Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation. PMID:27153105

  17. 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

  18. Methadone dose in heroin-dependent patients: role of clinical factors, comedications, genetic polymorphisms and enzyme activity

    PubMed Central

    Mouly, Stéphane; Bloch, Vanessa; Peoc'h, Katell; Houze, Pascal; Labat, Laurence; Ksouda, Kamilia; Simoneau, Guy; Declèves, Xavier; Bergmann, Jean Francois; Scherrmann, Jean-Michel; Laplanche, Jean-Louis; Lepine, Jean-Pierre; Vorspan, Florence

    2015-01-01

    Aims Methadone is characterized by wide intersubject variability regarding the dose needed to obtain full therapeutic response. We assessed the influence of sociodemographic, ethnic, clinical, metabolic and genotypic variables on methadone maintenance dose requirement in opioid-dependent responder patients. Methods Eighty-one stable patients (60 men and 21 women, 43.7 ± 8.1 years old, 63.1 ± 50.9 mg day−1 methadone), divided into quartiles with respect to the median daily dose, were enrolled and underwent clinical examination, treatment history and determination of liver/intestinal cytochrome P450 (CYP) 3A4 activity measured by the midazolam test, R,S-methadone trough concentration and clinically significant polymorphisms of the OPRM1, DRD2, COMT, ABCB1, CYP2B6, CYP3A5, CYP2C19 and CYP2D6 genes. Results Methadone maintenance dose was correlated to the highest dose ever used (r2 = 0.57, P < 0.0001). Fractioned methadone intake (odds ratio 4.87, 95% confidence interval 1.27–18.6, P = 0.02), bodyweight (odds ratio 1.57, 95% confidence interval 1.01–2.44, P = 0.04), history of cocaine dependence (80 vs. 44 mg day−1 in never-addict patients, P = 0.005) and ethnicity (Asian > Caucasian > African, P = 0.04) were independently associated with high-dose methadone in multiple regression analysis. A modest correlation was observed between liver/intestinal CYP3A4 activity and methadone dose at steady state (Spearman rank correlation coefficient [rs] = 0.21, P = 0.06) but not with highest dose ever used (rs = 0.15, P = 0.18) or dose-normalized R,S-methadone trough concentrations (rs = −0.05, P = 0.64). Concomitant CYP3A4 inhibitors only affected the relationship between methadone dose and R,S-methadone trough concentration. None of the genetic polymorphisms explored was predictive of the methadone maintenance dose. Conclusions Methadone maintenance dose was predicted by sociodemographic and clinical variables rather than genetic polymorphisms or liver/intestinal CYP

  19. Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch)

    PubMed Central

    Lavado, Ramon; Aparicio-Fabre, Rosaura; Schlenk, Daniel

    2013-01-01

    Phase I biotransformation enzymes are critically important in the disposition of xenobiotics within biota and are regulated by multiple environmental cues, particularly in anadromous fish species. Given the importance of these enzyme systems in xenobiotic/endogenous chemical bioactivation and detoxification, the current study was designed to better characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch) and the effects of salinity acclimation on those enzymes. Livers, gills and olfactory tissues were collected from coho salmon (Oncorhynchus kisutch) after they had undergone acclimation from freshwater to various salinity regimes of seawater (8, 16 and 32 g/L). Using immunoblot techniques coupled with testosterone hydroxylase catalytic activities, 4 orthologs of cytochrome P450 (CYP1A, CYP2K1, CYP2M1 and CYP3A27) were measured in each tissue. Also the expression of 2 transcripts of flavin-containing monooxygenases (FMO A and B) and associated activities were measured. With the exception of CYP1A, which was down-regulated in liver, protein expression of the other 3 enzymes was induced at higher salinity, with the greatest increase observed in CYP2M1 from olfactory tissues. In liver and gills, 6 - and 16 -hydroxylation of testosterone was also significantly increased after hypersaline acclimation. Similarly, FMO A was up-regulated in all 3 tissues in a salinity-dependent pattern, whereas FMO B mRNA was down-regulated. FMO-catalyzed benzydamine N-oxygenase and methyl p-tolyl sulfoxidation were significantly induced in liver and gills by hypersalinity, but was either unchanged or not detected in olfactory tissues. These data demonstrate thatenvironmental conditions may significantly alter the toxicity of environmental chemicals in salmon during freshwater/saltwater acclimation. PMID:23925894

  20. Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay.

    PubMed

    Kong, Wai Mun; Chik, Zamri; Ramachandra, Murali; Subramaniam, Umarani; Aziddin, Raja Elina Raja; Mohamed, Zahurin

    2011-01-01

    The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2. PMID:21876481

  1. 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

  2. Analysis of the impact of controlled release formulations on oral drug absorption, gut wall metabolism and relative bioavailability of CYP3A substrates using a physiologically-based pharmacokinetic model.

    PubMed

    Olivares-Morales, Andrés; Kamiyama, Yoshiteru; Darwich, Adam S; Aarons, Leon; Rostami-Hodjegan, Amin

    2015-01-25

    Controlled release (CR) formulations are usually designed to achieve similar exposure (AUC) levels as the marketed immediate release (IR) formulation. However, the AUC is often lower following CR compared to IR formulations. There are a few exceptions when the CR formulations have shown higher AUC. This study investigated the impact of CR formulations on oral drug absorption and CYP3A4-mediated gut wall metabolism. A review of the current literature on relative bioavailability (Frel) between CR and IR formulations of CYP3A substrates was conducted. This was followed by a systematic analysis to assess the impact of the release characteristics and the drug-specific factors (including metabolism and permeability) on oral bioavailability employing a physiologically-based pharmacokinetic (PBPK) modelling and simulation approach. From the literature review, only three CYP3A4 substrates showed higher Frel when formulated as CR. Several scenarios were investigated using the PBPK approach; in most of them, the oral absorption of CR formulations was lower as compared to the IR formulations. However, for highly permeable compounds that were CYP3A4 substrates the reduction in absorption was compensated by an increase in the fraction that escapes from first pass metabolism in the gut wall (FG), where the magnitude was dependent on CYP3A4 affinity. The systematic simulations of various interplays between different parameters demonstrated that BCS class 1 highly-cleared CYP3A4 substrates can display up to 220% higher relative bioavailability when formulated as CR compared to IR, in agreement with the observed data collected from the literature. The results and methodology of this study can be employed during the formulation development process in order to optimize drug absorption, especially for CYP3A4 substrates. PMID:25444842

  3. High-Throughput Analysis of Enzyme Activities

    SciTech Connect

    Guoxin Lu

    2007-12-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  4. 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

  5. Angiotensin Converting Enzyme Activity in Alopecia Areata

    PubMed Central

    Namazi, Mohammad Reza; Handjani, Farhad; Eftekhar, Ebrahim; Kalafi, Amir

    2014-01-01

    Background. Alopecia areata (AA) is a chronic inflammatory disease of the hair follicle. The exact pathogenesis of AA remains unknown, although recent studies support a T-cell mediated autoimmune process. On the other hand, some studies have proposed that the renin-angiotensin-aldosterone system (RAAS) may play a role in autoimmunity. Therefore, we assessed serum activity of angiotensin converting enzyme (ACE), a component of this system, in AA. Methods. ACE activity was measured in the sera of 19 patients with AA and 16 healthy control subjects. In addition, the relationship between severity and duration of the disease and ACE activity was evaluated. Results. Serum ACE activity was higher in the patient group (55.81 U/L) compared to the control group (46.41 U/L), but the difference was not statistically significant (P = 0.085). Also, there was no correlation between ACE activity and severity (P = 0.13) and duration of disease (P = 0.25) in the patient group. Conclusion. The increased serum ACE activity found in this study may demonstrate local involvement of the RAAS in the pathogenesis of AA. Assessment of ACE in a study with a larger sample size as well as in tissue samples is recommended in order to further evaluate the possible role of RAAS in AA. PMID:25349723

  6. Xyloketal B, a marine compound, acts on a network of molecular proteins and regulates the activity and expression of rat cytochrome P450 3a: a bioinformatic and animal study

    PubMed Central

    Su, Junhui; Chang, Cui; Xiang, Qi; Zhou, Zhi-Wei; Luo, Rong; Yang, Lun; He, Zhi-Xu; Yang, Hongtu; Li, Jianan; Bei, Yu; Xu, Jinmei; Zhang, Minjing; Zhang, Qihao; Su, Zhijian; Huang, Yadong; Pang, Jiyan; Zhou, Shu-Feng

    2014-01-01

    Natural compounds are becoming popular for the treatment of illnesses and health promotion, but the mechanisms of action and safety profiles are often unknown. Xyloketal B (XKB) is a novel marine compound isolated from the mangrove fungus Xylaria sp., with potent antioxidative, neuroprotective, and cardioprotective effects. However, its molecular targets and effects on drug-metabolizing enzymes are unknown. This study aimed to investigate the potential molecular targets of XKB using bioinformatic approaches and to examine the effect of XKB on the expression and activity of rat cytochrome P450 3a (Cyp3a) subfamily members using midazolam as a model probe. DDI-CPI, a server that can predict drug–drug interactions via the chemical–protein interactome, was employed to predict the targets of XKB, and the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze the pathways of the predicted targets of XKB. Homology modeling was performed using the Discovery Studio program 3.1. The activity and expression of rat hepatic Cyp3a were examined after the rats were treated with XKB at 7 and 14 mg/kg for 8 consecutive days. Rat plasma concentrations of midazolam and its metabolite 1′-OH-midazolam were determined using a validated high-performance liquid chromatographic method. Bioinformatic analysis showed that there were over 324 functional proteins and 61 related signaling pathways that were potentially regulated by XKB. A molecular docking study showed that XKB bound to the active site of human cytochrome P450 3A4 and rat Cyp3a2 homology model via the formation of hydrogen bonds. The in vivo study showed that oral administration of XKB at 14 mg/kg to rats for 8 days significantly increased the area under the plasma concentration-time curve (AUC) of midazolam, with a concomitant decrease in the plasma clearance and AUC ratio of 1′-OH-midazolam over midazolam. Further, oral administration of 14 mg/kg XKB for 8 days markedly reduced the

  7. 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

  8. Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor1

    PubMed Central

    Gräns, Johanna; Wassmur, Britt; Fernández-Santoscoy, María; Zanette, Juliano; Woodin, Bruce R.; Karchner, Sibel I.; Nacci, Diane E.; Champlin, Denise; Jayaraman, Saro; Hahn, Mark E.; Stegeman, John J.; Celander, Malin C.

    2015-01-01

    Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ~400 times higher, and the levels of non-dioxin-like PCBs ~3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations

  9. 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

  10. 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

  11. 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

  12. Pharmacokinetic and pharmacodynamic consequences of inhibition of terazosin metabolism via CYP3A1 and/or 3A2 by DA-8159, an erectogenic, in rats

    PubMed Central

    Oh, E Y; Bae, S K; Kwon, J W; You, M; Lee, D C; Lee, M G

    2007-01-01

    Background and purpose: Recently, orthostatic hypotension was observed in patients with benign prostatic hyperplasia who are taking vardenafil (a PDE 5 inhibitor) and terazosin (a long acting alpha blocker). Therefore, this study was performed with DA-8159 (a long acting PDE 5 inhibitor) and terazosin in rats to find whether or not pharmacokinetic and pharmacodynamic interactions between the two drugs were observed. Experimental approach: Pharmacokinetic and pharmacodynamic (changes in blood pressure) interactions between DA-8159 and terazosin were evaluated after simultaneous i.v. and p.o. administration of DA-8159 (30 mg kg−1) and terazosin (5 mg kg−1) to male Sprague–Dawley rats. Key results: After simultaneous i.v. and p.o. administration of terazosin and DA-8159, the total area under the plasma concentration–time curve from time zero to time infinity (AUC) of terazosin became significantly greater (57.4 and 75.4% increase for i.v. and p.o. administration, respectively) than those of without DA-8159. The blood pressure dropping effect was considerable after simultaneous p.o. administration of DA-8159 and terazosin compared with each drug alone. Conclusions and implications: The significantly greater AUC of terazosin after both simultaneous i.v. and p.o. administration of both drugs could be due to the hepatic (both i.v. and p.o.) and intestinal (p.o.) inhibition of the metabolism of terazosin via CYP3A1 and/or 3A2 by DA-8159, since both DA-8159 and terazosin are metabolized via CYP3A1 and/or 3A2 in rats. The blood pressure lowering effect after simultaneous p.o. administration of both drugs could be due to significant increase in plasma concentrations of terazosin. PMID:17351661

  13. 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

  14. Transfer of polychlorinated biphenyls and chlorinated pesticides from mother to pup in relation to cytochrome P450 enzyme activities in harp seals (Phoca groenlandica) from the gulf of St. Lawrence, Canada.

    PubMed

    Wolkers, Hans; Burkow, Ivan C; Hammill, Mike O; Lydersen, Christian; Witkamp, Renger F

    2002-01-01

    Congener-specific transfer of polychlorinated biphenyls (PCBs) and chlorinated pesticides from female to pup was studied in harp seals from eastern Canada. Possible effects on hepatic cytochrome P450 enzymes (CYP450) due to contaminant mobilization from blubber lipids in females and ingestion of contaminated milk in pups were studied. Contaminant transfer from blubber to milk in females favored the more polar compounds (lower chlorinated PCBs, toxaphenes, hexachlorocyclohexanes, and hexachlorobenzene) relative to more lipophilic compounds (higher chlorinated PCBs, dichlorodiphenyltrichloroethane [DDT], chlordane). In spite of substantial contaminant mobilization from blubber in females and ingestion of contaminated milk by pups, CYP450 activities were low in all animals. Possibly, increased plasma estradiol concentrations, involved in breeding after lactation, suppressed CYP450 directly. Although the pups were exposed to contaminants in milk, CYP450 activities were low, resulting in low contaminant metabolism. This was confirmed by similar contaminant patterns in milk and pups. A strong positive relation between CYP1A-like activities and body weight in the pups suggested not yet fully developed CYP1A enzymes. A negative association between CYP3A and pesticides in females and pups was hypothesized to be a result of metabolic inactivation of CYP450. The CYP450 enzyme activities were considered unsuitable indicators for contaminant mobilization and transfer in harp seals. PMID:11804067

  15. Molecular, cellular, and tissue impact of depleted uranium on xenobiotic-metabolizing enzymes.

    PubMed

    Gueguen, Yann; Rouas, Caroline; Monin, Audrey; Manens, Line; Stefani, Johanna; Delissen, Olivia; Grison, Stéphane; Dublineau, Isabelle

    2014-02-01

    Enzymes that metabolize xenobiotics (XME) are well recognized in experimental models as representative indicators of organ detoxification functions and of exposure to toxicants. As several in vivo studies have shown, uranium can alter XME in the rat liver or kidneys after either acute or chronic exposure. To determine how length or level of exposure affects these changes in XME, we continued our investigation of chronic rat exposure to depleted uranium (DU, uranyl nitrate). The first study examined the effect of duration (1-18 months) of chronic exposure to DU, the second evaluated dose dependence, from a level close to that found in the environment near mining sites (0.2 mg/L) to a supra-environmental dose (120 mg/L, 10 times the highest level naturally found in the environment), and the third was an in vitro assessment of whether DU exposure directly affects XME and, in particular, CYP3A. The experimental in vivo models used here demonstrated that CYP3A is the enzyme modified to the greatest extent: high gene expression changed after 6 and 9 months. The most substantial effects were observed in the liver of rats after 9 months of exposure to 120 mg/L of DU: CYP3A gene and protein expression and enzyme activity all decreased by more than 40 %. Nonetheless, no direct effect of DU by itself was observed after in vitro exposure of rat microsomal preparations, HepG2 cells, or human primary hepatocytes. Overall, these results probably indicate the occurrence of regulatory or adaptive mechanisms that could explain the indirect effect observed in vivo after chronic exposure. PMID:24146111

  16. Antioxidant enzymes activities in obese Tunisian children

    PubMed Central

    2013-01-01

    Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls), aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx). Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p < 0.05). Mean activities of anti-radical GPx and CAT enzymes were not affected by the BMI increase. Although, total cholesterol levels were statistically higher in the obese group, there was no significant association with the SOD activity. Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response. PMID:23360568

  17. 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

  18. Enzyme and root activities in surface-flow constructed wetlands.

    PubMed

    Kong, Ling; Wang, Yu-Bin; Zhao, Li-Na; Chen, Zhang-He

    2009-07-01

    Sixteen small-scale wetlands planted with four plant species were constructed for domestic wastewater purification. The objective of this study was to determine the correlations between contaminant removal and soil enzyme activity, root activity, and growth in the constructed wetlands. The results indicated that correlations between contaminant removal efficiency and enzyme activity varied depending on the contaminants. The removal efficiency of NH4+ was significantly correlated with both urease and protease activity in all wetlands, and the removal of total phosphorus and soluble reactive phosphorus was significantly correlated with phosphatase activity in most wetlands, while the removal of total nitrogen, NO3(-) , and chemical oxygen demand (COD) was significantly correlated with enzyme activity only in a few instances. Correlations between soil enzyme activity and root activity varied among species. Activities of all enzymes were significantly correlated with root activity in Vetiveria zizanioides and Phragmites australis wetlands, but not in Hymenocallis littoralis wetlands. Significant correlations between enzyme activity and root biomass and between enzyme activity and root growth were found mainly in Cyperus flabelliformis wetlands. Root activity was significantly correlated with removal efficiencies of all contaminants except NO3(-) and COD in V. zizanioides wetlands. Enzyme activities and root activity showed single-peak seasonal patterns. Activities of phosphatase, urease, and cellulase were significantly higher in the top layer of the substrate than in the deeper layers, and there were generally no significant differences between the deeper layers (deeper than 15 cm). PMID:19497608

  19. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish.

    PubMed

    Lin, Chun-Hung; Chou, Pei-Hsin; Chen, Pei-Jen

    2014-07-30

    Conazoles are a class of imidazole- or triazole-containing drugs commonly used as fungicides in agriculture and medicine. The broad application of azole drugs has led to the contamination of surface aquifers receiving the effluent of municipal or hospital wastewater or agricultural runoff. Several triazoles are rodent carcinogens; azole pollution is a concern to environmental safety and human health. However, the carcinogenic mechanisms associated with cytochrome P450 enzymes (CYPs) of conazoles remain unclear. We exposed adult medaka fish (Oryzias latipes) to continuous aqueous solutions of carcinogenic triadimefon and non-carcinogenic myclobutanil for 7 to 20 days at sub-lethal or environmentally relevant concentrations and assessed hepatic CYP activity and gene expression associated with CYP-mediated toxicity. Both triadimefon and myclobutanil induced hepatic CYP3A activity, but only triadimefon enhanced CYP1A activity. The gene expression of cyp3a38, cyp3a40, pregnane x receptor (pxr), cyp26b, retinoid acid receptor γ1 (rarγ1) and p53 was higher with triadimefon than myclobutanil. As well, yeast-based reporter gene assay revealed that 4 tested conazoles were weak agonists of aryl hydrocarbon receptor (AhR). We reveal differential CYP gene expression with carcinogenic and non-carcinogenic conazoles in a lower vertebrate, medaka fish. Liver CYP-enzyme induction may be a key event in conazole-induced tumorigenesis. This information is essential to evaluate the potential threat of conazoles to human health and fish populations in the aquatic environment. PMID:24962053

  20. Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?

    PubMed Central

    Kholodenko, B N; Brown, G C

    1996-01-01

    It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes