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Sample records for 2a6 2b6 2c9

  1. Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes.

    PubMed

    Srovnalova, Alzbeta; Svecarova, Michaela; Zapletalova, Michaela Kopecna; Anzenbacher, Pavel; Bachleda, Petr; Anzenbacherova, Eva; Dvorak, Zdenek

    2014-01-22

    Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 μM with IC50 = 32 μM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 μM with IC50 = 44 μM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected.

  2. Differential effects of nicotine treatment and ethanol self-administration on CYP2A6, CYP2B6 and nicotine pharmacokinetics in African green monkeys.

    PubMed

    Ferguson, C S; Miksys, S; Palmour, R M; Tyndale, R F

    2012-12-01

    In primates, nicotine is metabolically inactivated in the liver by CYP2A6 and possibly CYP2B6. Changes in the levels of these two enzymes may affect nicotine pharmacokinetics and influence smoking behaviors. This study investigated the independent and combined effects of ethanol self-administration and nicotine treatment (0.5 mg/kg b.i.d. s.c.) on hepatic CYP2A6 and CYP2B6 levels (mRNA, protein, and enzymatic activity), in vitro nicotine metabolism, and in vivo nicotine pharmacokinetics in monkeys. CYP2A6 mRNA and protein levels and in vitro coumarin (selective CYP2A6 substrate) and nicotine metabolism were decreased by nicotine treatment but unaffected by ethanol. CYP2B6 protein levels and in vitro bupropion (selective CYP2B6 substrate) metabolism were increased by ethanol but unaffected by nicotine treatment; CYP2B6 mRNA levels were unaltered by either treatment. Combined ethanol and nicotine exposure decreased CYP2A6 mRNA and protein levels, as well as in vitro coumarin and nicotine metabolism, and increased CYP2B6 protein levels and in vitro bupropion metabolism, with no change in CYP2B6 mRNA levels. Chronic nicotine resulted in higher nicotine plasma levels achieved after nicotine administration, consistent with decreased CYP2A6. Ethanol alone, or combined with nicotine, resulted in lower nicotine plasma levels by a mechanism independent of the change in these enzymes. Thus, nicotine can decrease hepatic CYP2A6, reducing the metabolism of its substrates, including nicotine, whereas ethanol can increase hepatic CYP2B6, increasing the metabolism of CYP2B6 substrates. In vivo nicotine pharmacokinetics are differentially affected by ethanol and nicotine, but when both drugs are used in combination the effect more closely resembles ethanol alone.

  3. Structural and Biophysical Characterization of Human Cytochromes P450 2B6 and 2A6 Bound to Volatile Hydrocarbons: Analysis and Comparison

    PubMed Central

    Wilderman, P. Ross; Liu, Jingbao; Jang, Hyun-Hee; Zhang, Qinghai; Stout, C. David; Halpert, James R.

    2015-01-01

    X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing –Cl and –NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6. PMID:25585967

  4. CYP2A6 and CYP2B6 genetic variation and its association with nicotine metabolism in South Western Alaska Native people

    PubMed Central

    Binnington, Matthew J.; Zhu, Andy Z.X.; Renner, Caroline C.; Lanier, Anne P.; Hatsukami, Dorothy K.; Benowitz, Neal L; Tyndale, Rachel F.

    2012-01-01

    Objectives Alaska Native people (AN) have a high prevalence of tobacco use and associated morbidity and mortality when compared to the general U.S. population. Variation in the CYP2A6 and CYP2B6 genes, encoding enzymes responsible for nicotine metabolic inactivation and procarcinogen activation, has not been characterized in AN and may contribute to the increased risk. Methods AN people (n = 400) residing in the Bristol Bay region of South Western Alaska were recruited for a cross-sectional study on tobacco use. They were genotyped for CYP2A6*1X2A, *1X2B, *1B, *2, *4, *7, *8, *9, *10, *12, *17, *35 and CYP2B6*4, *6, *9 and provided plasma and urine samples for measurement of nicotine and metabolites. Results CYP2A6 and CYP2B6 variant frequencies among the AN Yupik people (n=361) were significantly different from other ethnicities. Nicotine metabolism (as measured by the plasma and urinary ratio of metabolites trans-3’hydroxycotinine to cotinine [(3HC/COT)] was significantly associated with CYP2A6 (P< 0.001) but not CYP2B6 genotype (P = 0.95) when controlling for known covariates. Of note, plasma 3HC/COT ratios were high in the entire Yupik people, and among the Yupik CYP2A6 wild-type participants they were substantially higher than previously characterized racial/ethnic groups (P < 0.001 vs. Caucasians and African Americans). Conclusions Yupik AN people have a unique CYP2A6 genetic profile which associated strongly with in vivo nicotine metabolism. More rapid CYP2A6-mediated nicotine and nitrosamine metabolism in the Yupik people may modulate tobacco-related disease risk. PMID:22569203

  5. Pharmacogenetics of CYP2B6, CYP2A6 and UGT2B7 in HIV treatment in African populations: focus on efavirenz and nevirapine.

    PubMed

    Čolić, Antoinette; Alessandrini, Marco; Pepper, Michael S

    2015-05-01

    The CYP450 and UGT enzymes are involved in phase I and phase II metabolism of the majority of clinically prescribed drugs, including the non-nucleoside reverse transcriptase inhibitors, efavirenz and nevirapine, used in the treatment of HIV/AIDS. Variations in the activity of these enzymes due to gene polymorphisms can affect an individual's drug response or may lead to adverse drug reactions. There is an inter-ethnic distribution in the frequency of these polymorphisms, with African populations exhibiting higher genetic diversity compared to other populations. African specific alleles with clinical relevance have also emerged. Given the high prevalence of HIV/AIDS in sub-Saharan Africa, understanding the frequency of pharmacogenetically relevant alleles in populations of African origin, and their impact on efavirenz and nevirapine metabolism, is becoming increasingly critical. This review aims to investigate ethnic variation of CYP2B6, CYP2A6 and UGT2B7, and to understand the pharmacogenetic relevance when comparing frequencies in African populations to other populations worldwide.

  6. Optical Isomers of Atorvastatin, Rosuvastatin and Fluvastatin Enantiospecifically Activate Pregnane X Receptor PXR and Induce CYP2A6, CYP2B6 and CYP3A4 in Human Hepatocytes.

    PubMed

    Korhonova, Martina; Doricakova, Aneta; Dvorak, Zdenek

    2015-01-01

    Atorvastatin, fluvastatin and rosuvastatin are drugs used for treatment of hypercholesterolemia. They cause numerous drug-drug interactions by inhibiting and inducing drug-metabolizing cytochromes P450. These three statins exist in four optical forms, but they are currently used as enantiopure drugs, i.e., only one single enantiomer. There are numerous evidences that efficacy, adverse effects and toxicity of drugs may be enantiospecific. Therefore, we investigated the effects of optical isomers of atorvastatin, fluvastatin and rosuvastatin on the expression of drug-metabolizing P450s in primary human hepatocytes, using western blots and RT-PCR for measurement of proteins and mRNAs, respectively. The activity of P450 transcriptional regulators, including pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR), was assessed by gene reporter assays and EMSA. Transcriptional activity of AhR was not influenced by any statin tested. Basal transcriptional activity of GR was not affected by tested statins, but dexamethasone-inducible activity of GR was dose-dependently and enantioselectively inhibited by fluvastatin. Basal and ligand-inducible transcriptional activity of PXR was dose-dependently influenced by all tested statins, and the potency and efficacy between individual optical isomers varied depending on statin and optical isomer. The expression of CYP1A1 and CYP1A2 in human hepatocytes was not influenced by tested statins. All statins induced CYP2A6, CYP2B6 and CYP3A4, and the effects on CYP2C9 were rather modulatory. The effects varied between statins and enantiomers and induction potency decreased in order: atorvastatin (RR>RS = SR>SS) > fluvastatin (SR>RS = SS>RR) > rosuvastatin (only RS active). The data presented here might be of toxicological and clinical importance.

  7. Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6.

    PubMed

    Court, Michael H; Almutairi, Fawziah E; Greenblatt, David J; Hazarika, Suwagmani; Sheng, Hongyan; Klein, Kathrin; Zanger, Ulrich M; Bourgea, Joanne; Patten, Christopher J; Kwara, Awewura

    2014-07-01

    Efavirenz is commonly used to treat patients coinfected with human immunodeficiency virus and tuberculosis. Previous clinical studies have observed paradoxically elevated efavirenz plasma concentrations in patients with the CYP2B6*6/*6 genotype (but not the CYP2B6*1/*1 genotype) during coadministration with the commonly used four-drug antituberculosis therapy. This study sought to elucidate the mechanism underlying this genotype-dependent drug-drug interaction. In vitro studies were conducted to determine whether one or more of the antituberculosis drugs (rifampin, isoniazid, pyrazinamide, or ethambutol) potently inhibit efavirenz 8-hydroxylation by CYP2B6 or efavirenz 7-hydroxylation by CYP2A6, the main mechanisms of efavirenz clearance. Time- and concentration-dependent kinetics of inhibition by the antituberculosis drugs were determined using genotyped human liver microsomes (HLMs) and recombinant CYP2A6, CYP2B6.1, and CYP2B6.6 enzymes. Although none of the antituberculosis drugs evaluated at up to 10 times clinical plasma concentrations were found to inhibit efavirenz 8-hydroxylation by HLMs, both rifampin (apparent inhibition constant [Ki] = 368 μM) and pyrazinamide (Ki = 637 μM) showed relatively weak inhibition of efavirenz 7-hydroxylation. Importantly, isoniazid demonstrated potent time-dependent inhibition of efavirenz 7-hydroxylation in both HLMs (inhibitor concentration required for half-maximal inactivation [KI] = 30 μM; maximal rate constant of inactivation [kinact] = 0.023 min(-1)) and recombinant CYP2A6 (KI = 15 μM; kinact = 0.024 min(-1)) and also formed a metabolite intermediate complex consistent with mechanism-based inhibition. Selective inhibition of the CYP2B6.6 allozyme could not be demonstrated for any of the antituberculosis drugs using either recombinant enzymes or CYP2B6*6 genotype HLMs. In conclusion, the results of this study identify isoniazid as the most likely perpetrator of this clinically important drug-drug interaction through

  8. Preliminary investigation of the contribution of CYP2A6, CYP2B6, and UGT1A9 polymorphisms on artesunate-mefloquine treatment response in Burmese patients with Plasmodium falciparum malaria.

    PubMed

    Phompradit, Papichaya; Muhamad, Poonuch; Cheoymang, Anurak; Na-Bangchang, Kesara

    2014-08-01

    CYP2A6, CYP2B6, and UGT1A9 genetic polymorphisms and treatment response after a three-day course of artesunate-mefloquine was investigated in 71 Burmese patients with uncomplicated Plasmodium falciparum malaria. Results provide evidence for the possible link between CYP2A6 and CYP2B6 polymorphisms and plasma concentrations of artesunate/dihydroartemisinin and treatment response. In one patient who had the CYP2A6*1A/*4C genotype (decreased enzyme activity), plasma concentration of artesunate at one hour appeared to be higher, and the concentration of dihydroartemisinin was lower than for those carrying other genotypes (415 versus 320 ng/mL). The proportion of patients with adequate clinical and parasitologic response who had the CYP2B6*9/*9 genotype (mutant genotype) was significantly lower compared with those with late parasitologic failure (14.0% versus 19.0%). Confirmation through a larger study in various malaria-endemic areas is required before a definite conclusion on the role of genetic polymorphisms of these drug-metabolizing enzymes on treatment response after artesunate-based combination therapy can be made.

  9. Direct sequencing and comprehensive screening of genetic polymorphisms on CYP2 family genes (CYP2A6, CYP2B6, CYP2C8, and CYP2E1) in five ethnic populations.

    PubMed

    Kim, Jeong-Hyun; Cheong, Hyun Sub; Park, Byung Lae; Kim, Lyoung Hyo; Shin, Hee Jung; Na, Han Sung; Chung, Myeon Woo; Shin, Hyoung Doo

    2015-01-01

    Recently, CYP2A6, CYP2B6, CYP2C8, and CYP2E1 have been reported to play a role in the metabolic effect of pharmacological and carcinogenic compounds. Moreover, genetic variations of drug metabolism genes have been implicated in the interindividual variation in drug disposition and pharmacological response. To define the distribution of single nucleotide polymorphisms (SNPs) in these four CYP2 family genes and to discover novel SNPs across ethnic groups, 288 DNAs composed of 48 African-Americans, 48 European-Americans, 48 Japanese, 48 Han Chinese, and 96 Koreans were resequenced. A total of 143 SNPs, 26 in CYP2A6, 45 in CYP2B6, 29 in CYP2C8, and 43 in CYP2E1, were identified, including 13 novel variants. Notably, two SNPs in the regulatory regions, a promoter SNP rs2054675 and a nonsynonymous rs3745274 (p.172Q>H) in CYP2B6, showed significantly different minor allele frequencies (MAFs) among ethnic groups (minimum P = 4.30 × 10(-12)). In addition, rs2031920 in the promoter region of CYP2E1 showed a wide range of MAF between different ethnic groups, and even among other various ethnic groups based on public reports. Among 13 newly discovered SNPs in this study, 5 SNPs were estimated to have potential functions in further in silico analyses. Some differences in genetic variations and haplotypes of CYP2A6, CYP2B6, CYP2C8, and CYP2E1 were observed among populations. Our findings could be useful in further researches, such as genetic associations with drug responses.

  10. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

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

    PubMed

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

    2015-07-01

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

  12. Differences in Methadone Metabolism by CYP2B6 Variants.

    PubMed

    Gadel, Sarah; Friedel, Christina; Kharasch, Evan D

    2015-07-01

    Methadone is a long-acting opioid with considerable unexplained interindividual variability in clearance. Cytochrome P450 2B6 (CYP2B6) mediates clinical methadone clearance and metabolic inactivation via N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Retrospective studies suggest that individuals with the CYP2B6*6 allelic variant have higher methadone plasma concentrations. Catalytic activities of CYP2B6 variants are highly substrate- and expression-system dependent. This investigation evaluated methadone N-demethylation by expressed human CYP2B6 allelic variants in an insect cell coexpression system containing P450 reductase. Additionally, the influence of coexpressing cytochrome b5, whose role in metabolism can be inhibitory or stimulatory depending on the P450 isoform and substrate, on methadone metabolism, was evaluated. EDDP formation from therapeutic (0.25-1 μM) R- and S-methadone concentrations was CYP2B6.4 ≥ CYP2B6.1 ≥ CYP2B6.5 > CYP2B6.9 ≈ CYP2B6.6, and undetectable from CYP2B6.18. Coexpression of b5 had small and variant-specific effects at therapeutic methadone concentrations but at higher concentrations stimulated EDDP formation by CYP2B6.1, CYP2B6.4, CYP2B6.5, and CYP2B6.9 but not CYP2B6.6. In vitro intrinsic clearances were generally CYP2B6.4 ≥ CYP2B6.1 > CYP2B6.5 > CYP2B6.9 ≥ CYP2B6.6. Stereoselective methadone metabolism (S>R) was maintained with all CYP2B6 variants. These results show that methadone N-demethylation by CYP2B6.4 is greater compared with CYP2B6.1, whereas CYP2B6.9 and CYP2B6.6 (which both contain the 516G>T, Q172H polymorphism), are catalytically deficient. The presence or absence of b5 in expression systems may explain previously reported disparate catalytic activities of CYP2B6 variants for specific substrates. Differences in methadone metabolism by CYP2B6 allelic variants provide a mechanistic understanding of pharmacogenetic variability in clinical methadone metabolism and clearance.

  13. Imperatorin is a mechanism-based inactivator of CYP2B6.

    PubMed

    Zheng, Liwei; Cao, Jiaojiao; Lu, Dan; Ji, Lin; Peng, Ying; Zheng, Jiang

    2015-01-01

    Imperatorin (IMP) is the major active ingredient in many common medicinal herbs. We examined the irreversible inhibitory effect of IMP on CYP2B6. IMP produced a time- and concentration-dependent inactivation of CYP2B6. About 70% of activity of CYP2B6 was suppressed after its incubation with 1.5 μM IMP for 9 minutes. KI and kinact were found to be 0.498 μM and 0.079 min(-1), respectively. The loss of CYP2B6 activity required the presence of NADPH. Glutathione and catalase/superoxide dismutase showed little protection against the IMP-induced enzyme inactivation. Ticlopidine, a substrate of CYP2B6, showed protection of the enzyme against the inactivation induced by IMP. The estimated partition ratio of the inactivation was approximately 4. Additionally, a γ-ketoenal intermediate was identified in microsomal incubations with IMP. CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were found to be involved in bioactivation of IMP. In conclusion, IMP is a mechanism-based inactivator of CYP2B6. The formation of γ-ketoenal intermediate may account for the enzyme inactivation.

  14. Methadone N-demethylation by the common CYP2B6 allelic variant CYP2B6.6.

    PubMed

    Gadel, Sarah; Crafford, Amanda; Regina, Karen; Kharasch, Evan D

    2013-04-01

    The long-acting opioid methadone displays considerable unexplained interindividual pharmacokinetic variability. Methadone metabolism clinically occurs primarily by N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), catalyzed predominantly by CYP2B6. Retrospective studies suggest that the common allele variant CYP2B6*6 may influence methadone plasma concentrations. The catalytic activity of CYP2B6.6, encoded by CYP2B6*6, is highly substrate-dependent. This investigation compared methadone N-demethylation by CYP2B6.6 with that by wild-type CYP2B6.1. Methadone enantiomer and racemate N-demethylation by recombinant-expressed CYP2B6.6 and CYP2B6.1 was determined. At substrate concentrations (0.25-2 µM) approximating plasma concentrations occurring clinically, rates of methadone enantiomer N-demethylation by CYP2B6.6, incubated individually or as the racemate, were one-third to one-fourth those by CYP2B6.1. For methadone individual enantiomers and metabolism by CYP2B6.6 compared with CYP2B6.1, Vmax was diminished, Ks was greater and the in vitro intrinsic clearance was diminished 5- to 6-fold. The intrinsic clearance for R- and S-EDDP formation from racemic methadone was diminished approximately 6-fold and 3-fold for R- and S-methadone, respectively. Both CYP2B6.6 and CYP2B6.1 showed similar stereoselectivity (S>R-methadone). Human liver microsomes with diminished CYP2B6 content due to a CYP2B6*6 allele had lower rates of methadone N-demethylation. Results show that methadone N-demethylation catalyzed by CYP2B6.6, the CYP2B6 variant encoded by the CYP2B6*6 polymorphism, is catalytically deficient compared with wild-type CYP2B6.1. Diminished methadone N-demethylation by CYP2B6.6 may provide a mechanistic explanation for clinical observations of altered methadone disposition in individuals carrying the CYP2B6*6 polymorphism.

  15. Enantioselective inhibition of Cytochrome P450-mediated drug metabolism by a novel antithrombotic agent, S002-333: Major effect on CYP2B6.

    PubMed

    Bhateria, Manisha; Ramakrishna, Rachumallu; Puttrevu, Santosh Kumar; Saxena, Anil K; Bhatta, Rabi Sankar

    2016-08-25

    A significant number of new chemical entities (NCEs) fail in drug discovery due to inhibition of Cytochrome P450 (CYP) enzymes. Therefore, to avert costly drug failure at the clinical phase it becomes indispensable to evaluate the CYP inhibition profile of NCEs early in drug discovery. In light of these concerns, we envisioned to investigate the inhibitory effects of S002-333 [2-(4-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-b-carboxylic acid amide], a novel and potent antithrombotic agent, on nine major CYP enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4) of human liver microsomes (HLM). S002-333 exists as racemic mixture of S004-1032 (R-isomer) and S007-1558 (S-isomer), consequently, we further examined the enantioselective differences of S002-333 in the inhibition of human CYP enzymes. Of the CYP enzymes tested, CYP2B6-catalyzed bupropion 6-hydroxylation was inhibited by S002-333 (IC50 ∼ 9.25 ± 2.46 μM) in a stereoselective manner with (S)-isomer showing potent inhibition (IC50 ∼ 5.28 ± 1.25 μM) in contrast to (R)-isomer which showed negligible inhibition on CYP2B6 activity (IC50 > 50 μM). S002-333 and its (S)-isomer inhibited CYP2B6 activity in a non-competitive fashion with estimated Ki values of 10.1 ± 3.4 μM and 5.09 ± 1.05 μM, respectively. No shift in the IC50 value was observed for S002-333 and its isomers when preincubated for 30 min in the presence of NADPH suggesting that neither S002-333 nor its enantiomers are time-dependent inhibitors. Thus, the present findings signified that S002-333 is a potent stereoselective inhibitor of CYP2B6, whereas, inhibition for other CYPs was substantially negligible. These in vitro findings would be useful in deciding the development of S002-333 as a single-enantiomer or as a racemic mixture.

  16. Characterization of feline cytochrome P450 2B6.

    PubMed

    Okamatsu, Gaku; Komatsu, Tetsuya; Ono, Yuka; Inoue, Hiroki; Uchide, Tsuyoshi; Onaga, Takenori; Endoh, Daiji; Kitazawa, Takio; Hiraga, Takeo; Uno, Yasuhiro; Teraoka, Hiroki

    2017-02-01

    1. Little is known about drug metabolism in carnivores. Although the domestic cat (Felis catus) is an obligate carnivore and is the most common companion animal, usage and dosage of many drugs are determined according to information obtained from humans and dogs. We determined the complete cDNA sequence of CYP2B6 from the feline lung. 2. Feline CYP2B6 consists of 494 deduced amino acids, showing highest identity with the dog CYP2B ortholog, followed by those of horse, pig, primate and human. 3. Feline CYP2B6 transcripts were expressed predominantly in the lung and slightly in the small intestine but not in the liver without significant sex-dependent differences. Western blot analysis with an anti-human CYP2B6 antibody confirmed the presence of CYP2B protein in the lung but not in the liver. 4. Feline CYP2B6 proteins heterologously expressed in Escherichia coli metabolized several substrates specific to human CYP2B6, including 7-ethoxy-4-(trifluoromethyl) coumarin (EFC). The metabolic activity was strongly inhibited by medetomidine and atipamezole, potent inhibitors of canine CYP2B11 (now officially CYP2B6) as well as by ticlopidine and sertraline, inhibitors selective to human CYP2B6. 5. The results suggest that feline CYP2B6 is a functional CYP2B ortholog that plays a role in the local defense mechanism in the cat respiratory system and intestine.

  17. MOLECULAR CHARACTERIZATION OF CYP2B6 SUBSTRATES

    PubMed Central

    Ekins, Sean; Iyer, Manisha; Krasowski, Matthew D.; Kharasch, Evan D.

    2008-01-01

    CYP2B6 has not been as fully characterized at the molecular level as other members of the human cytochrome P450 family. As more widely used in vitro probes for characterizing the involvement of this enzyme in the metabolism of xenobiotics have become available, the number of molecules identified as CYP2B6 substrates has increased. In this study we have analyzed the available kinetic data generated by multiple laboratories with human recombinant expressed CYP2B6 and along with calculated molecular properties derived from the ChemSpider database, we have determined the molecular features that appear to be important for CYP2B6 substrates. In addition we have applied 2D and 3D QSAR methods to generate predictive pharmacophore and 2D models. For 28 molecules with Km data, the molecular weight (mean ± SD) is 253.78±74.03, ACD/logP is 2.68±1.51, LogDpH 5.5 is 1.51±1.43, LogDpH 7.4 is 2.02±1.25, hydrogen bond donor (HBD) count is 0.57 ±0.57, hydrogen bond acceptor (HBA) count is 2.57±1.37, rotatable bonds is 3.50±2.71 and total polar surface area (TPSA) is 27.63±19.42. A second set of 15 molecules without Km data possessed similar mean molecular property values. These properties are comparable to those of a set of 21 molecules used in a previous pharmacophore modeling study (Ekins et al., J Pharmacol Exp Ther 288 (1), 21–29, 1999). Only the LogD and HBD values were statistically significantly different between these different datasets. We have shown that CYP2B6 substrates are generally small hydrophobic molecules that are frequently central nervous system active, which may be important for drug discovery research. PMID:18537573

  18. Functional characterization of CYP2B6 allelic variants in demethylation of antimalarial artemether.

    PubMed

    Honda, Masashi; Muroi, Yuka; Tamaki, Yuichiro; Saigusa, Daisuke; Suzuki, Naoto; Tomioka, Yoshihisa; Matsubara, Yoichi; Oda, Akifumi; Hirasawa, Noriyasu; Hiratsuka, Masahiro

    2011-10-01

    Artemether (AM) is one of the most effective antimalarial drugs. The elimination half-life of AM is very short, and it shows large interindividual variability in pharmacokinetic parameters. The aim of this study was to identify cytochrome P450 (P450) isozymes responsible for the demethylation of AM and to evaluate functional differences between 26 CYP2B6 allelic variants in vitro. Of 14 recombinant P450s examined in this study, CYP2B6 and CYP3A4 were primarily responsible for production of the desmethyl metabolite dihydroartemisinin. The intrinsic clearance (V(max)/K(m)) of CYP2B6 was 6-fold higher than that of CYP3A4. AM demethylation activity was correlated with CYP2B6 protein levels (P = 0.004); however, it was not correlated with CYP3A4 protein levels (P = 0.27) in human liver microsomes. Wild-type CYP2B6.1 and 25 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.21 and CYP2B6.23-CYP2B6.27) were heterologously expressed in COS-7 cells. In vitro analysis revealed no enzymatic activity in 5 variants (CYP2B6.8, CYP2B6.12, CYP2B6.18, CYP2B6.21, and CYP2B6.24), lower activity in 7 variants (CYP2B6.10, CYP2B6.11, CYP2B6.14, CYP2B6.15, CYP2B6.16, CYP2B6.20, and CYP2B6.27), and higher activity in 4 variants (CYP2B6.2, CYP2B6.4, CYP2B6.6, and CYP2B6.19), compared with that of wild-type CYP2B6.1. In kinetic analysis, 3 variants (CYP2B6.2, CYP2B6.4, and CYP2B6.6) exhibited significantly higher V(max), and 3 variants (CYP2B6.14, CYP2B6.20 and CYP2B6.27) exhibited significantly lower V(max) compared with that of CYP2B6.1. This functional analysis of CYP2B6 variants could provide useful information for individualization of antimalarial drug therapy.

  19. In vivo individual variations in pharmacokinetics of efavirenz in cynomolgus monkeys genotyped for cytochrome P450 2C9.

    PubMed

    Iwasaki, Kazuhide; Kitsugi, Yusuke; Ikeda, Kanami; Yoshikawa, Takahiro; Hosaka, Shinya; Uehara, Shotaro; Uno, Yasuhiro; Utoh, Masahiro; Yamazaki, Hiroshi

    2016-09-01

    Cynomolgus monkeys are used frequently in preclinical studies for new drug development due to their evolutionary closeness to humans. An antiretroviral drug, efavirenz, is a typical probe substrate for human cytochrome P450 (P450) 2B6, but is mainly metabolized by cynomolgus monkey P450 2C9. In this study, plasma concentrations of efavirenz were assessed in six cynomolgus monkeys genotyped for P450 2C9 c.334 A > C (I112L) (three wild-type, one heterozygote and two homozygotes) by high performance liquid chromatography with tandem mass spectrometry. After intravenous administration at a dose of 1.0 mg/kg, biphasic plasma elimination curves of efavirenz were seen in these cynomolgus monkeys. The mean plasma concentration of the primary metabolite 8-hydroxyefavirenz (1 h after treatment, with hydrolysis by β-glucuronidase) in the wild-type group was significantly higher (4.0-fold) than the combined heterozygous and homozygous group mean. The area under the plasma concentration-time curve value of efavirenz in the homozygous group after oral administration at a dose of 2.0 mg/kg was significantly higher (2.0-fold) than the combined wild-type and heterozygous group. These results collectively indicated that P450 2C9 c.334 A > C (I112L) variation was associated with efavirenz metabolic clearance in vivo. Cynomolgus P450 2C9 polymorphism might account for interindividual variations of efavirenz metabolism in cynomolgus monkeys. Copyright © 2016 John Wiley & Sons, Ltd.

  20. The influence of sex, ethnicity, and CYP2B6 genotype on bupropion metabolism as an index of hepatic CYP2B6 activity in humans.

    PubMed

    Ilic, Katarina; Hawke, Roy L; Thirumaran, Ranjit K; Schuetz, Erin G; Hull, J Heyward; Kashuba, Angela D M; Stewart, Paul W; Lindley, Celeste M; Chen, Mei-Ling

    2013-03-01

    The effects of sex, ethnicity, and genetic polymorphism on hepatic CYP2B6 (cytochrome P450 2B6) expression and activity were previously demonstrated in vitro. Race/ethnic differences in CYP2B6 genotype and phenotype were observed only in women. To identify important covariates associated with interindividual variation in CYP2B6 activity in vivo, we evaluated these effects in healthy volunteers using bupropion (Wellbutrin SR GlaxoSmithKline, Research Triangle Park, NC) as a CYP2B6 probe substrate. A fixed 150-mg oral sustained-release dose of bupropion was administered to 100 healthy volunteers comprising four sex/ethnicity cohorts (n = 25 each): Caucasian men and Caucasian, African American, and Hispanic women. Blood samples were obtained at 0 and 6 hours postdose for the measurement of serum bupropion (BU) and hydroxybupropion (HB) concentrations. Whole blood was obtained at baseline for CYP2B6 genotyping. To characterize the relationship between CYP2B6 activity and ethnicity, sex, and genotype when accounting for serum BU concentrations (dose-adjusted log(10)-transformed), analysis of covariance model was fitted in which the dependent variable was CYP2B6 activity represented as the log(10)-transformed, metabolic ratio of HB to BU concentrations. Several CYP2B6 polymorphisms were associated with CYP2B6 activity. Evidence of dependence of CYP2B6 activity on ethnicity or genotype-by-ethnicity interactions was not detected in women. These results suggest that CYP2B6 genotype is the most important patient variable for predicting the level of CYP2B6 activity in women, when measured by the metabolism of bupropion. The bupropion metabolic ratio appears to detect known differences in CYP2B6 activity associated with genetic polymorphism, across different ethnic groups. Prospective studies will be needed to validate the use of bupropion as a probe substrate for clinical use.

  1. Phytoremediation of metolachlor by transgenic rice plants expressing human CYP2B6.

    PubMed

    Kawahigashi, Hiroyuki; Hirose, Sakiko; Ohkawa, Hideo; Ohkawa, Yasunobu

    2005-11-16

    We introduced the human cytochrome P450 gene CYP2B6 into rice plants (Oryza sativa L. cv. Nipponbare), and the CYP2B6-expressing rice plants became more tolerant to various herbicides than nontransgenic Nipponbare rice plants. In particular, CYP2B6 rice plants grown in soil showed tolerance to the chloroacetanilide herbicides alachlor and metolachlor. We evaluated the degradation of metolachlor by CYP2B6 rice plants to confirm the metabolic activity of the introduced CYP2B6. Although both CYP2B6 and nontransgenic Nipponbare rice plants could decrease the amount of metolachlor in plant tissue and culture medium, CYP2B6 rice plants could remove much greater amounts. In a greenhouse, the ability of CYP2B6 rice plants to remove metolachlor was confirmed in large-scale experiments, in which these plants appeared able to decrease residual quantities of metolachlor in water and soil.

  2. Pilot study of CYP2B6 genetic variation to explore the contribution of nitrosamine activation to lung carcinogenesis.

    PubMed

    Wassenaar, Catherine A; Dong, Qiong; Amos, Christopher I; Spitz, Margaret R; Tyndale, Rachel F

    2013-04-16

    We explored the contribution of nitrosamine metabolism to lung cancer in a pilot investigation of genetic variation in CYP2B6, a high-affinity enzymatic activator of tobacco-specific nitrosamines with a negligible role in nicotine metabolism. Previously we found that variation in CYP2A6 and CHRNA5-CHRNA3-CHRNB4 combined to increase lung cancer risk in a case-control study in European American ever-smokers (n = 860). However, these genes are involved in the pharmacology of both nicotine, through which they alter smoking behaviours, and carcinogenic nitrosamines. Herein, we separated participants by CYP2B6 genotype into a high- vs. low-risk group (*1/*1 + *1/*6 vs. *6/*6). Odds ratios estimated through logistic regression modeling were 1.25 (95% CI 0.68-2.30), 1.27 (95% CI 0.89-1.79) and 1.56 (95% CI 1.04-2.31) for CYP2B6, CYP2A6 and CHRNA5-CHRNA3-CHRNB4, respectively, with negligible differences when all genes were evaluated concurrently. Modeling the combined impact of high-risk genotypes yielded odds ratios that rose from 2.05 (95% CI 0.39-10.9) to 2.43 (95% CI 0.47-12.7) to 3.94 (95% CI 0.72-21.5) for those with 1, 2 and 3 vs. 0 high-risk genotypes, respectively. Findings from this pilot point to genetic variation in CYP2B6 as a lung cancer risk factor supporting a role for nitrosamine metabolic activation in the molecular mechanism of lung carcinogenesis.

  3. CYP2B6*6 is associated with increased breast cancer risk.

    PubMed

    Justenhoven, Christina; Pentimalli, Daniela; Rabstein, Sylvia; Harth, Volker; Lotz, Anne; Pesch, Beate; Brüning, Thomas; Dörk, Thilo; Schürmann, Peter; Bogdanova, Natalia; Park-Simon, Tjoung-Won; Couch, Fergus J; Olson, Janet E; Fasching, Peter A; Beckmann, Matthias W; Häberle, Lothar; Ekici, Arif; Hall, Per; Czene, Kamilla; Liu, Janjun; Li, Jingmei; Baisch, Christian; Hamann, Ute; Ko, Yon-Dschun; Brauch, Hiltrud

    2014-01-15

    The cytochrome P450 2B6 (CYP2B6) is involved in the metabolism of testosterone. Functional changes in this enzyme may influence endogenous hormone exposure, which has been associated with risk of breast cancer. To assess potential associations between two functional polymorphisms CYP2B6_516_G>T (rs3745274) and CYP2B6_785_A>G (rs2279343) and breast cancer risk, we established a specific matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay. The GENICA breast cancer case-control study showed associations between the variant genotypes CYP2B6_516_TT and CYP2B6_785_GG and breast cancer risk with odds ratios (ORs) of 1.34 (p = 0.001) and 1.31 (p = 0.002), respectively. A similar effect was observed for carriers of the CYP2B6_516_T allele in a validation study including four independent studies from Germany, Sweden and USA. In a pooled analysis of all five studies involving 4,638 breast cancer cases and 3,594 controls of European ancestry, carriers of the CYP2B6_516_G and the CYP2B6_785_G variant had an increased breast cancer risk with ORs of 1.10 (p = 0.027) and 1.10 (p = 0.031), respectively. We conclude that the genetic variants CYP2B6_516_G and CYP2B6_785_G (designated CYP2B6*6), which are known to decrease activity of the CYP2B6 enzyme, contribute to an increased breast cancer risk.

  4. Allele and genotype frequencies of CYP2B6 in a Turkish population.

    PubMed

    Yuce-Artun, Nazan; Kose, Gulcin; Suzen, H Sinan

    2014-06-01

    Increasing interest in cytochrome P450 2B6 (CYP2B6) genetic polymorphism was stimulated by revelations of a specific CYP2B6 genotype significantly affecting the metabolism of various drugs in common clinical use in terms of increasing drug efficacy and avoiding adverse drug reactions. The present study aimed to determine the frequencies of CYP2B6*4 CYP2B6*5, CYP2B6*6, CYP2B6*7 and CYP2B6*9 alleles in healthy Turkish individuals (n = 172). Frequencies of three single nucleotide polymorphisms were 516G>T (28%), 785A>G (33%), and 1459C>T (12%). The frequencies of CYP2B6*1, *4, *5, *6, *7, and *9 alleles were 54.3 (95% CI 49.04-59.56), 6.4% (95% CI 3.81-8.99), 11% (95% CI 7.69-14.31), 25.3% (95% CI 20.71-29.89), 0.87% (95% CI -0.11-1.85) and 2.0% (95% CI 0.52-3.48), respectively. Allele *6 was more frequent (25.3%) than the other variant alleles in Turkish subjects. The frequencies of CYP2B6*4, *5, *6, *7, and *9 alleles were similar to European populations but significantly different from that reported for Asian populations. This is the first study to document the frequencies of the CYP2B6*4, *5, *6, *7, *9 alleles in the healthy Turkish individuals and our results could provide clinically useful information on drug metabolism by CYP2B6 in Turkish population.

  5. CYP2B6 non-coding variation associated with smoking cessation is also associated with differences in allelic expression, splicing, and nicotine metabolism independent of common amino-acid changes.

    PubMed

    Bloom, A Joseph; Martinez, Maribel; Chen, Li-Shiun; Bierut, Laura J; Murphy, Sharon E; Goate, Alison

    2013-01-01

    The Cytochrome P450 2B6 (CYP2B6) enzyme makes a small contribution to hepatic nicotine metabolism relative to CYP2A6, but CYP2B6 is the primary enzyme responsible for metabolism of the smoking cessation drug bupropion. Using CYP2A6 genotype as a covariate, we find that a non-coding polymorphism in CYP2B6 previously associated with smoking cessation (rs8109525) is also significantly associated with nicotine metabolism. The association is independent of the well-studied non-synonymous variants rs3211371, rs3745274, and rs2279343 (CYP2B6*5 and *6). Expression studies demonstrate that rs8109525 is also associated with differences in CYP2B6 mRNA expression in liver biopsy samples. Splicing assays demonstrate that specific splice forms of CYP2B6 are associated with haplotypes defined by variants including rs3745274 and rs8109525. These results indicate differences in mRNA expression and splicing as potential molecular mechanisms by which non-coding variation in CYP2B6 may affect enzymatic activity leading to differences in metabolism and smoking cessation.

  6. Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain.

    PubMed

    Miksys, Sharon; Lerman, Caryn; Shields, Peter G; Mash, Deborah C; Tyndale, Rachel F

    2003-07-01

    CYP2B6 metabolizes drugs such as nicotine and bupropion, and many toxins and carcinogens. Nicotine induces CYP2B1 in rat brain and in humans polymorphic variation in CYP2B6 affects smoking cessation rates. The aim of this study was to compare CYP2B6 expression in brains of human smokers and non-smokers and alcoholics and non-alcoholics (n=26). CYP2B6 expression was brain region-specific, and was observed in both neurons and astrocytes. CYP2B6 levels were higher in brains of smokers and alcoholics, particularly in cerebellar Purkinje cells and hippocampal pyramidal neurons, cells known to be damaged in alcoholics. Significantly more (p<0.05) CYP2B6 protein was seen in four brain regions of smoking alcoholics compared to non-smoking non-alcoholics: hippocampus (5.8-fold), caudate nucleus (3.3-fold), putamen (3.0-fold) and cerebellar hemisphere (1.6-fold). The genetic variant C1459T (R487C) has been associated with reduced hepatic enzyme levels, stability and activity. Preliminary genotyping of this small sample (n=24) suggested that individuals with the CC genotype had higher brain CYP2B6 than those with the CT or TT genotype. Higher brain CYP2B6 activity in smokers and alcoholics may cause altered sensitivity to centrally acting drugs, increased susceptibility to neurotoxins and carcinogenic xenobiotics and contribute to central tolerance to nicotine.

  7. Polymorphic variants of cytochrome P450 2B6 (CYP2B6.4-CYP2B6.9) exhibit altered rates of metabolism for bupropion and efavirenz: a charge-reversal mutation in the K139E variant (CYP2B6.8) impairs formation of a functional cytochrome p450-reductase complex.

    PubMed

    Zhang, Haoming; Sridar, Chitra; Kenaan, Cesar; Amunugama, Hemali; Ballou, David P; Hollenberg, Paul F

    2011-09-01

    In this study, metabolism of bupropion, efavirenz, and 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) by CYP2B6 wild type (CYP2B6.1) and six polymorphic variants (CYP2B6.4 to CYP2B6.9) was investigated in a reconstituted system to gain a better understanding of the effects of the mutations on the catalytic properties of these naturally occurring variants. All six variants were successfully overexpressed in Escherichia coli, including CYP2B6.8 (the K139E variant), which previously could not be overexpressed in mammalian COS-1 cells (J Pharmacol Exp Ther 311:34-43, 2004). The steady-state turnover rates for the hydroxylation of bupropion and efavirenz and the O-deethylation of 7-EFC showed that these mutations significantly alter the catalytic activities of CYP2B6. It was found that CYP2B6.6 exhibits 4- and 27-fold increases in the K(m) values for the hydroxylation of bupropion and efavirenz, respectively, and CYP2B6.8 completely loses its ability to metabolize any of the substrates under normal turnover conditions. However, compared with CYP2B6.1, CYP2B6.8 retains 77% of its 7-EFC O-deethylase activity in the presence of tert-butyl hydroperoxide as an alternative oxidant, indicating that the heme and the active site are catalytically competent. Presteady-state measurements of the rate of electron transfer from NADPH-dependent cytochrome P450 reductase (CPR) to CYP2B6.8 using stopped-flow spectrophotometry revealed that CYP2B6.8 is incapable of accepting electrons from CPR. These observations provide conclusive evidence suggesting that the charge-reversal mutation in the K139E variant prevents CYP2B6.8 from forming a functional complex with CPR. Results from this work provide further insights to better understand the genotype-phenotype correlation regarding CYP2B6 polymorphisms and drug metabolism.

  8. Variation in CYP2A6 and tobacco dependence throughout adolescence and in young adult smokers

    PubMed Central

    Chenoweth, Meghan J.; Sylvestre, Marie-Pierre; Contreras, Gisele; Novalen, Maria; O’Loughlin, Jennifer; Tyndale, Rachel F.

    2015-01-01

    Background Smoking is influenced by genetic factors including variation in CYP2A6 and CYP2B6, which encode nicotine-metabolizing enzymes. In early adolescence, CYP2A6 slow nicotine metabolism was associated with higher dependence acquisition, but reduced cigarette consumption. Here we extend this work by examining associations of CYP2A6 and CYP2B6 with tobacco dependence acquisition in a larger sample of smokers followed throughout adolescence. Methods White participants from the Nicotine Dependence in Teens cohort that had ever inhaled (n=421) were followed frequently from age 12–18 years. Cox’s proportional hazards models compared the risk of ICD-10 tobacco dependence acquisition (score 3+) for CYP2A6 and CYP2B6 metabolism groups. Early smoking experiences, as well as amount smoked at end of follow-up, was also computed. At age 24 (N=162), we assessed concordance between self-reported cigarette consumption and salivary cotinine. Results In those who initiated inhalation during follow-up, CYP2A6 slow (vs. normal) metabolizers were at greater risk of dependence (hazards ratio (HR)=2.3; 95% CI=1.1, 4.8); CYP2B6 slow (vs. normal) metabolizers had non-significantly greater risk (HR=1.5; 95% CI=0.8, 2.6). Variation in CYP2A6 or CYP2B6 was not significantly associated with early smoking symptoms or cigarette consumption at end of follow-up. At age 24, neither gene was significantly associated with dependence status. Self-reported consumption was associated with salivary cotinine, a biomarker of tobacco exposure, acquired at age 24 (B=0.37; P<0.001). Conclusions Our findings extend previous work indicating that slow nicotine metabolism mediated by CYP2A6, and perhaps CYP2B6, increases risk for tobacco dependence throughout adolescence. PMID:26644138

  9. CYP2B6 poor metaboliser alleles involved in efavirenz and nevirapine metabolism: CYP2B6*9 and CYP2B6*18 distribution in HIV-exposed subjects from Dschang, Western Cameroon.

    PubMed

    Paganotti, Giacomo Maria; Russo, Gianluca; Sobze, Martin Sanou; Mayaka, George Bouting; Muthoga, Charles Waithaka; Tawe, Leabaneng; Martinelli, Axel; Romano, Rita; Vullo, Vincenzo

    2015-10-01

    The prescription of patients' tailored anti-infectious treatments is the ultimate goal of pharmacogenetics/genomics applied to antimicrobial treatments, providing a basis for personalized medicine. Despite the efforts to screen Africans for alleles underlying defective metabolism for a panel of different drugs, still more research is necessary to clarify the interplay between host genetic variation and treatments' response. HIV is a major infectious disease in sub-Saharan African countries, and the main prescribed anti-HIV combination therapy includes efavirenz (EFV) or nevirapine (NVP). The two drugs are both mainly metabolised by cytochrome P450 2B6 liver enzyme (CYP2B6). Defective variants of CYP2B6 gene, leading to higher drug exposure with subsequent possible side effects and low compliance, are well known. However, little is known about CYP2B6 alleles in Cameroon where only one study was done on this subject. The main objective of the present work is to assess, in a subset of HIV-exposed subjects from Dschang in West Cameroon, the prevalence of two SNPs in the CYP2B6 gene: 516G>T (rs3745274) and 983T>C (rs28399499), both associated to a defective EFV and NVP metabolism. We analyzed 168 DNA samples collected during two cross-sectional surveys performed in Dschang, West Cameroon. In the population studied the observed allele frequencies of 516G>T and 983T>C were 44.35% (95%CI, 36.84-51.86%) and 12.80% (95%CI, 7.75-17.85%), respectively. Moreover, concerning the CYP2B6 expected phenotypes, 28.57% of the population showed a poor metaboliser phenotype, while 27.38% and 44.05% showed an extensive (wild-type) and an intermediate metaboliser phenotype, respectively. Here we found that an important fraction of the subjects is carrying EFV/NVP poor metaboliser alleles. Our findings could help to improve the knowledge about the previewed efficacy of anti-HIV drug therapy in Cameroon. Finally, we designed a new method of detection for the 983T>C genetic variation that

  10. Global variation in CYP2C8–CYP2C9 functional haplotypes

    PubMed Central

    Speed, William C; Kang, Soonmo Peter; Tuck, David P; Harris, Lyndsay N; Kidd, Kenneth K

    2009-01-01

    We have studied the global frequency distributions of 10 single nucleotide polymorphisms (SNPs) across 132 kb of CYP2C8 and CYP2C9 in ∼2500 individuals representing 45 populations. Five of the SNPs were in noncoding sequences; the other five involved the more common missense variants (four in CYP2C8, one in CYP2C9) that change amino acids in the gene products. One haplotype containing two CYP2C8 coding variants and one CYP2C9 coding variant reaches an average frequency of 10% in Europe; a set of haplotypes with a different CYP2C8 coding variant reaches 17% in Africa. In both cases these haplotypes are found in other regions of the world at <1%. This considerable geographic variation in haplotype frequencies impacts the interpretation of CYP2C8/CYP2C9 association studies, and has pharmacogenomic implications for drug interactions. PMID:19381162

  11. Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos.

    PubMed

    D'Agostino, Jaime; Zhang, Haoming; Kenaan, Cesar; Hollenberg, Paul F

    2015-07-20

    Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro. Therefore, we characterized the MBI of CYP2B6 by CPS. CPS inactivated CYP2B6 in a time- and concentration-dependent manner with a kinact of 1.97 min(-1), a KI of 0.47 μM, and a partition ratio of 17.7. We further evaluated the ability of other organophosphate pesticides including chorpyrifos-methyl, diazinon, parathion-methyl, and azinophos-methyl to inactivate CYP2B6. These organophosphate pesticides were also potent MBIs of CYP2B6 characterized by similar kinact and KI values. The inactivation of CYP2B6 by CPS was accompanied by the loss of P450 detectable in the CO reduced spectrum and loss of detectable heme. High molecular weight aggregates were observed when inactivated CYP2B6 was run on SDS-PAGE gels indicating protein aggregation. Interestingly, we found that the rat homologue of CYP2B6, CYP2B1, was not inactivated by CPS despite forming CPO to a similar extent. On the basis of the locations of the Cys residues in the two proteins which could react with released sulfur during the metabolism of CPS, we investigated whether the C475 in CYP2B6, which is not conserved in CYP2B1, was the critical residue for inactivation by mutating it to a Ser. CYP2B6 C475S was inactivated to a similar extent as wild type CYP2B6 indicating that C475 is not likely the key difference between CYP2B1 and CYP2B6 with respect to inactivation. These results indicate that CPS and other organophosphate pesticides are potent MBIs of CYP2B6 which may have implications for the toxicity of these pesticides as well as the potential for pesticide-drug interactions.

  12. Association of CYP2C9*2 with Bosentan-Induced Liver Injury

    PubMed Central

    Markova, Svetlana M.; De Marco, Teresa; Bendjilali, Nasrine; Kobashigawa, Erin A.; Mefford, Joel; Sodhi, Jasleen; Le, Hoa; Zhang, Chenghong; Halladay, Jason; Rettie, Allan E.; Khojasteh, Cyrus; McGlothlin, Dana; Wu, Alan H.B.; Hsueh, Wen-Chi; Witte, John S.; Schwartz, Janice B.; Kroetz, Deanna L.

    2013-01-01

    Bosentan (Tracleer®) is an endothelin receptor antagonist prescribed for the treatment of pulmonary arterial hypertension (PAH). Its use is limited by drug-induced liver injury (DILI). To identify genetic markers of DILI, association analyses were performed on 56 Caucasian PAH patients receiving bosentan. Twelve functional polymorphisms in five genes (ABCB11, ABCC2, CYP2C9, SLCO1B1, SLCO1B3) implicated in bosentan pharmacokinetics were tested for associations with ALT, AST and DILI. After adjusting for BMI, CYP2C9*2 was the only polymorphism associated with ALT, AST and DILI (β = 2.16, P = 0.024; β = 1.92, P = 0.016; OR 95% CI = 2.29 - ∞, P = 0.003, respectively). Bosentan metabolism in vitro by CYP2C9*2 was significantly reduced compared to CYP2C9*1 and was comparable to CYP2C9*3. These results suggest that CYP2C9*2 is a potential genetic marker for prediction of bosentan-induced liver injury and warrants investigation for the optimization of bosentan treatment. PMID:23863877

  13. Association of CYP2C9*2 with bosentan-induced liver injury.

    PubMed

    Markova, S M; De Marco, T; Bendjilali, N; Kobashigawa, E A; Mefford, J; Sodhi, J; Le, H; Zhang, C; Halladay, J; Rettie, A E; Khojasteh, C; McGlothlin, D; Wu, A H B; Hsueh, W-C; Witte, J S; Schwartz, J B; Kroetz, D L

    2013-12-01

    Bosentan (Tracleer) is an endothelin receptor antagonist prescribed for the treatment of pulmonary arterial hypertension (PAH). Its use is limited by drug-induced liver injury (DILI). To identify genetic markers of DILI, association analyses were performed on 56 Caucasian PAH patients receiving bosentan. Twelve functional polymorphisms in five genes (ABCB11, ABCC2, CYP2C9, SLCO1B1, and SLCO1B3) implicated in bosentan pharmacokinetics were tested for associations with alanine aminotransferase (ALT), aspartate aminotransferase (AST), and DILI. After adjusting for body mass index, CYP2C9*2 was the only polymorphism associated with ALT, AST, and DILI (β = 2.16, P = 0.024; β = 1.92, P = 0.016; odds ratio 95% CI = 2.29-∞, P = 0.003, respectively). Bosentan metabolism by CYP2C9*2 in vitro was significantly reduced compared with CYP2C9*1 and was comparable to that by CYP2C9*3. These results suggest that CYP2C9*2 is a potential genetic marker for prediction of bosentan-induced liver injury and warrants investigation for the optimization of bosentan treatment.

  14. CYP2B6 18492T->C polymorphism compromises efavirenz concentration in coinfected HIV and tuberculosis patients carrying CYP2B6 haplotype *1/*1.

    PubMed

    Manosuthi, Weerawat; Sukasem, Chonlaphat; Thongyen, Supeda; Nilkamhang, Samruay; Manosuthi, Sukanya; Sungkanuparph, Somnuek

    2014-01-01

    Data regarding the effect of the CYP2B6 18492T→C polymorphism on plasma efavirenz concentrations and 96-week virologic responses in patients coinfected with HIV and tuberculosis (TB) are still unavailable. A total of 139 antiretroviral-naive HIV-infected adults with active TB were prospectively enrolled to receive efavirenz 600 mg-tenofovir 300 mg-lamivudine 300 mg. Eight single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped. Seven SNPs, including 64C→T, 499C→G, 516G→T, 785A→G, 1375A→G, 1459C→T, and 21563C→T, were included for CYP2B6 haplotype determination. The CYP2B6 18492T→C polymorphism was studied in 48 patients who carried haplotype *1/*1. At 12 and 24 weeks after antiretroviral therapy, plasma efavirenz concentrations at 12 h after dosing were measured. Plasma HIV RNA was monitored every 12 weeks for 96 weeks. Of 48 patients {body weight [mean±standard deviation (SD)], 56±10 kg}, 77% received a rifampin-containing anti-TB regimen. No drug resistance-associated mutation was detected at baseline. The frequencies of the wild type (18492TT) and the heterozygous (18492TC) and homozygous (18492CC) mutants of the CYP2B6 18492T→C polymorphism were 39%, 42%, and 19%, respectively. At 12 weeks, mean (±SD) efavirenz concentrations of patients who carried the 18492TT, 18492TC, and 18492CC mutants were 2.8±1.6, 1.7±0.9, and 1.4±0.5 mg/liter, respectively (P=0.005). At 24 weeks, the efavirenz concentrations of the corresponding groups were 2.4±0.8, 1.7±0.8, and 1.2±0.4 mg/liter, respectively (P=0.003). A low efavirenz concentration was independently associated with 18492T→C (β=-0.937, P=0.004) and high body weight (β=-0.032, P=0.046). At 96 weeks, 19%, 17%, and 28% of patients carrying the 18492TT, 18492TC, and 18492CC mutants, respectively, had plasma HIV RNA levels of >40 copies/ml and developed efavirenz-associated mutations (P=0.254). In summary, the CYP2B6 18492T→C polymorphism compromises efavirenz concentrations in

  15. Transgenic rice containing human CYP2B6 detoxifies various classes of herbicides.

    PubMed

    Hirose, Sakiko; Kawahigashi, Hiroyuki; Ozawa, Kenjirou; Shiota, Noriaki; Inui, Hideyuki; Ohkawa, Hideo; Ohkawa, Yasunobu

    2005-05-04

    The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.

  16. Re-engineering of CYP2C9 to probe acid-base substrate selectivity.

    PubMed

    Tai, Guoying; Dickmann, Leslie J; Matovic, Nicholas; DeVoss, James J; Gillam, Elizabeth M J; Rettie, Allan E

    2008-10-01

    A common feature of many CYP2C9 ligands is their weak acidity. As revealed by crystallography, the structural basis for this behavior involves a charge-pairing interaction between an anionic moiety on the substrate and an active site R108 residue. In the present study we attempted to re-engineer CYP2C9 to better accept basic ligands by charge reversal at this key residue. We expressed and purified the R108E and R108E/D293N mutants and compared their ability with that of native CYP2C9 to interact with (S)-warfarin, diclofenac, pyrene, propranolol, and ibuprofen amine. As expected, the R108E mutant maintained all the native enzyme's pyrene 1-hydroxylation activity, but catalytic activity toward diclofenac and (S)-warfarin was abrogated. In contrast, the double mutant displayed much less selectivity in its behavior toward these control ligands. Neither of the mutants displayed significant enhancement of propranolol metabolism, and all three preparations exhibited a type II (inhibitor) rather than type I (substrate) spectrum with ibuprofen amine, although binding became progressively weaker with the single and double mutants. Collectively, these data underscore the importance of the amino acid at position 108 in the acid substrate selectivity of CYP2C9, highlight the accommodating nature of the CYP2C9 active site, and provide a cautionary note regarding facile re-engineering of these complex cytochrome P450 active sites.

  17. Psoralen, a mechanism-based inactivator of CYP2B6.

    PubMed

    Ji, Lin; Lu, Dan; Cao, Jiaojiao; Zheng, Liwei; Peng, Ying; Zheng, Jiang

    2015-10-05

    Furanocoumarin compound psoralen (PRN) is a major active ingredient found in herbaceous plants. PRN has been used for the treatment of various dermal diseases in China. We evaluated the inhibitory effect of PRN on cytochrome P450 2B6 (CYP2B6) and found that PRN induced a time-, concentration-, and NADPH-dependent inactivation of CYP2B6 with the values of KI and kinact being 110.2 μM and 0.200 min(-1), respectively. Ticlopidine, a CYP2B6 substrate, prevented the enzyme from the inactivation induced by PRN. Exogenous nucleophile glutathione (GSH) and catalase/superoxide dismutase showed limited protection of CYP2B6 from the inactivation. The estimated partition ratio of the inactivation was approximately 400. GSH trapping experiments indicates that an epoxide or/and γ-ketoenal intermediate was formed in microsomal incubations with PRN. In summary, PRN was characterized as a mechanism-based inactivator of CYP2B6.

  18. Inhibitory Effects of Garcinia cambogia Extract on CYP2B6 Enzyme Activity.

    PubMed

    Yu, Jun Sang; Choi, Min Sun; Park, Jong Suk; Rehman, Shaheed Ur; Nakamura, Katsunori; Yoo, Hye Hyun

    2017-03-13

    This study assessed the inhibitory effects of Garcinia cambogia extract on the cytochrome P450 enzymes in vitro. G. cambogia extract was incubated with cytochrome P450 isozyme-specific substrates in human liver microsomes and recombinant CYP2B6 isozyme, and the formation of the marker metabolites was measured to investigate the inhibitory potential on cytochrome P450 enzyme activities. The results showed that G. cambogia extract has significant inhibitory effects on CYP2B6 activity in a concentration-dependent manner. Furthermore, the inhibition was potentiated following preincubation with NADPH, indicating that G. cambogia extract is a time-dependent inhibitor of CYP2B6. Meanwhile, hydroxycitric acid, the major bioactive ingredient of G. cambogia extract, did not exhibit significant inhibition effects on cytochrome P450 enzyme activities. G. cambogia extract could modulate the pharmacokinetics of CYP2B6 substrate drugs and lead to interactions with those drugs. Therefore, caution may be required with respect to concomitant intake of dietary supplements containing G. cambogia extract with CYP2B6 substrates.

  19. Prevalence of CYP2B6 polymorphisms in Argentinians: the role of genetic testing.

    PubMed

    Scibona, P; Vazquez, C; Cajal, A R; Argibay, P F; Belloso, W H

    2015-12-11

    CYP2B6 is a highly polymorphic isoenzyme involved in the metabolism of many drugs including cyclophosphamide, bupropion, and efavirenz. A single nucleotide polymorphism (SNP) in CYP2B6 (516G>T) resulted in decreased expression and function associated with the CYP2B6*6 haplotype. Among the clinical implications of this phenotype, decreased activation of cyclophosphamide and increased plasma levels of efavirenz associated with increased central nervous system toxicity have been reported. The frequency of the CYP2B6 (516G>T) SNP has been studied in several different populations, but there is no data regarding distribution among Argentinians. In this study, 102 DNA samples from healthy volunteers were analyzed using a polymerase chain reaction-restriction fragment length polymorphism reaction specific for the CYP2B6 (516G>T) SNP. Our results showed a prevalence of 71.08% for the G allele and 28.92% for the T allele. This was distributed as 52.9% for the GG genotype (reduced dosage required), 36.6% for the GT genotype (normal dosage range), and 10.8% for the TT genotype (high drug toxicity). There was no preferential gender distribution observed. The relatively high prevalence of the TT genotype in our population supports the clinical use of genotyping as an additional tool in personalized medicine.

  20. Dosing algorithms for vitamin K antagonists across VKORC1 and CYP2C9 genotypes.

    PubMed

    Baranova, E V; Verhoef, T I; Ragia, G; le Cessie, S; Asselbergs, F W; de Boer, A; Manolopoulos, V G; Maitland-van der Zee, A H

    2017-03-01

    Essentials Prospective studies of pharmacogenetic-guided (PG) coumarin dosing produced varying results. EU-PACT acenocoumarol and phenprocoumon trials compared PG and non-PG dosing algorithms. Sub-analysis of EU-PACT identified differences between trial arms across VKORC1-CYP2C9 groups. Adjustment of the PG algorithm might lead to a higher benefit of genotyping.

  1. Single Heteroatom Substitutions in the Efavirenz Oxazinone Ring Impact Metabolism by CYP2B6.

    PubMed

    Cox, Philip M; Bumpus, Namandjé N

    2016-12-06

    Previously, we observed that the oxazinone ring is important for cytochrome P450 2B6 (CYP2B6) activity toward efavirenz ((4S)-6-chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one), a CYP2B6 substrate used to treat HIV. To further understand the structural characteristics of efavirenz that render it a CYP2B6 substrate, we tested the importance of each heteroatom of the oxazinone ring. We assembled a panel of five analogues: 6-chloro-4-(2-cyclopropylethynyl)-1,4-dihydro-2-methyl-4-(trifluoromethyl)-2H-3,1-benzoxazine (1), (4S)-6-chloro-4-[(1E)-2-cyclopropylethenyl]-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (2), (4S)-6-chloro-4-(2-cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinazolinone (3), 6-chloro-4-(cyclopropylethynyl)-3,4-dihydro-4-(trifluoromethyl)-2(1H)-quinolinone (4), and 6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-4H-benzo[d][1,3]dioxin-2-one (5). The metabolism of compounds 1-5 was investigated using human liver microsomes, individual P450s, and mass spectrometry or UV/Vis absorbance detection. Steady-state analysis of CYP2B6 metabolism of 1-5 showed KM values ranging from 0.3- to 3.9-fold different from that observed for efavirenz (KM : 3.6±1.7 μm). The lowest KM values, approximating 1 μm, were observed for the metabolism of 1, whereas the greatest KM value, 14±6.4 μm, was found for 4. Our work reveals that analogues with heteroatom changes in the oxazinone ring are still CYP2B6 substrates, although the changes in KM suggest altered substrate binding.

  2. High plasma efavirenz concentration and CYP2B6 polymorphisms in Thai HIV-1 infections.

    PubMed

    Sukasem, Chonlaphat; Chamnanphon, Montri; Koomdee, Napatrupron; Puangpetch, Apichaya; Santon, Siwalee; Jantararoungtong, Thawinee; Prommas, Santirat; Chantratita, Wasun; Manosuthi, Weerawat

    2013-01-01

      Efavirenz is mainly metabolized by cytochrome P450 2B6 (CYP2B6). This study aimed to examine the frequencies of CYP2B6 and the association between CYP2B6 polymorphisms and plasma efavirenz concentrations in an HIV-1 infected Thai population. Mid-dose plasma efavirenz concentration was determined at 12 weeks following the initiation of an antiretroviral therapy (tenofovir, lamivudine and efavirenz) in 100 Thai adults with HIV-1 infection using high-performance liquid chromatography. Candidate CYP2B6 polymorphisms (c.64C>T, c.499C>G, c.516G>T, c.785A>G, c.1375A>G, c.1459C>T) were conducted by real-time PCR-based allelic discrimination. The most frequent polymorphisms among this cohort were the CYP2B6 c.785A>G and c.516G>T, which had a frequency of 0.36 and 0.32, respectively. From the cases observed, two single nucleotide polymorphisms (SNPs) (c.516G>T and c.785A>G) were significantly associated with high efavirenz plasma levels (p < 0.05). The most frequent haplotypic combinations were *1/*6, *1/*1, *1/*2 and *6/*6 at a frequency of 42.0%, 32.0%, 8.0% and 7.0%, respectively. Increased plasma concentrations of efavirenz were present in individuals with CYP2B6 *6/*6 [7.210 mg/L; interquartile range (IQR), 5.020-9.260] when compared to those with CYP2B6*1/*1 (1.570 mg/L; IQR, 1.295-2.670), p < 0.001. In our study, the impact of SNPs which are correlated with a high level of efavirenz plasma concentrations was found. The genetic configuration of SNPs which are associated with high plasma efavirenz levels may be useful in optimizing the efavirenz dose that is used in HIV-1 infected patients.

  3. CYP2B6*6 and CYP2B6*18 Predict Long-Term Efavirenz Exposure Measured in Hair Samples in HIV-Positive South African Women.

    PubMed

    Röhrich, Carola R; Drögemöller, Britt I; Ikediobi, Ogechi; van der Merwe, Lize; Grobbelaar, Nelis; Wright, Galen E B; McGregor, Nathaniel; Warnich, Louise

    2016-06-01

    Long-term exposure to efavirenz (EFV) measured in hair samples may predict response to antiretroviral treatment (ART). Polymorphisms in CYP2B6 are known to alter EFV levels. The aim of this study was to assess the relationship between CYP2B6 genotype, EFV levels measured in hair, and virological outcomes on ART in a real-world setting. We measured EFV levels in hair from HIV-positive South African females who had been receiving EFV-based treatment for at least 3 months from the South African Black (SAB) (n = 81) and Cape Mixed Ancestry (CMA) (n = 53) populations. Common genetic variation in CYP2B6 was determined in 15 individuals from each population using bidirectional Sanger sequencing. Prioritized variants (n = 16) were subsequently genotyped in the entire patient cohort (n = 134). The predictive value of EFV levels in hair and selected variants in CYP2B6 on virological treatment outcomes was assessed. Previously described alleles (CYP2B6*2, CYP2B6*5, CYP2B6*6, CYP2B6*17, and CYP2B6*18), as well as two novel alleles (CYP2B6*31 and CYP2B6*32), were detected in this study. Compared to noncarriers, individuals homozygous for CYP2B6*6 had ∼109% increased EFV levels in hair (p = .016) and CYP2B6*18 heterozygotes demonstrated 82% higher EFV hair levels (p = .0006). This study confirmed that alleles affecting CYP2B6 metabolism and subsequent EFV exposure are present at significant frequencies in both the SAB and CMA populations. Furthermore, this study demonstrated that the use of hair samples for testing EFV concentrations may be a useful tool in determining long-term drug exposure in resource-limited countries.

  4. Equine cytochrome P450 2B6 — Genomic identification, expression and functional characterization with ketamine

    SciTech Connect

    Peters, L.M.; Demmel, S.; Pusch, G.; Buters, J.T.M.; Zielinski, J.; Leeb, T.; Mevissen, M.; Schmitz, A.

    2013-01-01

    Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug–drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V{sub max} for S-/and R-norketamine formation was 0.49 and 0.45 nmol/h/mg cellular protein and K{sub m} was 3.41 and 2.66 μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC{sub 50} of 5.63 and 6.26 μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP

  5. Genotype-phenotype correlation of cytochrome P450 2C9 polymorphism in Indian National Capital Region.

    PubMed

    Varshney, Ekta; Saha, Nilanjan; Tandon, Monika; Shrivastava, Vikesh; Ali, Shakir

    2013-12-01

    Identification of polymorphism of cytochrome P450 2C9 (CYP2C9) enzymes in different ethnic populations is important to understand the differences in clinical responses to drugs. This study determines the CYP2C9 genetic polymorphism in Indian National Capital Region and correlates the phenotype-genotype. Losartan (25 mg) was administered to 107 volunteers to assess CYP2C9 activity, and, on the basis of results, volunteers were categorized as rapid and poor metabolizers. Molecular typing of CYP2C9*1 (wild type), CYP2C9*2, and CYP2C9*3 (the most common variant) was carried out by single-base primer extension technology for 37 subjects, of which 9 were poor metabolizers, and 28 were rapid metabolizers. 14.28 % of the studied population was identified as poor metabolizer for the category of drugs metabolized by CYP2C9. Significant difference was observed between the mean ratio (drug/metabolite) of poor (11.38 ± 5.88) and rapid (1.18 ± 1.11) drug metabolizers. The study suggests that phenotyping of CYP2C9 is desirable before enrollment of subjects for clinical trials or for deciding drug dose regimen as 14.28 % of study population was found to be poor metabolizer for the category of drugs metabolized by CYP2C9. This study establishes phenotype-genotype correlation, and proposes to use genotyping or phenotyping to evaluate the status of drug metabolizing capacity of CYP2C9 as a primary screening procedure before enrolling subjects in clinical trials or in clinical practice.

  6. Photoelectron Spectroscopy Study of [Ta2B6]-: a Hexagonal Bipyramdial Cluster

    NASA Astrophysics Data System (ADS)

    Jian, Tian; Li, Weili; Romanescu, Constantin; Wang, Lai-Sheng

    2014-06-01

    It has been a long-sought goal in cluster science to discover stable atomic clusters as building blocks for cluster-assembled nanomaterials, as exemplified by the fullerenes and their subsequent bulk syntheses.[1,2] Clusters have also been considered as models to understand bulk properties, providing a bridge between molecular and solid-state chemistry.[3] Herein we report a joint photoelectron spectroscopy and theoretical study on the [Ta2B6]- and [Ta2B6] clusters.[4] The photoelectron spectrum of [Ta2B6]- displays a simple spectral pattern and a large HOMO-LUMO gap, suggesting its high symmetry. Theoretical calculations show that both the neutral and anion are D6h pyramidal. The chemical bonding analyses for [Ta2B6] revealed the nature of the B6 and Ta interactions and uncovered strong covalent bonding between B6 and Ta. The D6h-[TaB6Ta] gaseous cluster is reminiscent of the structural pattern in the ReB6X6Re core in the [(Cp*Re)2B6H4Cl2] and the TiB6Ti motif in the newly synthesized Ti7Rh4Ir2B8 solid-state compound.[5,6] The current work provides an intrinsic link between a gaseous cluster and motifs for solid materials. Continued investigations of the transition-metal boron clusters may lead to the discovery of new structural motifs involving pure boron clusters for the design of novel boride materials. Reference [1] H.W. Kroto, J. R. Heath, S. C. OBrien, R. F. Curl, R. E. Smalley, Nature 1985, 318, 162 - 163. [2] W. Krtschmer, L. D. Lamb, K. Fostiropoulos, D. R. Huffman, Nature 1990, 347, 354 - 358. [3] T. P. Fehlner, J.-F. Halet, J.-Y. Saillard, Molecular Clusters: A Bridge to Solid-State Chemitry, Cambridge University Press, UK, 2007. [4] W. L. Li, L. Xie, T. Jian, C. Romanescu, X. Huang, L.-S. Wang, Angew. Chem. Int. Ed. 2014, 126, 1312 - 1316. [5] B. Le Guennic, H. Jiao, S. Kahlal, J.-Y. Saillard, J.-F. Halet, S. Ghosh, M. Shang, A. M. Beatty, A. L. Rheingold, T. P. Fehlner, J. Am. Chem. Soc. 2004, 126, 3203 - 3217. [6] B. P. T. Fokwa, M. Hermus, Angew

  7. The CYP2B6*6 allele significantly alters the N-demethylation of ketamine enantiomers in vitro.

    PubMed

    Li, Yibai; Coller, Janet K; Hutchinson, Mark R; Klein, Kathrin; Zanger, Ulrich M; Stanley, Nathan J; Abell, Andrew D; Somogyi, Andrew A

    2013-06-01

    Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 μM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 μM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

  8. Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole.

    PubMed

    Chan, Jeannine; Oshiro, Tyler; Thomas, Sarah; Higa, Allyson; Black, Stephen; Todorovic, Aleksandar; Elbarbry, Fawzy; Harrelson, John P

    2016-04-01

    Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (K(I) = 18.0 µM; k(inact) = 0.056 minute(-1)), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes).

  9. Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

    PubMed Central

    Chan, Jeannine; Oshiro, Tyler; Thomas, Sarah; Higa, Allyson; Black, Stephen; Todorovic, Aleksandar; Elbarbry, Fawzy

    2016-01-01

    Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 µM). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP2E1; IC50 values for P450s 1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 were 15.8-fold higher or more. t-CA is a type I ligand for CYP2A6 (KS = 14.9 µM). Inhibition of CYP2A6 by t-CA was metabolism-dependent; inhibition required NADPH and increased with time. Glutathione lessened the extent of inhibition modestly and statistically significantly. The carbon monoxide binding spectrum was dramatically diminished after exposure to NADPH and t-CA, suggesting degradation of the heme or CYP2A6 apoprotein. Using a static model and mechanism-based inhibition parameters (KI = 18.0 µM; kinact = 0.056 minute−1), changes in the area under the concentration-time curve (AUC) for nicotine and letrozole were predicted in the presence of t-CA (0.1 and 1 µM). The AUC fold-change ranged from 1.1 to 3.6. In summary, t-CA is a potential source of pharmacokinetic variability for CYP2A6 substrates due to metabolism-dependent inhibition, especially in scenarios when exposure to t-CA is elevated due to high dietary exposure, or when cinnamon is used as a treatment of specific disease states (e.g., diabetes). PMID:26851241

  10. Pharmacogenetic testing of CYP2C9 and VKORC1 alleles for warfarin.

    PubMed

    Flockhart, David A; O'Kane, Dennis; Williams, Marc S; Watson, Michael S; Flockhart, David A; Gage, Brian; Gandolfi, Roy; King, Richard; Lyon, Elaine; Nussbaum, Robert; O'Kane, Dennis; Schulman, Kevin; Veenstra, David; Williams, Marc S; Watson, Michael S

    2008-02-01

    American College of Medical Genetics statements and guidelines are designed primarily as an educational resource for medical geneticists and other health care professionals to help them provide quality medical genetic services. Adherence to these standards and guidelines does not necessarily ensure a successful medical outcome. These statements and guidelines should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the health care professional should apply his or her own professional judgment to the specific clinical circumstances presented by the individual patient or specimen. It may be prudent, however, to document in the patient's record the rationale for any significant deviation from these standards and guidelines. Warfarin (Coumadin) is a potent drug that when used judiciously and monitored closely, leads to substantial reductions in morbidity and mortality from thromboembolic events. However, even with careful monitoring, initiation of warfarin dosing is associated with highly variable responses between individuals and challenges achieving and maintaining levels within the narrow therapeutic range that can lead to adverse drug events. Variants of two genes, CYP2C9 and VKORC1, account for 30-50% of the variability in dosing of warfarin; thus, many believe that testing of these genes will aid in warfarin dosing recommendations. Evidence about this test is evolving rapidly, as is its translation into clinical practice. In an effort to address this situation, a multidisciplinary expert group was organized in November 2006 to evaluate the role of CYP2C9 and VKORC1 testing in altering warfarin-related therapeutic goals and reduction of adverse drug events. A recently completed Rapid-ACCE (Analytical, Clinical Validity, Clinical Utility, and Ethical, Legal, and Social Implications

  11. [Relationship of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced influenza-virus-associated encephalopathy].

    PubMed

    Funato, Tadao; Kozawa, Kanoko; Kaku, Mitsuo

    2002-02-01

    The mechanism causing influenza-virus-associated encephalopathy is unclear, even though diclofenac metabolites may induce this pathogenesis. CYP2C9 is known as the major cytochrome P450 gene product that catalyzes diclofenac in human liver. It is uncertain whether the mutation of CYP2C9 is associated the pharmacologic effects of diclofenac in influenza infection. Therefore, we applied a simple and rapid procedure involving real-time fluorescence allele-specific PCR(TaqMan-ASA) assay and denaturing HPLC assay to detect the mutation of CYP2C9 gene. A single-base mutation in the CYP2C9 gene was found in one of thirty subjects in the healthy population. We suggest that this mutation in the CYP2C9 gene may be related to diclofenac-induced influenza-virus-associated encephalopathy.

  12. Effect of CYP2B6*6 and CYP2C19*2 genotype on chlorpyrifos metabolism.

    PubMed

    Crane, Alice L; Klein, Kathrin; Zanger, Ulrich M; Olson, James R

    2012-03-11

    Chlorpyrifos (CPF) is a widely used organophosphorus (OP) pesticide. CPF is bioactivated by cytochrome P450s (CYPs) to the potent cholinesterase inhibitor chlorpyrifos oxon (CPF-O) or detoxified to 3,5,6-trichloro-2-pyridinol (TCPy). Human CYP2B6 has the highest reported Vmax)/Km (intrinsic clearance--CL(int)) for bioactivation while CYP2C19 has the highest reported CL(int) for detoxification of CPF. In this study, 22 human liver microsomes (HLMs) genotyped for common variants of these enzymes (CYP2B6*6 and CYP2C19*2) were incubated with 10 μM and 0.5 μM CPF and assayed for metabolite production. While no differences in metabolite production were observed in homozygous CYP2C19*2 HLMs, homozygous CYP2B6*6 specimens produced significantly less CPF-O than wild-type specimens at 10 μM (mean 144 and 446 pmol/min/mg, respectively). This correlated with reduced expression of CYP2B6 protein (mean 4.86 and 30.1 pmol/mg, for CYP2B6*6 and *1, respectively). Additionally, CYP2B6*1 and CYP2B6*6 were over-expressed in mammalian COS-1 cells to assess for the first time the impact of the CYP2B6*6 variant on the kinetic parameters of CPF bioactivation. The Vmax for CYP2B6*6 (1.05×10⁵ pmol/min/nmol CYP2B6) was significantly higher than that of CYP2B6*1 (4.13×10⁴ pmol/min/nmol CYP2B6) but the K(m) values did not differ (1.97 μM for CYP2B6*6 and 1.84 μM for CYP2B6*1) resulting in CL(int) rates of 53.5 and 22.5 nL/min/nmol CYP2B6 for *6 and *1, respectively. These data suggest that CYP2B6*6 has increased specific activity but reduced capacity to bioactivate CPF in HLMs compared to wild-type due to reduced hepatic protein expression, indicating that individuals with this genotype may be less susceptible to CPF toxicity.

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

    PubMed Central

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

    2013-01-01

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

  14. Clinical drugs undergoing polymorphic metabolism by human cytochrome P450 2C9 and the implication in drug development.

    PubMed

    He, S-M; Zhou, Z-W; Li, X-T; Zhou, S-F

    2011-01-01

    CYP2C9 metabolizes more than 100 clinically used drugs including phenytoin, S-warfarin, tolbutamide, glipizide, diclofenac, and losartan with varying contributions. CYP2C9 is considered one of the most important CYPs, with substrate specificity typical of many new chemical entities (i.e. lipophilic bases). A large interindividual variation has been identified for the CYP2C9 activity and for the clinical response to the therapeutics metabolised by the enzyme. So far, at least 33 variants of CYP2C9 (*2 through to *34) have been identified. CYP2C9 is one of the clinically significant drug metabolising enzymes that demonstrates genetic variants with significant phenotype and clinical outcomes. This review updates our current knowledge on the polymorphic metabolism of drugs by CYP2C9 and discusses its implications in drug development. The authors have searched through computer-based literatures by full text search in Medline (via Pubmed), ScienceDirect, Genetics Abstracts (CSA), SCOPUS, Chemical Abstracts, Current Contents Connect (ISI), Cochrance Library, CINAHL (EBSCO), CrossRef Search and Embase (all from inception to October 31 2010). A comprehensive literature search has identified 32 drugs that are subject to CYP2C9-mediated polymorphic metabolism. Drugs that are subject to polymorphic metabolism with clinical significance include nine nonsteroidal anti-inflammatory agents, six sulfonylurea antidiabetic drugs and, most critically, three oral coumarin anticoagulants. Polymorphisms in CYP2C9 have the potential to affect the clearance and clinical response of CYP2C9 substrate drugs with low therapeutic indices such as warfarin, phenytoin, and certain antidiabetic drugs. Warfarin has served as a model drug of how pharmacogenetics can be employed to achieve maximum efficacy and minimum toxicity. Minimizing interindividual variability in drug exposure due to CYP2C9 polymorphisms is an important goal in drug development and discovery.

  15. Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells.

    PubMed

    Li, Linhao; Li, Daochuan; Heyward, Scott; Wang, Hongbing

    2016-01-01

    CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

  16. New potent and selective cytochrome P450 2B6 (CYP2B6) inhibitors based on three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis

    PubMed Central

    Korhonen, L E; Turpeinen, M; Rahnasto, M; Wittekindt, C; Poso, A; Pelkonen, O; Raunio, H; Juvonen, R O

    2007-01-01

    Background and purpose: The cytochrome P450 2B6 (CYP2B6) enzyme metabolises a number of clinically important drugs. Drug-drug interactions resulting from inhibition or induction of CYP2B6 activity may cause serious adverse effects. The aims of this study were to construct a three-dimensional structure-activity relationship (3D-QSAR) model of the CYP2B6 protein and to identify novel potent and selective inhibitors of CYP2B6 for in vitro research purposes. Experimental approach: The inhibition potencies (IC50 values) of structurally diverse chemicals were determined with recombinant human CYP2B6 enzyme. Two successive models were constructed using Comparative Molecular Field Analysis (CoMFA). Key results: Three compounds proved to be very potent and selective competitive inhibitors of CYP2B6 in vitro (IC50<1 μM): 4-(4-chlorobenzyl)pyridine (CBP), 4-(4-nitrobenzyl)pyridine (NBP), and 4-benzylpyridine (BP). A complete inhibition of CYP2B6 activity was achieved with 0.1 μM CBP, whereas other CYP-related activities were not affected. Forty-one compounds were selected for further testing and construction of the final CoMFA model. The created CoMFA model was of high quality and predicted accurately the inhibition potency of a test set (n=7) of structurally diverse compounds. Conclusions and implications: Two CoMFA models were created which revealed the key molecular characteristics of inhibitors of the CYP2B6 enzyme. The final model accurately predicted the inhibitory potencies of several structurally unrelated compounds. CBP, BP and NBP were identified as novel potent and selective inhibitors of CYP2B6 and CBP especially is a suitable inhibitor for in vitro screening studies. PMID:17325652

  17. Characterization of CYP2B6 in a CYP2B6-humanized mouse model: inducibility in the liver by phenobarbital and dexamethasone and role in nicotine metabolism in vivo.

    PubMed

    Liu, Zhihua; Li, Lei; Wu, Hong; Hu, Jing; Ma, Jun; Zhang, Qing-Yu; Ding, Xinxin

    2015-02-01

    The aim of this study was to further characterize the expression and function of human CYP2B6 in a recently generated CYP2A13/2B6/2F1-transgenic (TG) mouse model, in which CYP2B6 is expressed selectively in the liver. The inducibility of CYP2B6 by phenobarbital (PB) and dexamethasone (DEX), known inducers of CYP2B6 in human liver, was examined in the TG mice, as well as in TG/Cyp2abfgs-null (or "CYP2B6-humanized") mice. Hepatic expression of CYP2B6 mRNA and protein was greatly induced by PB or DEX treatment in both TG and TG/Cyp2abfgs-null mice. Function of the transgenic CYP2B6 was first studied using bupropion as a probe substrate. In PB-treated mice, the rates of hepatic microsomal hydroxybupropion formation (at 50 μM bupropion) were >4-fold higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice (for both male and female mice); the rate difference was accompanied by a 5-fold higher catalytic efficiency in the TG/Cyp2abfgs-null mice and was abolished by an antibody to CYP2B6. The ability of CYP2B6 to metabolize nicotine was then examined, both in vitro and in vivo. The rates of hepatic microsomal cotinine formation from nicotine were significantly higher in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice, pretreated with PB or DEX. Furthermore, systemic nicotine metabolism was faster in TG/Cyp2abfgs-null than in Cyp2abfgs-null mice. Thus, the transgenic CYP2B6 was inducible and functional, and, in the absence of mouse CYP2A and CYP2B enzymes, it contributed to nicotine metabolism in vivo. The CYP2B6-humanized mouse will be valuable for studies on in vivo roles of hepatic CYP2B6 in xenobiotic metabolism and toxicity.

  18. Allele and genotype frequencies of CYP2B6 and CYP2C19 polymorphisms in Egyptian agricultural workers.

    PubMed

    Ellison, Corie A; Abou El-Ella, Soheir S; Tawfik, Maha; Lein, Pamela J; Olson, James R

    2012-01-01

    Genetic variability in cytochrome P-450 (CYP) has the potential to modify pharmacological and toxicological responses to many chemicals. Both CYP2B6 and CYP2C19 are pharmacologically and toxicologically relevant due to their ability to metabolize multiple drugs and environmental contaminants, including the organophosphorus (OP) pesticide chlorpyrifos. The aim of this study was to determine the prevalence of CYP2B6 and CYP2C19 variants in an indigenous Egyptian population (n = 120) that was shown to be occupationally exposed to chlorpyrifos. Further, the genotyping data was compared for Egyptians with previously studied populations to determine between population differences. Allelic frequencies were CYP2B6 1459C > T (3.8%), CYP2B6 785A > G (30.4%), CYP2B6 516G > T (28.8%), CYP2C19 681G > A (3.8%), and CYP2C19 431G > A (0%). The most prevalent CYP2B6 genotype combinations were CYP2B6 *1/*1 (44%), *1/*6 (38%), *6/*6 (8%), and *1/*5 (6%). The frequency of the CYP2C19 genotype combinations were CYP2C19 *1/*1 (93%), *1/*2 (6%), and *2/*2 (1%). The frequency of the CYP2B6 516G > T and CYP2B6 785A > G polymorphisms in this Egyptian cohort is similar to that found North American and European populations but significantly different from that reported for West African populations, while that of CYP2B6 1459C > T is similar to that found in Africans and African Americans. The observed frequency of CYP2C19 681G > A in Egyptians is similar to that of African pygmies but significantly different from other world populations, while CYP2C19 431 G > A was significantly different from that of African pygmies but similar to other world populations.

  19. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    PubMed Central

    Zhu, Ge-Jian; Yu, Ying-Nian; Li, Xin; Qian, Yu-Li

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 (CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. METHODS: After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography (HPLC). RESULTS: The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild typeCYP2C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 ± 0.109 μmol•min-1 ·g-1 S9 protein or 8.62 ± 2.02 mol•min-1 ·mol-1 CYP, but was undetectable in parental CHL cell. CONCLUSION: The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL-CYP2C9, efficiently expressing the protein of CYP2C9, was established. PMID:11925616

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2012-02-13

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

  2. CYP2B6 gene single-nucleotide polymorphisms in an Italian population sample and relationship with nicotine dependence.

    PubMed

    Riccardi, Laura Natalia; Carano, Francesco; Bini, Carla; Ceccardi, Stefania; Ferri, Gianmarco; Pelotti, Susi

    2015-02-01

    The extensively polymorphic CYP2B6 gene metabolizes endogenous and exogenous compounds, among which are nicotine and bupropion, although its contribution to the systemic metabolism of nicotine still remains controversial. In the present study, the distribution of the CYP2B6 variant and genotype frequencies were analyzed in a sample of 202 Italian individuals who were also invited to answer the Fagerström test for nicotine dependence (FTND), in an effort to assess the involvement of CYP2B6 polymorphisms in nicotine dependence. Eight single-nucleotide polymorphisms of CYP2B6 were tested and seven different variants were identified showing frequencies similar to the European population. The reduced activity of the CYP2B6*6 variant was significantly (p=0.025) distributed among the nicotine-dependent individuals compared to non-nicotine dependents. Also, the CYP2B6*1/*6 genotype achieved statistical significance (p=0.016) within the nicotine-dependent individuals. The high occurrence of CYP2B6*6 carriers among nicotine-dependent individuals may suggest a possible involvement in nicotine dependence, with a potential impact on smoking cessation treatments tailored to the individual smoker's genotype.

  3. Chlorpropamide 2-hydroxylation is catalysed by CYP2C9 and CYP2C19 in vitro: chlorpropamide disposition is influenced by CYP2C9, but not by CYP2C19 genetic polymorphism

    PubMed Central

    Shon, Ji-Hong; Yoon, Young-Ran; Kim, Min-Jung; Kim, Kyoung-Ah; Lim, Young-Chae; Liu, Kwang-Hyeon; Shin, Dong-Hoon; Lee, Chung Han; Cha, In-June; Shin, Jae-Gook

    2005-01-01

    Aims We evaluated the involvement of cytochrome P450 (CYP) isoforms 2C9 and 2C19 in chlorpropamide 2-hydroxylation in vitro and in chlorpropamide disposition in vivo. Methods To identify CYP isoforms(s) that catalyse 2-hydroxylation of chlorpropamide, the incubation studies were conducted using human liver microsomes and recombinant CYP isoforms. To evaluate whether genetic polymorphisms of CYP2C9 and/or CYP2C19 influence the disposition of chlorpropamide, a single oral dose of 250 mg chlorpropamide was administered to 21 healthy subjects pregenotyped for CYP2C9 and CYP2C19. Results In human liver microsomal incubation studies, the formation of 2-hydroxychlorpropamide (2-OH-chlorpropamide), a major chlorpropamide metabolite in human, has been best described by a one-enzyme model with estimated Km and Vmax of 121.7 ± 19.9 µm and 16.1 ± 5.0 pmol min−1 mg−1 protein, respectively. In incubation studies using human recombinant CYP isoforms, however, 2-OH-chlorpropamide was formed by both CYP2C9 and CYP2C19 with similar intrinsic clearances (CYP2C9 vs. CYP2C19: 0.26 vs. 0.22 µl min−1 nmol−1 protein). Formation of 2-OH-chlorpropamide in human liver microsomes was significantly inhibited by sulfaphenazole, but not by S-mephenytoin, ketoconazole, quinidine, or furafylline. In in vivo clinical trials, eight subjects with the CYP2C9*1/*3 genotype exhibited significantly lower nonrenal clearance [*1/*3 vs.*1/*1: 1.8 ± 0.2 vs. 2.4 ± 0.1 ml h−1 kg−1, P < 0.05; 95% confidence interval (CI) on the difference 0.2, 1.0] and higher metabolic ratios (of chlorpropamide/2-OH-chlorpropamide in urine: *1/*3 vs. *1/*1: 1.01 ± 0.19 vs. 0.56 ± 0.08, P < 0.05; 95% CI on the difference −0.9, −0.1) than did 13 subjects with CYP2C9*1/*1 genotype. In contrast, no differences in chlorpropamide pharmacokinetics were observed for subjects with the CYP2C19 extensive metabolizer vs. poor metabolizer genotypes. Conclusions These results suggest that chlorpropamide disposition is

  4. Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans

    PubMed Central

    Carlile, David J; Hakooz, Nancy; Bayliss, Martin K; Houston, J Brian

    1999-01-01

    Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) μl min−1 mg−1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1–5 μm is substantially lower than the value determined in microsomes based on total concentrations (56 μm), correction for the in vitro binding reduces this value to 20 μm and 6 μm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. PMID:10383540

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

    PubMed Central

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

    2010-01-01

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

  6. PREVALENCE OF COMBINATORIAL CYP2C9 AND VKORC1 GENOTYPES IN PUERTO RICANS: IMPLICATIONS FOR WARFARIN MANAGEMENT IN HISPANICS

    PubMed Central

    Duconge, Jorge; Cadilla, Carmen L.; Windemuth, Andreas; Kocherla, Mohan; Gorowski, Krystyna; Seip, Richard L.; Bogaard, Kali; Renta, Jessica Y.; Piovanetti, Paola; D’Agostino, Darrin; Santiago-Borrero, Pedro J.; Ruaño, Gualberto

    2010-01-01

    Polymorphisms in the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes significantly alter the effective warfarin dose. We determined the frequencies of alleles, single carriers, and double carriers of single nucleotide polymorphisms (SNPs) in the CYP2C9 and VKORC1 genes in a Puerto Rican cohort and gauged the impact of these polymorphisms on warfarin dosage using a published algorithm. A total of 92 DNA samples were genotyped using Luminex® x-MAP technology. The polymorphism frequencies were 6.52%, 5.43% and 28.8% for CYP2C9 *2, *3 and VKORC1-1639 G>A polymorphisms, respectively. The prevalence of combinatorial genotypes was 16% for carriers of both the CYP2C9 and VKORC1 polymorphisms, 9% for carriers of CYP2C9 polymorphisms, 35% for carriers of the VKORC1 polymorphism, and the remaining 40% were non-carriers for either gene. Based on a published warfarin dosing algorithm, single, double and triple carriers of functionally deficient polymorphisms predict reductions of 1.0–1.6, 2.0–2.9, and 2.9–3.7 mg/day, respectively, in warfarin dose. Overall, 60% of the population carried at least a single polymorphism predicting deficient warfarin metabolism or responsiveness and 13% were double carriers with polymorphisms in both genes studied. Combinatorial genotyping of CYP2C9 and VKORC1 can allow for individualized dosing of warfarin among patients with gene polymorphisms, potentially reducing the risk of stroke or bleeding. PMID:20073138

  7. Pharmacokinetics of chlorpheniramine, phenytoin, glipizide and nifedipine in an individual homozygous for the CYP2C9*3 allele.

    PubMed

    Kidd, R S; Straughn, A B; Meyer, M C; Blaisdell, J; Goldstein, J A; Dalton, J T

    1999-02-01

    Genetic polymorphisms in the cytochrome P450 (CYP) family are widely known to contribute to interindividual differences in the pharmacokinetics of many drugs. Several alleles for the CYP2C9 gene have been reported. Individuals homozygous for the Leu359 variant (CYP2C9*3) have been shown to have significantly lower drug clearances compared with Ile359 (CYP2C9*1) homozygous individuals. A male Caucasian who participated in six bioavailability studies in our laboratory over a period of several years showed extremely low clearance of two drugs: phenytoin and glipizide (both substrates of CYP2C9), but not for nifedipine (a CYP3A4 substrate) and chlorpheniramine (a CYP2D6 substrate). His oral clearance of phenytoin was 21% of the mean of the other 11 individuals participating in the study, and his oral clearance of glipizide, a second generation sulfonylurea structurally similar to tolbutamide, was only 188% of the mean of the other 10 individuals. However, his oral clearance of nifedipine and chlorpheniramine did not differ from individuals in other studies performed at our laboratories. An additional blood sample was obtained from this individual to determine if he possessed any of the known CYP2C9 or CYP2C19 allelic variants that would account for his poor clearance of the CYP2C9 substrates (phenytoin and glipizide) compared with the CYP3A4 (nifedipine) and CYP2D6 (chlorpheniramine) substrates. The results of the genotype testing showed that this individual was homozygous for the CYP2C9*3 allele and did not possess any of the known defective CYP2C19 alleles. This study establishes that the Leu359 mutation is responsible for the phenytoin and glipizide/tolbutamide poor metabolizer phenotype.

  8. Mechanism-Based Inactivation of Cytochrome P450 2B6 by Methadone through Destruction of Prosthetic Heme

    PubMed Central

    Amunugama, Hemali T.; Zhang, Haoming

    2012-01-01

    Methadone is a μ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a KI = 10.0 μM and kinact = 0.027 min−1. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone. PMID:22685215

  9. Mechanism-based inactivation of cytochrome P450 2B6 by methadone through destruction of prosthetic heme.

    PubMed

    Amunugama, Hemali T; Zhang, Haoming; Hollenberg, Paul F

    2012-09-01

    Methadone is a μ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a K(I) = 10.0 μM and k(inact) = 0.027 min⁻¹. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone.

  10. NCOA6 differentially regulates the expression of the CYP2C9 and CYP3A4 genes

    PubMed Central

    Surapureddi, Sailesh; Rana, Ritu; Goldstein, Joyce A

    2011-01-01

    CYP2Cs and CYP3A4 sub family of enzymes of the Cytochrome P-450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR-HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors. PMID:21292004

  11. Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity.

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Ohnishi, Ryoko; Hatano, Tsutomu

    2010-01-01

    Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug-food or drug-herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver-Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver-Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC(50) values of 0.07 and 0.03 microM, respectively. The K(i) value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.

  12. High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6.

    PubMed

    Twist, Greyson P; Gaedigk, Roger; Leeder, J Steven; Gaedigk, Andrea

    2013-06-01

    High-resolution melt (HRM) analysis using 'release-on-demand' dyes, such as EvaGreen(®) has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African-Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

  13. Adverse events in a newborn on valproate therapy due to loss-of-function mutations in CYP2C9

    PubMed Central

    Nagy, Andrea; Bűdi, Tamás; Temesvári, Manna; Szever, Zsuzsa; Szabó, Pál Tamás; Monostory, Katalin

    2015-01-01

    An increased risk of valproate-induced toxicity has been reported in children, particularly in those younger than 2 years of age. Significant variations in valproate pharmacokinetics and shifts in the metabolic pathways towards CYP2C9-dependent metabolism seem to play some role in the age-related differences in the incidence of adverse events. We present the case of a premature patient with moderate hemorrhage in the subependymal region (grade II — intraventricular hemorrhage without ventricular dilatation), several myoclonic episodes in her right upper arm (series of jerks lasting milliseconds), and epileptiform abnormalities on the EEG (localized spike-and-wave in the left frontal region with preserved background activity who was treated with valproate. Serious side effects, consisting of bone marrow depression, hyperammonemia, and serum alkaline phosphatase elevation, were observed seventeen days after the beginning of valproate therapy. The toxic symptoms were likely the consequence of a reduced ability to metabolize valproate. The patient was demonstrated to carry two loss-of-function mutations in CYP2C9 (CYP2C9*3/*3) resulting in exaggerated blood concentrations of valproate. The present case highlights the importance of assaying inborn errors in CYP2C9 gene in pediatric patients to avoid valproate-evoked serious side effects. PMID:26543813

  14. Pharmacogenetic relevance of the CYP2C9*3 allele in a tenoxicam bioequivalence study performed on Spaniards.

    PubMed

    Peiró, A M; Novalbos, J; Zapater, P; Moreu, R; López-Rodríguez, R; Rodríguez, V; Abad-Santos, F; Horga, J F

    2009-01-01

    We performed a study to quantify CYP2C9 and CYP2C8 alleles influence on the variability observed in tenoxicam pharmacokinetic (PK) and implication in a bioequivalence study design performed on Spaniards. Eighteen healthy volunteers were included in an open, randomized, crossover, phase I bioequivalence study. Significant increases were found in CYP2C9*3 alleles vs. *1 and *2 in AUC(0-infinity) (median (min-max)): 256 (230-516) vs. 150 (100-268) and 169 (124-197) microg h/mL (p<0.01) and half-life time (t1/2) 102 (79-36) vs. 56 (45-94) and 64 (60-80)h (p<0.01). Non-significant differences were observed in C(max) 1.9 (1.8-2.9) vs. 2.4 (1.7-3.4), 2.5 (1.6-2.9) microg/mL or in according to CYP2C8 alleles presence. CYP2C9*3 allele is associated to a longer elimination time of tenoxicam. PK parameters calculated in bioequivalence studies (AUC(0-infinity), t1/2) may be influenced by the presence of CYP2C9*3 allele resulting in a high variability. Thus, bioequivalence studies of tenoxicam formulations should be designed considering genotype profile.

  15. Prediction of Warfarin Dose Reductions in Puerto Rican Patients, Based on Combinatorial CYP2C9 and VKORC1 Genotypes

    PubMed Central

    Valentin, Isa Ivette; Vazquez, Joan; Rivera-Miranda, Giselle; Seip, Richard L; Velez, Meredith; Kocherla, Mohan; Bogaard, Kali; Cruz-Gonzalez, Iadelisse; Cadilla, Carmen L; Renta, Jessica Y; Felliu, Juan F; Ramos, Alga S; Alejandro-Cowan, Yirelia; Gorowski, Krystyna; Ruaño, Gualberto; Duconge, Jorge

    2012-01-01

    BACKGROUND The influence of CYP2C9 and VKORC1 polymorphisms on warfarin dose has been investigated in white, Asian, and African American populations but not in Puerto Rican Hispanic patients. OBJECTIVE To test the associations between genotypes, international normalized ratio (INR) measurements, and warfarin dosing and gauge the impact of these polymorphisms on warfarin dose, using a published algorithm. METHODS A retrospective warfarin pharmacogenetic association study in 106 Puerto Rican patients was performed. DNA samples from patients were assayed for 12 variants in both CYP2C9 and VKORC1 loci by HILOmet PhyzioType assay. Demographic and clinical nongenetic data were retrospectively collected from medical records. Allele and genotype frequencies were determined and Hardy-Weinberg equilibrium (HWE) was tested. RESULTS Sixty-nine percent of patients were carriers of at least one polymorphism in either the CYP2C9 or the VKORC1 gene. Double, triple, and quadruple carriers accounted for 22%, 5%, and 1%, respectively. No significant departure from HWE was found. Among patients with a given CYP2C9 genotype, warfarin dose requirements declined from GG to AA haplotypes; whereas, within each VKORC1 haplotype, the dose decreased as the number of CYP2C9 variants increased. The presence of these loss-of-function alleles was associated with more out-of-range INR measurements (OR = 1.38) but not with significant INR >4 during the initiation phase. Analyses based on a published pharmacogenetic algorithm predicted dose reductions of up to 4.9 mg/day in carriers and provided better dose prediction in an extreme subgroup of highly sensitive patients, but also suggested the need to improve predictability by developing a customized model for use in Puerto Rican patients. CONCLUSIONS This study laid important groundwork for supporting a prospective pharmacogenetic trial in Puerto Ricans to detect the benefits of incorporating relevant genomic information into a customized DNA

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

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Hatano, Tsutomu

    2010-01-01

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

  17. Polymorphisms of VKORC1 and CYP2C9 are associated with warfarin sensitivity in Chinese population

    PubMed Central

    Jia, LiQun; Wang, Zanxin; Men, Jianlong; Cai, Heng; Wei, Minxin

    2017-01-01

    Objective Warfarin is a commonly prescribed anticoagulant for prevention of thromboembolic events. Wide inter-individual dose variation, narrow therapeutic range and risk of serious bleeding result in difficulties in achieving the therapeutic effect. The present study was designed to clarify the real biological significance of the polymorphisms of VKORC1 and cytochrome P450 2C9 (CYP2C9) in warfarin metabolism. Methods A total of 214 patients with warfarin anticoagulant therapy were selected. During follow-up of anticoagulation, warfarin dosage and associated international normalized ratio values were recorded. Genetic polymorphisms of VKORC1 promoter and from exon 1 to exon 3 and CYP2C9 exon 4 sequence were detected by polymerase chain reaction and gene sequencing. Results Five polymorphisms were identified in this research, which were VKORC1 1173C>T (intron 1), 1542G>C (intron 2), 2255C>T (intron 2), 3730C>T (3′-downstream) and CYP2C9 exon 4 −65G>C. VKORC1 1173CT, 1542GC, 2255CT and 3730CT polymorphisms were detected in same patients, but CYP2C9 exon 4 −65GC carriers were different from them. VKORC1 1173CT, 1542GC, 2255CT, 3730CT carriers and CYP2C9 exon 4 −65GC carriers had significantly higher warfarin daily dosage than others (3.2±0.6 vs 3.1±1.1 vs 2.6±0.8 mg/day). Logistic regression analysis revealed VKORC1 1173CT, 1542GC, 2255CT, 3730CT carrier status (odds ratio [OR] =3.233, 95% confidence interval [CI]: 1.259–8.303, P=0.015) and obesity with body mass index >27 kg/m2 (OR =1.223, 95% CI: 1.097–1.363, P<0.001) to have independent and statistically significant contributions to high warfarin dosage. Conclusion In general, in VKORC1 1173CT, 1542GC, 2255CT and 3730CT carriers and in obese patients, warfarin maintenance doses were significantly higher than in the others.

  18. 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.

  19. CYP2B6 SNPs are associated with methadone dose required for effective treatment of opioid addiction.

    PubMed

    Levran, Orna; Peles, Einat; Hamon, Sara; Randesi, Matthew; Adelson, Miriam; Kreek, Mary Jeanne

    2013-07-01

    Adequate methadone dosing in methadone maintenance treatment (MMT) for opioid addiction is critical for therapeutic success. One of the challenges in dose determination is the inter-individual variability in dose-response. Methadone metabolism is attributed primarily to cytochrome P450 enzymes CYP3A4, CYP2B6 and CYP2D6. The CYP2B6*6 allele [single nucleotide polymorphisms (SNPs) 785A>G (rs2279343) and 516G>T (rs3745274)] was associated with slow methadone metabolism. To explore the effects of CYP2B6*6 allele on methadone dose requirement, it was genotyped in a well-characterized sample of 74 Israeli former heroin addicts in MMT. The sample is primarily of Middle Eastern/European ancestry, based on ancestry informative markers (AIMs). Only patients with no major co-medication that may affect methadone metabolism were included. The stabilizing daily methadone dose in this sample ranges between 13 and 260mg (mean 140±52mg). The mean methadone doses required by subjects homozygous for the variant alleles of the CYP2B6 SNPs 785A>G and 516G>T (88, 96mg, respectively) were significantly lower than those of the heterozygotes (133, 129mg, respectively) and the non-carriers (150, 151mg, respectively) (nominal P=0.012, 0.048, respectively). The results remain significant after controlling for age, sex and the ABCB1 SNP 1236C>T (rs1128503), which was previously shown to be associated with high methadone dose requirement in this population (P=0.006, 0.030, respectively). An additional 77 CYP2B6, CYP3A4 and CYP2D6 SNPs were genotyped. Of these, 24 SNPs were polymorphic and none showed significant association with methadone dose. Further studies are necessary to replicate these preliminary findings in additional subjects and other populations.

  20. VKORC1 and CYP2C9 polymorphisms related to adverse events in case-control cohort of anticoagulated patients

    PubMed Central

    Misasi, Silvia; Martini, Giuliana; Paoletti, Oriana; Calza, Stefano; Scovoli, Giovanni; Marengoni, Alessandra; Testa, Sophie; Caimi, Luigi; Marchina, Eleonora

    2016-01-01

    Abstract Vitamin K antagonists (VKAs) are highly effective but have a narrow therapeutic index and require routine monitoring of the INR. The primary aim of pharmacogenetics (PGx) is to optimize patient care, achieving drug treatments that are personalized according to the genetic profile of each patient. The best-characterized genes involved in VKA PGx involve pharmacokinetics (VKORC1) and pharmacodynamics (CYP2C9) of VKA metabolism. The role of these genes in clinical outcomes (bleeding and thrombosis) during oral anticoagulant (OAC) therapy is controversial. The aim of the present study was to evaluate any potential association between genotype VKORC1 and CYP2C9 and adverse events (hemorrhagic and/or thrombotic), during initiation and long-term VKA treatment, in Caucasian patients. Furthermore, we aimed to determine if the concomitant prescription of other selected drugs affected the association between genotype and adverse events. We performed a retrospective, matched case-control study to determine associations between multiple gene variants, drug intake, and any major adverse effects in anticoagulated patients, monitored in 2 Italian anticoagulation clinics. Our results show that anticoagulated patients have a high risk of adverse events if they are carriers of 1 or more genetic polymorphisms in the VKORC1 (rs9923231) and CYP2C9 (rs1799853 and rs1057910) genes. Information on CYP2C9 and VKORC1 variants may be useful to identify individualized oral anticoagulant treatment for each patient, improve management and quality of VKA anticoagulation control, and monitor drug surveillance in pharmacovigilance programs. PMID:28033245

  1. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    PubMed

    Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

    2013-12-01

    Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

  2. [FREQUENCY OF POLYMORPHISM OF VKORC1 AND CYP2C9 GENES IN TWO REGIONS OF GEORGIA].

    PubMed

    Jokhadze, T; Kakauridze, N; Buadze, T; Gaiozishvili, M; Lezhava, T

    2016-01-01

    The aim of the research was to study the frequency of VKROC1 and CYP2C9 genes different alleles for healthy donors and for patients with thrombosis, in two regions of Georgia - in Samegrelo and in Tbilisi and to reveal the interdependence of the studied genes products in the treatment of thrombosis with warfarin. Warfarin is an anticoagulant, causing the inactivation of the VKORC1 gene product, which is one of the clotting factors. The protein product of CYP2C9 gene is involved in the metabolism of warfarin. Genotyping of blood samples for studied genes alleles was carried out using a tube scanner (ESE Quant Tube Scaner), allowing to identify SNPs. In the studied group of patients with thrombosis from Samegrelo region the wild-type homozygotes by the gene VKORC1 were - 90%; heterozygotes - 10%; mutant homozygotes have not met at all. In the studied group of patients with thrombosis from Tbilisi, also predominated homozygous wild type (60%); heterozygotes were - 40%; mutant homozygotes were not met. The genotypes of healthy donors fromTbilisi does not differed from the same indicator of of Samegrelo (homozygous "wild" AA - 37%; genotype AB - 47%; and mutant genotype - BB - 16%). In patients with thrombosis, from Samegrelo, wild-tipe homozygotes and heterozygotes by CYP2C9 gene were almost the same rate (51% and 49% -, respectively); mutant homozygotes were not revealed. In patients from Tbilisi, the frequency of wild-type homozygotes was 70%, heterozygotes and mutant homozygotes was 20% and 10% - respectively. The ratio of the frequencies of CYP2C9 gene alleles in healthy donors from Tbilisi and Samegrelo is not different - wild-type homozygotes - 77%; heterozygotes - 23%; mutant homozygotes in both regions were not met. VKORC1 and / or CYP2C9 genes polymorphisms are presented in a number of clinical dosing algorithms and in prospective clinical trials. It is revealed the significant variation of genotypes in patients with thrombosis (in both studied regions), which

  3. The effect of ritonavir on human CYP2B6 catalytic activity: heme modification contributes to the mechanism-based inactivation of CYP2B6 and CYP3A4 by ritonavir.

    PubMed

    Lin, Hsia-lien; D'Agostino, Jaime; Kenaan, Cesar; Calinski, Diane; Hollenberg, Paul F

    2013-10-01

    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 K(I) of 0.9 μM, a k(inact) 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 (K(i) = 0.33 μM) and a type II ligand (spectral dissociation constant-K(s) = 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.

  4. CCAAT/Enhancer-binding Protein α (C/EBPα) and Hepatocyte Nuclear Factor 4α (HNF4α) Synergistically Cooperate with Constitutive Androstane Receptor to Transactivate the Human Cytochrome P450 2B6 (CYP2B6) Gene

    PubMed Central

    Benet, Marta; Lahoz, Agustín; Guzmán, Carla; Castell, José V.; Jover, Ramiro

    2010-01-01

    The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/enhancer-binding protein α (C/EBPα), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor, the CYP2B6 expression and inducibility by CITCO are restored in human hepatoma HepG2 cells at levels similar to those in cultured human hepatocytes. Moreover, several other phase I and II genes are simultaneously activated, which suggests that this is an effective approach to endow dedifferentiated human hepatoma cells with a particular metabolic competence and response to inducers. In order to gain insight into the molecular mechanism, we examined the cooperation of these three transcription factors on the CYP2B6 5′-flanking region. We show new CYP2B6-responsive sequences for C/EBPα and HNF4α and a novel synergistic regulatory mechanism whereby C/EBPα, HNF4α, and constitutive androstane receptor bind and cooperate through proximal and distal response elements to confer a maximal level of expression. The results obtained from human liver also suggest that important differences in the expression and binding of C/EBPα and HNF4α could account for the large interindividual variability of the hepatic CYP2B6 enzyme, which metabolizes commonly used drugs. PMID:20622021

  5. Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation.

    PubMed

    Pearce, Robin E; Gaedigk, Roger; Twist, Greyson P; Dai, Hongying; Riffel, Amanda K; Leeder, J Steven; Gaedigk, Andrea

    2016-07-01

    Although CYP2B6 catalyzes the biotransformation of many drugs used clinically for children and adults, information regarding the effects of development on CYP2B6 expression and activity are scarce. Utilizing a large panel of human liver samples (201 donors: 24 fetal, 141 pediatric, and 36 adult), we quantified CYP2B6 mRNA and protein expression levels, characterized CYP2B6 (bupropion hydroxylase) activity in human liver microsomes (HLMs), and performed an extensive genotype analysis to differentiate CYP2B6 haplotypes such that the impact of genetic variation on these parameters could be assessed. Fetal livers contained extremely low levels of CYP2B6 mRNA relative to postnatal samples and fetal HLMs did not appear to catalyze bupropion hydroxylation; however, fetal CYP2B6 protein levels were not significantly different from postnatal levels. Considerable interindividual variation in CYP2B6 mRNA expression, protein levels, and activity was observed in postnatal HLMs (mRNA, ∼40,000-fold; protein, ∼300-fold; activity, ∼600-fold). The extremely wide range of interindividual variability in CYP2B6 expression and activity was significantly associated with age (P < 0.01) following log transformation of the data. Our data suggest that CYP2B6 activity appears as early as the first day of life, increases through infancy, and by 1 year of age, CYP2B6 levels and activity may approach those of adults. Surprisingly, CYP2B6 interindividual variability was not significantly associated with genetic variation in CYP2B6, nor was it associated with differences in gender or ethnicity, suggesting that factors other than these are largely responsible for the wide range of variability in CYP2B6 expression and activity observed among a large group of individuals/samples.

  6. CYP2B6 pharmacogenetics-based in vitro-in vivo extrapolation of efavirenz clearance by physiologically based pharmacokinetic modeling.

    PubMed

    Xu, Cong; Quinney, Sara K; Guo, Yingying; Hall, Stephen D; Li, Lang; Desta, Zeruesenay

    2013-12-01

    Efavirenz is mainly cleared by CYP2B6. The CYP2B6*6 allele is associated with lower efavirenz clearance. Efavirenz clearance was predictable using in vitro data for carriers of the CYP2B6*1/*1 genotype, but the prediction in carriers of the CYP2B6*6 allele was poor. To test the hypothesis that incorporation of mechanism of reduced efavirenz metabolism by the CYP2B6*6 allele can predict the genetic effect on efavirenz pharmacokinetics, in vitro-in vivo extrapolation of efavirenz clearance was performed by physiologically based pharmacokinetic modeling (Simcyp Simulator; Simcyp Ltd., Sheffield, UK) using data obtained from expressed CYP2B6.1 and CYP2B6.6 as well as human liver microsomes (HLMs) with CYP2B6*1/*1, *1/*6, and *6/*6 genotypes. Simulated pharmacokinetics of a single 600-mg oral dose of efavirenz for individuals with each genotype was compared with data observed in healthy subjects genotyped for the CYP2B6*6 allele (n = 20). Efavirenz clearance for carriers of the CYP2B6*1/*1 genotype was predicted reasonably well using HLM data, but the clearance in carriers of the CYP2B6*6 allele was underpredicted using both expressed and HLM systems. Improved prediction of efavirenz clearance was obtained from expressed CYP2B6 after recalculating intersystem extrapolation factors for CYP2B6.1 and CYP2B6.6 based on in vitro intrinsic clearance of bupropion 4-hydroxylation. These findings suggest that genetic effect on both CYP2B6 protein expression and catalytic efficiency needs to be taken into account for the prediction of pharmacokinetics in individuals carrying the CYP2B6*6/*6 genotype. Expressed CYP2B6 proteins may be a reliable in vitro system to predict effect of the CYP2B6*6 allele on the metabolism of CYP2B6 substrates.

  7. CYP2B6 Pharmacogenetics–Based In Vitro–In Vivo Extrapolation of Efavirenz Clearance by Physiologically Based Pharmacokinetic Modeling

    PubMed Central

    Xu, Cong; Quinney, Sara K.; Guo, Yingying; Hall, Stephen D.; Li, Lang

    2013-01-01

    Efavirenz is mainly cleared by CYP2B6. The CYP2B6*6 allele is associated with lower efavirenz clearance. Efavirenz clearance was predictable using in vitro data for carriers of the CYP2B6*1/*1 genotype, but the prediction in carriers of the CYP2B6*6 allele was poor. To test the hypothesis that incorporation of mechanism of reduced efavirenz metabolism by the CYP2B6*6 allele can predict the genetic effect on efavirenz pharmacokinetics, in vitro–in vivo extrapolation of efavirenz clearance was performed by physiologically based pharmacokinetic modeling (Simcyp Simulator; Simcyp Ltd., Sheffield, UK) using data obtained from expressed CYP2B6.1 and CYP2B6.6 as well as human liver microsomes (HLMs) with CYP2B6*1/*1, *1/*6, and *6/*6 genotypes. Simulated pharmacokinetics of a single 600-mg oral dose of efavirenz for individuals with each genotype was compared with data observed in healthy subjects genotyped for the CYP2B6*6 allele (n = 20). Efavirenz clearance for carriers of the CYP2B6*1/*1 genotype was predicted reasonably well using HLM data, but the clearance in carriers of the CYP2B6*6 allele was underpredicted using both expressed and HLM systems. Improved prediction of efavirenz clearance was obtained from expressed CYP2B6 after recalculating intersystem extrapolation factors for CYP2B6.1 and CYP2B6.6 based on in vitro intrinsic clearance of bupropion 4-hydroxylation. These findings suggest that genetic effect on both CYP2B6 protein expression and catalytic efficiency needs to be taken into account for the prediction of pharmacokinetics in individuals carrying the CYP2B6*6/*6 genotype. Expressed CYP2B6 proteins may be a reliable in vitro system to predict effect of the CYP2B6*6 allele on the metabolism of CYP2B6 substrates. PMID:23846872

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

    PubMed

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

    2015-04-01

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

  9. Metformin represses drug-induced expression of CYP2B6 by modulating the constitutive androstane receptor signaling.

    PubMed

    Yang, Hui; Garzel, Brandy; Heyward, Scott; Moeller, Timothy; Shapiro, Paul; Wang, Hongbing

    2014-02-01

    Metformin is currently the most widely used drug for the treatment of type 2 diabetes. Mechanistically, metformin interacts with many protein kinases and transcription factors that alter the expression of numerous downstream target genes governing lipid metabolism, cell proliferation, and drug metabolism. The constitutive androstane receptor (CAR, NR1i3), a known xenobiotic sensor, has recently been recognized as a novel signaling molecule, in that its activation could be regulated by protein kinases in addition to the traditional ligand binding. We show that metformin could suppress drug-induced expression of CYP2B6 (a typical target gene of CAR) by modulating the phosphorylation status of CAR. In human hepatocytes, metformin robustly suppressed the expression of CYP2B6 induced by both indirect (phenobarbital) and direct CITCO [6-(4-chlorophenyl)imidazo[2,1-b]1,3thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime] activators of human CAR. Mechanistic investigation revealed that metformin specifically enhanced the phosphorylation of threonine-38 of CAR, which blocks CAR nuclear translocation and activation. Moreover, we showed that phosphorylation of CAR by metformin was primarily an AMP-activated protein kinase- and extracellular signal-regulated kinase 1/2-dependent event. Additional two-hybrid and coimmunoprecipitation assays demonstrated that metformin could also disrupt CITCO-mediated interaction between CAR and the steroid receptor coactivator 1 or the glucocorticoid receptor-interacting protein 1. Our results suggest that metformin is a potent repressor of drug-induced CYP2B6 expression through specific inhibition of human CAR activation. Thus, metformin may affect the metabolism and clearance of drugs that are CYP2B6 substrates.

  10. Cytochrome P450 2C9 *2 and *3 polymorphisms and the dose and effect of sulfonylurea in type II diabetes mellitus.

    PubMed

    Becker, M L; Visser, L E; Trienekens, P H; Hofman, A; van Schaik, R H N; Stricker, B H Ch

    2008-02-01

    Sulfonylurea hypoglycemics are mainly metabolized by the cytochrome P450 2C9 (CYP2C9) enzyme. The CYP2C9*2 and *3 polymorphisms encode proteins with less enzymatic activity and are correlated with elevated serum levels of sulfonylurea, as demonstrated in healthy volunteers. In this study, the effect of these variants is described for patients with diabetes mellitus treated with sulfonylurea. Associations between CYP2C9 polymorphisms, prescribed doses of sulfonylurea, and change in glucose levels after the start of sulfonylurea therapy were assessed in all patients with incident diabetes mellitus starting on sulfonylurea therapy in the Rotterdam Study, a population-based cohort study of 7,983 elderly people. In CYP2C9*3 allele carriers using tolbutamide, the prescribed dose was lower compared to patients with the wild-type CYP2C9 genotype. No differences in the prescribed dose were found in tolbutamide users with the CYP2C9*1/*2 or CYP2C9*2/*2 genotype compared to wild-type patients or in patients using other sulfonylurea. In CYP2C9*3 allele carriers, the mean decrease in fasting serum glucose levels after the start of tolbutamide therapy was larger than in patients with the wild-type genotype, although not statistically significant. Patients with diabetes mellitus who are carriers of a CYP2C9*3 allele require lower doses of tolbutamide to regulate their serum glucose levels compared to patients with the wild-type genotype.

  11. In silico prediction of efavirenz and rifampicin drug–drug interaction considering weight and CYP2B6 phenotype

    PubMed Central

    Rekić, Dinko; Röshammar, Daniel; Mukonzo, Jackson; Ashton, Michael

    2011-01-01

    AIMS This study aimed to test whether a pharmacokinetic simulation model could extrapolate nonclinical drug data to predict human efavirenz exposure after single and continuous dosing as well as the effects of concomitant rifampicin and further to evaluate the weight-based dosage recommendations used to counteract the rifampicin–efavirenz interaction. METHODS Efavirenz pharmacokinetics were simulated using a physiologically based pharmacokinetic model implemented in the Simcyp™ population-based simulator. Physicochemical and metabolism data obtained from the literature were used as input for prediction of pharmacokinetic parameters. The model was used to simulate the effects of rifampicin on efavirenz pharmacokinetics in 400 virtual patients, taking into account bodyweight and CYP2B6 phenotype. RESULTS Apart from the absorption phase, the simulation model predicted efavirenz concentration–time profiles reasonably well, with close agreement with clinical data. The simulated effects of rifampicin co-administration on efavirenz treatment showed only a minor decrease of 16% (95% confidence interval 13–19) in efavirenz area under the concentration–time curve, of the same magnitude as what has been clinically observed (22%). Efavirenz exposure depended on CYP2B6 phenotype and bodyweight. Increasing the efavirenz dose during concomitant rifampicin was predicted to be most successful in patients over 50 kg regardless of CYP2B6 status. CONCLUSIONS Our findings, although based on a simulation approach using limited in vitro data, support the current recommendations for using a 50 kg bodyweight cut-off for efavirenz dose increment when co-treating with rifampicin. PMID:21395646

  12. Generation and characterization of a CYP2A13/2B6/2F1-transgenic mouse model.

    PubMed

    Wei, Yuan; Wu, Hong; Li, Lei; Liu, Zhihua; Zhou, Xin; Zhang, Qing-Yu; Weng, Yan; D'Agostino, Jaime; Ling, Guoyu; Zhang, Xiuling; Kluetzman, Kerri; Yao, Yunyi; Ding, Xinxin

    2012-06-01

    CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (∼100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (∼0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (∼0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

  13. Q172H replacement overcomes effects on the metabolism of cyclophosphamide and efavirenz caused by CYP2B6 variant with Arg262.

    PubMed

    Ariyoshi, Noritaka; Ohara, Miyuki; Kaneko, Mayumi; Afuso, Sakino; Kumamoto, Takuya; Nakamura, Hiroyoshi; Ishii, Itsuko; Ishikawa, Tsutomu; Kitada, Mitsukazu

    2011-11-01

    There are a number of reports indicating that CYP2B6*6 (c.516G>T and c.785A>G) is responsible for decreased clearance of efavirenz (EFV), although increased disposition of cyclophosphamide (CPA) in individuals with this polymorphism was observed. Thus, we hypothesized that the effects of the two single nucleotide polymorphisms (SNPs) of CYP2B6*6 on the metabolism of drugs might be considerably different between these two agents. To clarify this possibility, we expressed two major variants of this enzyme, CYP2B6.6 (Q172H and K262R) and CYP2B6.4 (K262R), and investigated metabolic activities of these variants toward EFV and CPA. Kinetic analyses clearly indicated that CYP2B6.4 possessed enhanced metabolic activity toward EFV compared with that of the wild-type enzyme (CYP2B6.1), whereas CPA was metabolized less efficiently by CYP2B6.4 than by CYP2B6.1. On the other hand, CYP2B6.6 showed a completely opposite character, suggesting that Q172H gives inverse effects on metabolic activities of CYP2B6 affected by K262R. Although it is recognized that effects of amino acid change in cytochrome P450 on the metabolic activity depend on substrates, this study revealed SNPs giving an opposite effect on the metabolism of two clinically important drugs currently used. Furthermore, this study provides the first evidence that Q172H can reverse the direction of the effect caused by K262R in CYP2B6 on the metabolism of certain drugs.

  14. Evaluation of CYP2B6 Induction and Prediction of Clinical Drug-Drug Interactions: Considerations from the IQ Consortium Induction Working Group-An Industry Perspective.

    PubMed

    Fahmi, Odette A; Shebley, Mohamad; Palamanda, Jairam; Sinz, Michael W; Ramsden, Diane; Einolf, Heidi J; Chen, Liangfu; Wang, Hongbing

    2016-10-01

    Drug-drug interactions (DDIs) due to CYP2B6 induction have recently gained prominence and clinical induction risk assessment is recommended by regulatory agencies. This work aimed to evaluate the potency of CYP2B6 versus CYP3A4 induction in vitro and from clinical studies and to assess the predictability of efavirenz versus bupropion as clinical probe substrates of CYP2B6 induction. The analysis indicates that the magnitude of CYP3A4 induction was higher than CYP2B6 both in vitro and in vivo. The magnitude of DDIs caused by induction could not be predicted for bupropion with static or dynamic models. On the other hand, the relative induction score, net effect, and physiologically based pharmacokinetics SimCYP models using efavirenz resulted in improved DDI predictions. Although bupropion and efavirenz have been used and are recommended by regulatory agencies as clinical CYP2B6 probe substrates for DDI studies, CYP3A4 contributes to the metabolism of both probes and is induced by all reference CYP2B6 inducers. Therefore, caution must be taken when interpreting clinical induction results because of the lack of selectivity of these probes. Although in vitro-in vivo extrapolation for efavirenz performed better than bupropion, interpretation of the clinical change in exposure is confounded by the coinduction of CYP2B6 and CYP3A4, as well as the increased contribution of CYP3A4 to efavirenz metabolism under induced conditions. Current methods and probe substrates preclude accurate prediction of CYP2B6 induction. Identification of a sensitive and selective clinical substrate for CYP2B6 (fraction metabolized > 0.9) is needed to improve in vitro-in vivo extrapolation for characterizing the potential for CYP2B6-mediated DDIs. Alternative strategies and a framework for evaluating the CYP2B6 induction risk are proposed.

  15. Acenocoumarol sensitivity and pharmacokinetic characterization of CYP2C9 *5/*8,*8/*11,*9/*11 and VKORC1*2 in black African healthy Beninese subjects.

    PubMed

    Allabi, Aurel Constant; Horsmans, Yves; Alvarez, Jean-Claude; Bigot, André; Verbeeck, Roger K; Yasar, Umit; Gala, Jean-Luc

    2012-06-01

    This study aimed at investigating the contribution of CYP2C9 and VKORC1 genetic polymorphisms to inter-individual variability of acenocoumarol pharmacokinetics and pharmacodynamics in Black Africans from Benin. Fifty-one healthy volunteers were genotyped for VKORC1 1173C>T polymorphism. All of the subjects had previously been genotyped for CYP2C9*5, CYP2C9*6, CYP2C9*8, CYP2C9*9 and CYP2C9*11 alleles. Thirty-six subjects were phenotyped with a single 8 mg oral dose of acenocoumarol by measuring plasma concentrations of (R)- and (S)-acenocoumarol 8 and 24 h after the administration using chiral liquid-chromatography tandem mass-spectrometry. International normalized ratio (INR) values were determined prior to and 24 h after the drug intake. The allele frequency of VKORC1 variant (1173C>T) was 1.96% (95% CI 0.0-4.65%). The INR values did not show statistically significant difference between the CYP2C9 genotypes, but were correlated with body mass index and age at 24 h post-dosing (P < 0.05). At 8 h post dose, the (S)-acenocoumarol concentrations in the CYP2C9*5/*8 and CYP2C9*9/*11 genotypes were about 1.9 and 5.1 fold higher compared with the CYP2C9*1/*1 genotype and 2.2- and 6.0-fold higher compared with the CYP2C9*1/*9 group, respectively. The results indicated that pharmacodynamic response to acenocoumarol is highly variable between the subjects. This variability seems to be associated with CYP2C9*5/*8 and *9/*11 variant and demographic factors (age and weight) in Beninese subjects. Significant association between plasma (S)-acenocoumarol concentration and CYP2C9 genotypes suggested the use of (S)-acenocoumarol for the phenotyping purpose. Larger number of subjects is needed to study the effect of VKORC1 1173C>T variant due to its low frequency in Beninese population.

  16. Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples with Single Substrates.

    PubMed

    Erratico, Claudio A; Deo, Anand K; Bandiera, Stelvio M

    2015-01-01

    Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.

  17. Combined effect of CYP2B6 and NAT2 genotype on plasma efavirenz exposure during rifampin-based antituberculosis therapy in the STRIDE study.

    PubMed

    Luetkemeyer, Anne F; Rosenkranz, Susan L; Lu, Darlene; Grinsztejn, Beatriz; Sanchez, Jorge; Ssemmanda, Michael; Sanne, Ian; McIlleron, Helen; Havlir, Diane V; Haas, David W

    2015-06-15

    In STRIDE, slow metabolizer CYP2B6 and NAT2 genotypes were each associated with increased plasma efavirenz concentrations during antituberculosis therapy. Concentrations were greater on therapy than off therapy in 58% with CYP2B6 and 93% with NAT2 slow metabolizer genotypes. Individuals with slow metabolizer genotypes in both genes had markedly elevated concentrations.

  18. CYP2C9*2 allele increases risk for hypoglycemia in POR*1/*1 type 2 diabetic patients treated with sulfonylureas.

    PubMed

    Ragia, G; Tavridou, A; Elens, L; Van Schaik, R H N; Manolopoulos, V G

    2014-01-01

    It is previously shown that carriers of the defective allele CYP2C9*3 that leads to impaired sulfonylurea metabolism are at increased sulfonylurea-induced hypoglycemia risk due to diminished drug metabolism, whereas no effect of CYP2C9*2 allele was found. Recently, a polymorphism in P450 oxidoreductase (POR) gene, assigned as POR*28 allele, was associated with increased CYP2C9 activity. The aim of this study was to assess i) the effect of POR*28 allele on sulfonylurea-induced hypoglycemia risk and ii) the association of CYP2C9*2 allele with hypoglycemia risk in non-carriers of POR*28 allele. The study group consisted of 176 patients with diagnosed type 2 diabetes mellitus (T2DM) treated with sulfonylureas, of whom 92 patients had experienced at least one drug-associated hypoglycemic event (cases), while 84 had never experienced a hypoglycemic event (controls). POR*28 allele was detected by use of real-time TaqMan PCR. POR*28 allele was not associated with sulfonyl-urea-induced hypoglycemia. In POR*1/*1 patients, CYP2C9*1/*2 genotype was more common in cases than in controls (32.7 vs. 14.3%, p=0.041). In a model adjusted for age, BMI, duration of T2DM and renal function, and POR*1/*1 entered as a selection variable, CYP2C9*2 allele increased the hypoglycemia risk in response to sulfonylurea (odds ratio: 3.218, p=0.031). In conclusion, our results suggest that POR*28 allele is masking the association of CYP2C9*2 allele with sulfonyl-urea-induced hypoglycemia. Therefore, POR*28 allele is an important source of CYP2C9 activity variability and combined with CYP2C9 gene poly-morphisms may explain individual variability in the effect of sulfonylureas.

  19. Polymorphisms and haplotypes of the CYP2B6 detoxification gene in the predisposition of Acute Myeloid Leukemia (AML) and induction of its cytogenetic abnormalities.

    PubMed

    Daraki, Aggeliki; Kakosaiou, Katerina; Zachaki, Sophia; Sambani, Constantina; Aleporou-Marinou, Vassiliki; Kollia, Panagoula; Manola, Kalliopi N

    2016-11-01

    CYP2B6 is a polymorphic detoxification gene which plays a vital role in the degradation of genotoxic compounds. In this study we hypothesized that inadequate detoxification due to CYP2B6 polymorphisms may contribute to AML. To evaluate the potential impact of CYP2B6 polymorphisms on AML development and induction of its specific chromosomal abnormalities we studied C(777)A and A(785)G polymorphisms for the first time in AML. Furthermore, we investigated the co-existence of the above polymorphisms with G(516)T polymorphism to determine the CYP2B6 high-risk haplotypes in AML susceptibility. Our study included 619 AML patients and 430 healthy donors. Concerning C(777)A CYP2B6 polymorphism, no significant difference was found between patients and controls. However, A(785)G CYP2B6 polymorphism showed a statistically higher frequency of the variant genotypes in patients (48.2%), mainly in secondary AML patients (49.1%) than in controls (26.1%). Moreover, an increased frequency of the variant genotypes was found in those with abnormal karyotypes, especially with -7/del(7q), -5/del(5q), +8, inv(16) and t(8;21). The combination of the three CYP2B6 polymorphisms (G(516)T, C(777)A & A(785)G) revealed seven haplotypes. Four out of six haplotypes with at least one mutant allele were significantly associated with an increased risk for AML. Interestingly, T516A777G785 haplotype, where the three mutant alleles co-existed, had ~3-fold increased risk to be found in patients than controls. The association between haplotypes and cytogenetic aberrations revealed a positive correlation between specific CYP2B6 haplotypes and AML cytogenetic abnormalities. Our data suggest that A(785)G CYP2B6 gene polymorphism and specific CYP2B6 haplotypes may contribute to AML and its specific chromosomal aberrations.

  20. Prevalence of poor and rapid metabolizers of drugs metabolized by CYP2B6 in North Indian population residing in Indian national capital territory.

    PubMed

    Varshney, Ekta; Saha, Nilanjan; Tandon, Monika; Shrivastava, Vikesh; Ali, Shakir

    2012-01-01

    Identification of poor and rapid metabolizers for the category of drugs metabolized by cytochrome P450 2B6 (CYP2B6) is important for understanding the differences in clinical responses of drugs metabolized by this enzyme. This study reports the prevalence of poor and rapid metabolizers in North Indian population residing in the National Capital Territory. The prevalence of poor and rapid metabolizers was determined in the target population for the category of drugs metabolized by CYP2B6 by measuring plasma bupropion, a drug metabolized by CYP2B6, and its metabolite. Bupropion (75 mg) was administered to 107 volunteers, and the drug (bupropion) and its metabolite (hydroxybupropion) were determined simultaneously by LCMS/MS in the plasma. CYP2B6 activity was measured as hydroxybupropion/bupropion ratio, and volunteers were categorized as rapid or poor metabolizers on the basis of cutoff value of log (hydroxybupropion/bupropion). Significant differences were observed between the mean metabolite/drug ratio of rapid metabolizers (Mean = 0.59) and poor metabolizers (Mean = 0.26) with p<0.0001. Results indicate that 20.56% individuals in the target population were poor metabolizers for the category of drugs metabolized by CYP2B6. Cutoff value defined in this study can be used as a tool for evaluating the status of CYP2B6 using bupropion as a probe drug. The baseline information would be clinically useful before administering the drugs metabolized by this isoform.

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

    PubMed Central

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

    2016-01-01

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

  2. Involvement of CAR and PXR in the transcriptional regulation of CYP2B6 gene expression by ingredients from herbal medicines.

    PubMed

    Xu, Cong; Luo, Mengyue; Jiang, Huidi; Yu, Lushan; Zeng, Su

    2015-01-01

    1. Induction of hepatic drug-metabolizing enzymes can affect drug efficacy and cause toxicity. However, so far, limited information is available regarding the molecular mechanism how herbal medicines induce human CYP2B6, which metabolizes many of the clinically used therapeutics and activates several pro-carcinogens or toxicants. Accumulated evidence suggests that the human constitutive androstane receptor (hCAR) and the human pregnane X receptor (hPXR) play important roles in trans-activation of CYP2B6. In this study, we investigated the effects of 68 Chinese herbal ingredients on the receptor specificity of hPXR/hCAR-mediated CYP2B6 induction by luciferase reporter gene assays in transiently transfected HepG2 cells and on the expression of CYP2B6 in LS174T cells. 2. The HepG2 cells were transiently transfected with human CYP2B6 luciferase promoter reporter plasmids along with hPXR or hCAR3. The results indicated that apigenin (Api), curcumol (Cur) and praeruptorin A (Pra A) were identified as potent activators of hPXR, and Pra A was also a ligand of hCAR. 3. Furthermore, CYP2B6 mRNA expression in LS174T cells treated with the three herbal ingredients was determined by real-time polymerase chain reaction. By combining western blot and LC-MS/MS, CYP2B6 protein expression and catalytic activity induced by the three herbal ingredients were measured. 4. Our observation showed Api and Cur up-regulated CYP2B6 expression by transactivation of hPXR, and Pra A acted as the ligand of both hPXR and hCAR to induce CYP2B6 expression.

  3. Potential Contribution of Cytochrome P450 2B6 to Hepatic 4-Hydroxycyclophosphamide Formation In Vitro and In VivoS⃞

    PubMed Central

    Raccor, Brianne S.; Claessens, Adam J.; Dinh, Jean C.; Park, Julie R.; Hawkins, Douglas S.; Thomas, Sushma S.; Makar, Karen W.; McCune, Jeannine S.

    2012-01-01

    Results from retrospective studies on the relationship between cytochrome P450 (P450) 2B6 (CYP2B6) genotype and cyclophosphamide (CY) efficacy and toxicity in adult cancer patients have been conflicting. We evaluated this relationship in children, who have faster CY clearance and receive different CY-based regimens than adults. These factors may influence the P450s metabolizing CY to 4-hydroxycyclophosphamide (4HCY), the principal precursor to CY's cytotoxic metabolite. Therefore, we sought to characterize the in vitro and in vivo roles of hepatic CYP2B6 and its main allelic variants in 4HCY formation. CYP2B6 is the major isozyme responsible for 4HCY formation in recombinant P450 Supersomes. In human liver microsomes (HLM), 4HCY formation correlated with known phenotypic markers of CYP2B6 activity, specifically formation of (S)-2-ethyl-1,5-dimethyl-3,3-diphenyl pyrrolidine and hydroxybupropion. However, in HLM, CYP3A4/5 also contributes to 4HCY formation at the CY concentrations similar to plasma concentrations achieved in children (0.1 mM). 4HCY formation was not associated with CYP2B6 genotype at low (0.1 mM) or high (1 mM) CY concentrations potentially because CYP3A4/5 and other isozymes also form 4HCY. To remove this confounder, 4HCY formation was evaluated in recombinant CYP2B6 enzymes, which demonstrated that 4HCY formation was lower for CYP2B6.4 and CYP2B6.5 compared with CYP2B6.1. In vivo, CYP2B6 genotype was not directly related to CY clearance or ratio of 4HCY/CY areas under the curve in 51 children receiving CY-based regimens. Concomitant chemotherapy agents did not influence 4HCY formation in vitro. We conclude that CYP2B6 genotype is not consistently related to 4HCY formation in vitro or in vivo. PMID:21976622

  4. Effect of diurnal variation, CYP2B6 genotype and age on the pharmacokinetics of nevirapine in African children

    PubMed Central

    Bienczak, Andrzej; Cook, Adrian; Wiesner, Lubbe; Mulenga, Veronica; Kityo, Cissy; Kekitiinwa, Addy; Walker, A. Sarah; Owen, Andrew; Gibb, Diana M.; Burger, David; McIlleron, Helen; Denti, Paolo

    2017-01-01

    Objectives To characterize the effects of CYP2B6 polymorphisms, diurnal variation and demographic factors on nevirapine pharmacokinetics in African children. Methods Non-linear mixed-effects modelling conducted in NONMEM 7.3 described nevirapine plasma concentration–time data from 414 children aged 0.3–15 years. Results Nevirapine pharmacokinetics was best described using a one-compartment disposition model with elimination through a well-stirred liver model accounting for a first-pass effect and transit-compartment absorption. Intrinsic clearance was affected by diurnal variation (characterized using a cosine function with peak amplitude 29% at 12 noon) and CYP2B6 metabolizer status [extensive metabolizer (EM) 516GG|983TT, reference; intermediate metabolizer (IM) 516GT|983TT or 516GG|983TC, 17% lower; slow metabolizer (SM) 516TT|983TT or 516GT|983TC, 50% lower; ultra-slow metabolizer (USM) 516GG|983CC, 68% lower]. Age was found to affect pre-hepatic bioavailability: 31.7% lower at birth and increasing exponentially. Median (90% CI) evening Cmin values in the different metabolizer groups were 5.01 (3.01–7.47), 6.55 (3.65–13.32), 11.59 (5.44–22.71) and 12.32 (12.32–27.25) mg/L, respectively. Evening Cmin values were <3 mg/L in 43% of EM weighing <6 kg and 26% of IM weighing <6 kg, while 73% of SM and 88% of USM in all weight-bands had evening Cmin values >8 mg/L. Cmin was not markedly affected by administration time, but was altered by unequal splitting of the daily dose. Conclusions Diurnal variation does not greatly affect nevirapine exposure. However, when daily doses cannot be split equally, the larger dose should be given in the morning. To achieve homogeneous exposures, nevirapine doses for SM and USM should be reduced by 50%, and children weighing <6 kg with EM or IM metabolizer status should receive the same dose as children weighing 6–10 kg. PMID:27707991

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

    PubMed Central

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

    2007-01-01

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

  6. Interethnic differences in the relevance of CYP2C9 genotype and environmental factors for diclofenac metabolism in Hispanics from Cuba and Spain.

    PubMed

    Llerena, A; Alvarez, M; Dorado, P; González, I; Peñas-LLedó, E; Pérez, B; Cobaleda, J; Calzadilla, L R

    2014-06-01

    The aims of this study were to evaluate the diclofenac metabolism in Hispanics from Cuba and Spain and its relation to ethnicity, CYP2C9 genotypes and environmental factors. Diclofenac hydroxylation capacity (concentration ratios of diclofenac/metabolites in 8-h urine) was studied in 160 Cuban (classified as 76 Cuban-Whites-CWs and 84 Cuban-Mestizos-CMs) and 148 Spaniard (SPs) healthy volunteers. Diclofenac and its main metabolites, 4'-hydroxy (OH), 3'-OH and 5-OH diclofenac, and CYP2C9*2 to *6 and *8 alleles were also determined in 132 and 128 CWs and CMs, respectively. Gender, tobacco, caffeine and ethanol consumption were also evaluated. The mean diclofenac/4'-OH diclofenac ratio was higher in CMs (0.72±0.25) than in CWs (0.64±0.20; P<0.05) and SPs (0.57±0.26; P<0.001). The mean diclofenac/4'-OH diclofenac ratio was higher (P<0.05) in subjects with CYP2C9*1/*3 (0.77±0.19; n=22) and CYP2C9*1/*8 (0.93±0.33; n=4) genotypes than with CYP2C9*1/*1 (0.65±0.24; n=90). Environmental factors did not seem to influence the diclofenac metabolism in these populations. The present findings show for the first time interethnic differences between Hispanic groups in urinary diclofenac/4'-OH diclofenac ratios, and the relevance of CYP2C9*3 and CYP2C9*8 alleles.

  7. Estradiol induces cytochrome P450 2B6 expression at high concentrations: Implication in estrogen-mediated gene regulation in pregnancy

    PubMed Central

    Koh, Kwi Hye; Jurkovic, Steve; Yang, Kyunghee; Choi, Su-Young; Jung, Jin Woo; Kim, Kwang Pyo; Zhang, Wei; Jeong, Hyunyoung

    2012-01-01

    Pregnancy alters the rate and extent of drug metabolism, but little is known about the underlying molecular mechanism. We have found that 17β-estradiol (E2) upregulates expression of the major drug-metabolizing enzyme CYP2B6 in primary human hepatocytes. Results from promoter reporter assays in HepG2 cells revealed that E2 activates constitutive androstane receptor (CAR) and enhances promoter activity of CYP2B6, for which high concentrations of E2 reached during pregnancy were required. E2 triggered nuclear translocation of CAR in primary rat hepatocytes that were transiently transfected with human CAR as well as in primary human hepatocytes, further confirming transactivation of CAR by E2. E2-activated estrogen receptor (ER) also enhanced CYP2B6 promoter activity. The DNA-binding domain of ER was not required for the induction of CYP2B6 promoter activity by E2, suggesting involvement of a non-classical mechanism of ER action. Results from deletion and mutation assays as well as electrophorectic mobility shift and supershift assays revealed that two AP-1 binding sites (−1782/−1776 and −1664/−1658 of CYP2B6) are critical for ER-mediated activation of the CYP2B6 promoter by E2. Concurrent activation of both ER and CAR by E2 enhanced CYP2B6 expression in a synergistic manner. Our data demonstrate that at high concentrations reached during pregnancy, E2 activates both CAR and ER that synergistically induce CYP2B6 expression. These results illustrate pharmacological activity of E2 that would likely become prominent during pregnancy. PMID:22484313

  8. Clinical application of a new warfarin-dosing regimen based on the CYP2C9 and VKORC1 genotypes in atrial fibrillation patients

    PubMed Central

    JIANG, NIAN-XIN; GE, JUN-WEI; XIAN, YU-QIONG; HUANG, SHAO-YING; LI, YAN-SONG

    2016-01-01

    The polymorphisms of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) are important genetic factors for warfarin dose determinations. The present study aimed to investigate the contribution of the CYP2C9 and VKORC1 genotypes to warfarin dose requirement in atrial fibrillation (AF) patients, and to evaluate the clinical application of a warfarin-dosing algorithm. A total of 122 AF patients with a target international normalized ratio of 2.0 to 3.0 were included to determine the genotypes of CYP2C9 (rs1057910) and VKORC1 (rs9923231). A warfarin-dosing algorithm was developed based on age, height, and the CYP2C9 and VKORC1 genotypes of AF patients. The results indicated that the mean warfarin daily dose requirement was lower in the CYP2C9*1/*3 genotype compared with those in the homozygous wild-type CYP2C9*1/*1 patients (P<0.05), and was higher in patients with the VKORC1 AG and GG genotypes compared with those with the AA genotype (P<0.05). The multivariate regression model showed that age, height, and the CYP2C9 and VKORC1 genotypes were the best variables for estimating warfarin dose (R2=56.4%). A new warfarin-dosing algorithm was developed and its validity was confirmed in a second cohort of AF patients. During the 50-day follow-up, 63.3% (19/30) of control group patients and 86.7% (26/30) of patients in the experimental group acquired the warfarin maintenance dose. Among all the patients who acquired the warfarin maintenance dose, the mean time elapse from initiation until warfarin maintenance dose was significantly less in the experimental group (25.8±1.7 day) compared to the control group (33.1±1.9 day) (P<0.05). There was significant linear correlation between predicted warfarin maintenance dose and actual dose (r=0.822, P<0.01). In conclusion, a new warfarin-dosing algorithm was developed based on the CYP2C9 and VKORC1 genotypes, and it can shorten the time elapse from initiation until warfarin maintenance dose in AF patients

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

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

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

    PubMed Central

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

    2010-01-01

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

  12. Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines.

    PubMed

    Scheer, Nico; Kapelyukh, Yury; Chatham, Lynsey; Rode, Anja; Buechel, Sandra; Wolf, C Roland

    2012-12-01

    Compared with rodents and many other animal species, the human cytochrome P450 (P450) Cyp2c gene cluster varies significantly in the multiplicity of functional genes and in the substrate specificity of its enzymes. As a consequence, the use of wild-type animal models to predict the role of human CYP2C enzymes in drug metabolism and drug-drug interactions is limited. Within the human CYP2C cluster CYP2C9 is of particular importance, because it is one of the most abundant P450 enzymes in human liver, and it is involved in the metabolism of a wide variety of important drugs and environmental chemicals. To investigate the in vivo functions of cytochrome P450 Cyp2c genes and to establish a model for studying the functions of CYP2C9 in vivo, we have generated a mouse model with a deletion of the murine Cyp2c gene cluster and a corresponding humanized model expressing CYP2C9 specifically in the liver. Despite the high number of functional genes in the mouse Cyp2c cluster and the reported roles of some of these proteins in different biological processes, mice deleted for Cyp2c genes were viable and fertile but showed certain phenotypic alterations in the liver. The expression of CYP2C9 in the liver also resulted in viable animals active in the metabolism and disposition of a number of CYP2C9 substrates. These mouse lines provide a powerful tool for studying the role of Cyp2c genes and of CYP2C9 in particular in drug disposition and as a factor in drug-drug interaction.

  13. Altered CYP2C9 Activity Following Modulation of CYP3A4 Levels in Human Hepatocytes: an Example of Protein-Protein Interactions

    PubMed Central

    Tweedie, Donald J.; Chan, Tom S.; Tracy, Timothy S.

    2014-01-01

    Cytochrome P450 (P450) protein-protein interactions resulting in modulation of enzyme activities have been well documented using recombinant isoforms. This interaction has been less clearly demonstrated in a more physiologic in vitro system such as human hepatocytes. As an expansion of earlier work (Subramanian et al., 2010), in which recombinant CYP2C9 activity decreased with increasing levels of CYP3A4, the current study modulated CYP3A4 content in human hepatocytes to determine the impact on CYP2C9. Modulation of CYP3A4 levels in situ was enabled by the use of a long-term human hepatocyte culture model (HepatoPac) shown to retain phenotypic hepatocyte function over a number of weeks. The extended period of culture allowed time for knockdown of CYP3A4 protein by small interfering RNA (siRNA) with subsequent recovery, as well as upregulation through induction with a recovery period. CYP3A4 gene silencing resulted in a 60% decrease in CYP3A4 activity and protein levels with a concomitant 74% increase in CYP2C9 activity, with no change in CYP2C9 mRNA levels. Upon removal of siRNA, both CYP2C9 and CYP3A4 activities returned to pre-knockdown levels. Importantly, modulation of CYP3A4 protein levels had no impact on cytochrome P450 reductase activities or levels. However, the possibility for competition for limiting reductase cannot be ruled out. Interestingly, lowering CYP3A4 levels also increased UDP-glucuronosyltransferase 2B7 activity. These studies clearly demonstrate that alterations in CYP3A4 levels can modulate CYP2C9 activity in situ and suggest that further studies are warranted to evaluate the possible clinical consequences of these findings. PMID:25157098

  14. Investigation of the mechanisms underlying the differential effects of the K262R mutation of P450 2B6 on catalytic activity

    PubMed Central

    Bumpus, Namandjé N.; Hollenberg, Paul F.

    2008-01-01

    Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262 to arginine mutant of P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We previously reported that the mutant enzyme was not able to metabolize 17-α-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Similarly, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme. PMID:18621926

  15. 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.

  16. Application of HC-AFW1 Hepatocarcinoma Cells for Mechanistic Studies: Regulation of Cytochrome P450 2B6 Expression by Dimethyl Sulfoxide and Early Growth Response 1.

    PubMed

    Petzuch, Barbara; Groll, Nicola; Schwarz, Michael; Braeuning, Albert

    2015-11-01

    Various exogenous compounds, for example, the drugs bupropione and propofol, but also various cytostatics, are metabolized in the liver by the enzyme cytochrome P450 (P450) CYP2B6. Transcription from the CYP2B6 gene is regulated mainly via the transcription factors constitutive androstane receptor (CAR) and pregnane-X-receptor (PXR). Most hepatic cell lines express no or only low levels of CYP2B6 because of loss of these two regulators. Dimethyl sulfoxide (DMSO) is frequently used in liver cell cultivation and is thought to affect the expression of various P450 isoforms by inducing or preserving cellular differentiation. We studied the effects of up to 1.5% of DMSO as cell culture medium supplement on P450 expression in hepatocarcinoma cells from line HC-AFW1. DMSO did not induce differentiation of the HC-AFW1 cell line, as demonstrated by unaltered levels of selected mRNA markers important for hepatocyte differentiation, and also by the lack of a DMSO effect on a broader spectrum of P450s. By contrast, CYP2B6 mRNA was strongly induced by DMSO. This process was independent of CAR or PXR activation. Interestingly, elevated transcription of CYP2B6 was accompanied by a simultaneous induction of early growth response 1 (EGR1), a transcription factor known to influence the expression of CYP2B6. Expression of wild-type EGR1 or of a truncated, dominant-negative EGR1 mutant was able to mimic or attenuate the DMSO effect, respectively. These findings demonstrate that EGR1 is involved in the regulation of CYP2B6 by DMSO in HC-AFW1 cells.

  17. Investigational small-molecule drug selectively suppresses constitutive CYP2B6 activity at the gene transcription level: physiologically based pharmacokinetic model assessment of clinical drug interaction risk.

    PubMed

    Zamek-Gliszczynski, Maciej J; Mohutsky, Michael A; Rehmel, Jessica L F; Ke, Alice B

    2014-06-01

    The glycogen synthase kinase-3 inhibitor LY2090314 specifically impaired CYP2B6 activity during in vitro evaluation of cytochrome P450 (P450) enzyme induction in human hepatocytes. CYP2B6 catalytic activity was significantly decreased following 3-day incubation with 0.1-10 μM LY2090314, on average by 64.3% ± 5.0% at 10 μM. These levels of LY2090314 exposure were not cytotoxic to hepatocytes and did not reduce CYP1A2 and CYP3A activities. LY2090314 was not a time-dependent CYP2B6 inhibitor, did not otherwise inhibit enzyme activity at concentrations ≤10 μM, and was not metabolized by CYP2B6. Thus, mechanism-based inactivation or other direct interaction with the enzyme could not explain the observed reduction in CYP2B6 activity. Instead, LY2090314 significantly reduced CYP2B6 mRNA levels (Imax = 61.9% ± 1.4%; IC50 = 0.049 ± 0.043 μM), which were significantly correlated with catalytic activity (r(2) = 0.87, slope = 0.77; Imax = 57.0% ± 10.8%, IC50 = 0.057 ± 0.027 μM). Direct inhibition of constitutive androstane receptor by LY2090314 is conceptually consistent with the observed CYP2B6 transcriptional suppression (Imax = 100.0% ± 10.8% and 57.1% ± 2.4%; IC50 = 2.5 ± 1.2 and 2.1 ± 0.4 μM for isoforms 1 and 3, respectively) and may be sufficiently extensive to overcome the weak but potent activation of pregnane X receptor by ≤10 μM LY2090314 (19.3% ± 2.2% of maximal rifampin response, apparent EC50 = 1.2 ± 1.1 nM). The clinical relevance of these findings was evaluated through physiologically based pharmacokinetic model simulations. CYP2B6 suppression by LY2090314 is not expected clinically, with a projected <1% decrease in hepatic enzyme activity and <1% decrease in hydroxybupropion exposure following bupropion coadministration. However, simulations showed that observed CYP2B6 suppression could be clinically relevant for a drug with different pharmacokinetic properties from LY2090314.

  18. Differential regulation of hepatic CYP2B6 and CYP3A4 genes by constitutive androstane receptor but not pregnane X receptor.

    PubMed

    Faucette, Stephanie R; Sueyoshi, Tatsuya; Smith, Cornelia M; Negishi, Masahiko; Lecluyse, Edward L; Wang, Hongbing

    2006-06-01

    Accumulated evidence suggests that cross-talk between the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) results in shared transcriptional activation of CYP2B and CYP3A genes. Although most data imply symmetrical cross-regulation of these genes by rodent PXR and CAR, the actual selectivities of the corresponding human receptors are unknown. The objective of this study was to evaluate the symmetry of human (h) PXR and hCAR cross-talk by comparing the selectivities of these receptors for CYP2B6 and CYP3A4. Human hepatocyte studies revealed nonselective induction of both CYP2B6 and CYP3A4 by hPXR activation but marked preferential induction of CYP2B6 by selective hCAR activation. Gel shift assays demonstrated that hPXR exhibited strong and relatively equal binding to all functional response elements in both CYP2B6 and CYP3A4 genes, whereas hCAR displayed significantly weak binding to the CYP3A4 proximal ER6 motif. In cell-based transfection assays, hCAR displayed greater activation of CYP2B6 reporter gene expression compared with CYP3A4 with constructs containing both proximal and distal regulatory elements. Furthermore, in agreement with binding observations, transfection assays using promoter constructs containing repeats of CYP2B6 DR4 and CYP3A4 ER6 motifs revealed an even greater difference in reporter activation by hCAR. In contrast, hPXR activation resulted in less discernible differences between CYP2B6 and CYP3A4 reporter gene expression. These results suggest asymmetrical cross-regulation of CYP2B6 and CYP3A4 by hCAR but not hPXR in that hCAR exhibits preferential induction of CYP2B6 relative to CYP3A4 because of its weak binding and functional activation of the CYP3A4 ER6.

  19. Novel CAR-mediated mechanism for synergistic activation of two distinct elements within the human cytochrome P450 2B6 gene in HepG2 cells.

    PubMed

    Swales, Karen; Kakizaki, Satoru; Yamamoto, Yukio; Inoue, Kaoru; Kobayashi, Kaoru; Negishi, Masahiko

    2005-02-04

    The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at -1732/-1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (-256/-233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription.

  20. Association of the CYP2B6 c.516G>T polymorphism with high blood propofol concentrations in women from northern Greece.

    PubMed

    Mastrogianni, Orthodoxia; Gbandi, Emma; Orphanidis, Amvrosios; Raikos, Nikolaos; Goutziomitrou, Evangelia; Kolibianakis, Efstratios M; Tarlatzis, Basil C; Goulas, Antonis

    2014-01-01

    Cytochrome P450 2B6 (CYP2B6) is responsible for the initial biotransformation of profol, an extensively metabolized intravenous anesthetic. In this study we examined the effect of the apparently functional CYP2B6 c.516G>T polymorphism on the distribution of propofol concentrations, quantified by GC/MS analysis following a single bolus dose, in the blood of 44 Greek women undergoing oocyte retrieval. Univariate analysis using age, height, weight and smoking status as covariates, as well as the Mann-Whitney non-parametric test, revealed a strong trend of association of the T allele with high propofol concentrations determined in whole blood, shortly after a single bolus dose. Propofol concentrations which were higher than one standard deviation of the mean were almost invariably associated with carriage of the T allele.

  1. Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes.

    PubMed

    Yeung, John H K; Or, Penelope M Y

    2011-10-15

    Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20μM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K(i) value of 14.2μM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K(i) value of 0.32μM. In human CYP2C9 isoform, the K(i) value of PSP was 29.5μM and the K(i) value of sulfaphenazole was 0.04μM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K(i) values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction.

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

    PubMed Central

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

    2012-01-01

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

  3. Ethanol self-administration and nicotine treatment induce brain levels of CYP2B6 and CYP2E1 in African green monkeys.

    PubMed

    Ferguson, Charmaine S; Miksys, Sharon; Palmour, Roberta M; Tyndale, Rachel F

    2013-09-01

    CYP2B6 and CYP2E1 are enzymes responsible for the metabolism of many centrally acting drugs, toxins and endogenous compounds. Human smokers and alcoholics have elevated levels of CYP2B6 and CYP2E1 in certain brain regions, which may contribute to altered drug efficacy, neurotoxicity and metabolic tolerance. The objective of this study was to determine the effects of ethanol self-administration and nicotine treatment, alone and in combination, on brain CYP2B6 and CYP2E1 levels in monkeys. Monkeys were randomized into four groups (N = 10/group): an ethanol-only group, a nicotine-only group, an ethanol + nicotine group and a control (no drug) group. Ethanol (10% alcohol in sucrose solution) was voluntarily self-administered by the monkeys and nicotine was given as subcutaneous injections (0.5 mg/kg bid). Immunocytochemistry revealed induction of both CYP2B6 and CYP2E1 protein in certain brain regions and cells within monkey brain as a result of ethanol self-administration, nicotine treatment and combined exposure to both drugs. Immunoblotting analyses demonstrated CYP2B6 induction by ethanol in the caudate, putamen and cerebellum (1.5-3.2 fold, P < 0.05), and CYP2E1 induction by nicotine in the frontal cortex and putamen (1.6-2.0 fold, P < 0.05). Combined ethanol and nicotine exposure induced CYP2B6 in the caudate, putamen, thalamus and cerebellum (1.4-2.4 fold, P < 0.05), and CYP2E1 in the frontal cortex and putamen (1.5-1.8, P < 0.05). CYP2B6 and CYP2E1 mRNA levels were unaffected by ethanol or nicotine exposure. In summary, ethanol and nicotine can induce CYP2B6 and CYP2E1 protein in the primate brain, which could potentially result in altered sensitivity to centrally acting drugs and toxins.

  4. Rational Engineering of Cytochromes P450 2B6 and 2B11 for Enhanced Stability: Insights Into Structural Importance of Residue 334

    PubMed Central

    Talakad, Jyothi C.; Wilderman, P. Ross; Davydov, Dmitri R.; Kumar, Santosh; Halpert, James R.

    2009-01-01

    Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques. PMID:19944064

  5. Molecular modelling of CYP2B6 based on homology with the CYP2C5 crystal structure: analysis of enzyme-substrate interactions.

    PubMed

    Lewis, David F V; Lake, Brian G; Dickins, Maurice; Goldfarb, Peter S

    2002-01-01

    The results of homology modelling of CYP2B6 based on the CYP2C5 crystal structure is described in terms of substrates and inhibitors binding within the putative active site. In general these results are in agreement with currently available evidence from substrate metabolism, mode of inhibitor action and site-directed mutagenesis experiments within the CYP2B subfamily of enzymes. Consequently, the model based on the CYP2C5 template represents an advance on those models produced from bacterial P450s, such as CYP101 and CYP102. Quantitative Structure-Activity Relationships (QSARs) for substrates binding to CYP2B6 indicate a key role for hydrogen bonding, and lipophilic character, as determined by the log P parameter (where P is the octanol/water partition coefficient), is also of importance for explaining the variation in experimental binding affinity for CYP2B6 substrates. It is possible to estimate the binding energies for typical CYP2B6 substrates based on their properties and interactions with the enzyme, which show good concordance with experimental data in the form of apparent Km values.

  6. Interaction of quercetin and its metabolites with warfarin: Displacement of warfarin from serum albumin and inhibition of CYP2C9 enzyme.

    PubMed

    Poór, Miklós; Boda, Gabriella; Needs, Paul W; Kroon, Paul A; Lemli, Beáta; Bencsik, Tímea

    2017-04-01

    Flavonoids are ubiquitous molecules in nature with manifold pharmacological effects. Flavonoids interact with several proteins, and thus potentially interfere with the pharmacokinetics of various drugs. Though much is known about the protein binding characteristics of flavonoid aglycones, the behaviour of their metabolites, which are extensively formed in the human body has received little attention. In this study, the interactions of the flavonoid aglycone quercetin and its main metabolites with the albumin binding of the oral anticoagulant warfarin were investigated by fluorescence spectroscopy and ultrafiltration. Furthermore, the inhibitory effects of these flavonoids on CYP2C9 enzyme were tested because the metabolic elimination of warfarin is catalysed principally by this enzyme. Herein, we demonstrate that each tested flavonoid metabolite can bind to human serum albumin (HSA) with high affinity, some with similar or even higher affinity than quercetin itself. Quercetin metabolites are able to strongly displace warfarin from HSA suggesting that high quercetin doses can strongly interfere with warfarin therapy. On the other hand, tested flavonoids showed no or weaker inhibition of CYP2C9 compared to warfarin, making it very unlikely that quercetin or its metabolites can significantly inhibit the CYP2C9-mediated inactivation of warfarin.

  7. Effect of mid-dose efavirenz concentrations and CYP2B6 genotype on viral suppression in patients on first-line antiretroviral therapy.

    PubMed

    Orrell, Catherine; Bienczak, Andrzej; Cohen, Karen; Bangsberg, David; Wood, Robin; Maartens, Gary; Denti, Paolo

    2016-06-01

    The therapeutic range for efavirenz plasma concentrations is unclear and some studies found no correlation with viral non-suppression. Efavirenz concentrations are variable, driven in part by polymorphisms in CYP2B6. We hypothesised that efavirenz mid-dosing concentrations, together with CYP2B6 metaboliser genotype, could predict viral non-suppression. Participants starting first-line efavirenz-based antiretroviral therapy were monitored for 48 weeks. HIV-RNA and efavirenz mid-dose interval concentrations were determined at Weeks 16 and 48. CYP2B6 metaboliser genotype status was determined by 516G→T and 983T→C polymorphisms. Cox proportional hazards modelling was used to predict viral non-suppression and to determine the most predictive efavirenz mid-dosing concentration threshold. In total, 180 participants were included. Median efavirenz concentrations were 2.3 mg/L (IQR 1.6-4.6 mg/L) and 2.2 mg/L (IQR 1.5-3.9 mg/L) at Weeks 16 and 48, respectively. Moreover, 49 (27.2%), 84 (46.7%) and 39 (21.7%) participants had extensive, intermediate or slow CYP2B6 metaboliser genotype, respectively. Log2 efavirenz concentrations [adjusted hazard ratio (aHR) = 0.77, 95% CI 0.67-0.89] and baseline CD4 cell count (aHR = 0.994, 95% CI 0.989-0.998), but not CYP2B6 genotype, were predictive of viral non-suppression. For every doubling of efavirenz concentration there was a 23% decrease in the hazard of non-suppression. A threshold of 0.7 mg/L was found to be the efavirenz mid-dosing concentration that was most predictive of non-suppression. Mid-dosing efavirenz concentrations are predictive of viral non-suppression, but the currently recommended lower therapeutic limit (1 mg/L) is higher than our finding. Knowledge of CYP2B6 metaboliser genotype is not required for prediction of virological outcomes.

  8. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  9. CYP2B6 Genotype Guided Dosing of Propofol Anesthesia in the Elderly based on Nonparametric Population Pharmacokinetic Modeling and Simulations

    PubMed Central

    Eugene, Andy R.

    2017-01-01

    Objective The primary aim of this article is to test the hypothesis that nonparametric pharmacometric modeling will accurately identify CYP2B6 genotype subgroups based on data from a study that reported results based on parametric pharmacokinetics (PK). Methods Propofol concentration-time data were originally reported in the Kansaku et al. 2011 publication. Nonparametric Nonlinear Mixed Effects Modeling (NLME) was conducted using the PMETRICS R package while population pharmacokinetic model parameters were estimated using a FORTRAN compiler. Finally, model-based dosing simulations were conducted in the MATLAB Simbiology. Results A total of 51 patients were included in the final PK analysis. A two-compartment gamma multiplicative error model adequately described the propofol concentration-time data. The precision of the goodness-of-fit plots resulted in an R2 of 0.927 and an R2 of 0.992 for the population prediction and individual predictions, respectively. Neither the UGT1A9 nor the CYP2B6 G516T gene variants resulted in statistically significant PK parameter differences while the CYP2B6 A785G gene variants resulted in statistically significant differences for the elimination rate. Model-based dosing-simulations comparing patients with the CYP2B6 AA & AG genotypes to both GG genotypes and patients from a multicenter trial suggest a 50% decrease in propofol infusion dose, to 25mg/kg/min, be made to result in approximately equivalent drug exposures. Conclusion Based on the pharmacometric modeling and simulation, if no dosage adjustments are made for the elderly CYP2B6 AA and AG genotypes, a 250% higher propofol blood exposure will be evident within 1-hour from the start of the infusion. Thus, based on the pharmacokinetic model, genotyping elderly patients for the CYP2B6 AA and AG gene variants will decrease the total propofol blood exposure during anesthesia and sedation when an infusion dose adjustment is made to 25mg/kg/min. PMID:28154789

  10. Synthesis and optical characterization of LiKB4O7, Li2B6O10, and LiCsB6O10 glasses

    SciTech Connect

    Adamiv, V.; Teslyuk, I.; Dyachok, Ya.; Romanyuk, G.; Krupych, O.; Mys, O.; Martynyuk-Lototska, I.; Burak, Ya.; Vlokh, R.

    2010-10-01

    In the current work we report on the synthesis of LiKB4O7, Li2B6O10, and LiCsB6O10 borate glasses. The results for their piezo-optic, acousto-optic, acoustic, elastic, refractive, optical transmission, and optical resistance properties are also presented. It is shown that some of these glasses represent efficient acousto-optic materials that are transparent down to the vacuum ultraviolet range and highly resistant to laser radiation.

  11. CYP2B6*6 is an independent determinant of inferior response to fludarabine plus cyclophosphamide in chronic lymphocytic leukemia.

    PubMed

    Johnson, Gillian G; Lin, Ke; Cox, Trevor F; Oates, Melanie; Sibson, David R; Eccles, Richard; Lloyd, Bryony; Gardiner, Laura-Jayne; Carr, Daniel F; Pirmohamed, Munir; Strefford, Jonathan C; Oscier, David G; Gonzalez de Castro, David; Else, Monica; Catovsky, Daniel; Pettitt, Andrew R

    2013-12-19

    Fludarabine plus cyclophosphamide (FC) is the chemotherapy backbone of modern chronic lymphocytic leukemia (CLL) treatment. CYP2B6 is a polymorphic cytochrome P450 isoform that converts cyclophosphamide to its active form. This study investigated the possible impact of genetic variation in CYP2B6 on response to FC chemotherapy in CLL. Available DNA samples from the LRF CLL4 trial, which compared chlorambucil, fludarabine, and FC, were screened by TaqMan real-time polymerase chain reaction assays for CYP2B6 SNPs c.516G>T and c.785A>G, which define the most common variant allele (*6). Among the 455 samples successfully genotyped, 265 (58.2%), 134 (29.5%), and 29 (6.4%) were classified as *1/*1, *1/*6, and *6/*6, respectively. Patients expressing at least one *6 allele were significantly less likely to achieve a complete response (CR) after FC (odds ratio 0.27; P = .004) but not chlorambucil or fludarabine. Analysis of individual response indicators confirmed that this inferior response resulted from impaired cytoreduction rather than delayed hemopoietic recovery. Multivariate analysis controlling for age, gender, stage, IGHV mutational status, 11q deletion, and TP53 deletion/mutation identified CYP2B6*6 and TP53 mutation/deletion as the only independent determinants of CR attainment after FC. Our study provides the first demonstration that host pharmacogenetics can influence therapeutic response in CLL. This trial is registered as an International Standard Randomised Control Trial, number NCT 58585610 at www.clinicaltrials.gov.

  12. Inhibition of bupropion metabolism by selegiline: mechanism-based inactivation of human CYP2B6 and characterization of glutathione and peptide adducts.

    PubMed

    Sridar, Chitra; Kenaan, Cesar; Hollenberg, Paul F

    2012-12-01

    Selegiline, the R-enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6. The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation (7-EFC) activity of CYP2B6 as well as bupropion metabolism. The inactivations were time-, concentration-, and NADPH-dependent and were characterized by K(I) values of 0.14 and 0.6 μM, k(inact) values of 0.022 and 0.029 min⁻¹, and t(½) values of 31.5 and 24 min, respectively. In standard inhibition assays, selegiline increased the K(m) of CYP2B6 for bupropion from 10 to 92 μM and decreased the k(cat) by ∼50%. The reduced carbon-monoxide difference spectrum revealed over a 50% loss in the cytochrome P450 spectrum in the inactivated sample, with no loss in heme, and there was ∼70% loss in enzyme activity. Trapping of the reactive metabolite using GSH led to the identification of a GSH-selegiline conjugate with a m/z 528 that could be explained by hydroxylation of selegiline followed by the addition of glutathione to the propargyl moiety after oxygenation to form the ketene intermediate. Liquid chromatography-tandem mass spectrometry analysis of the labeled protein following digestion with trypsin revealed the peptide ⁶⁴DVFTVHLGPR⁷³ as the peptide modified by the reactive metabolite of selegiline and the site of adduct formation is Asp64.

  13. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

    PubMed

    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation.

  14. Synthesis and optical characterization of LiKB4O7, Li2B6O10, and LiCsB6O10 glasses.

    PubMed

    Adamiv, V; Teslyuk, I; Dyachok, Ya; Romanyuk, G; Krupych, O; Mys, O; Martynyuk-Lototska, I; Burak, Ya; Vlokh, R

    2010-10-01

    In the current work we report on the synthesis of LiKB(4)O(7), Li(2)B(6)O(10), and LiCsB(6)O(10) borate glasses. The results for their piezo-optic, acousto-optic, acoustic, elastic, refractive, optical transmission, and optical resistance properties are also presented. It is shown that some of these glasses represent efficient acousto-optic materials that are transparent down to the vacuum ultraviolet range and highly resistant to laser radiation.

  15. Sleep quality in efavirenz-treated Chinese HIV patients - comparing between GT and GG genotype of CYP2B6-516 G/T polymorphisms.

    PubMed

    Lee, Shui Shan; To, Kin Wang; Lee, Man Po; Wong, Ngai Sze; Chan, Denise P C; Li, Patrick C K; Cheung, Siu Wai; Chan, Raphael C Y

    2014-03-01

    Seventy-two adult Chinese HIV-positive treatment-naïve patients were recruited in a study to evaluate prospectively the associations between CYP2B6 516 G/T polymorphisms and sleep quality following treatment with an efavirenz-based regimen. Overall, the patients gave an allelic frequency of 0.3 for CYP2B6 516 T, and a genotype frequency of 9.4% for TT. Compared to GG, GT gave a higher median value of plasma efavirenz level at four weeks (3.77 mg/L vs 2.59 mg/L, p < 0.001) and 12 months (3.57 mg/L vs 2.97 mg/L, p = 0.026). Using generalised estimating equations analysis to track the variance over time, there was poorer Pittsburgh Sleep Quality Index in GT compared to GG, while GT was associated with a higher efavirenz level of >4 mg/L. There was however no difference in the component sleep scores nor was there direct association between sleep quality and plasma efavirenz levels. The results suggested that CYP2B6 genotype was associated with different patterns of sleep problems, further investigation of which is warranted with the objective of optimizing therapy with efavirenz-based regimens.

  16. The use of mechanistic DM-PK-PD modelling to assess the power of pharmacogenetic studies –CYP2C9 and warfarin as an example

    PubMed Central

    Dickinson, Gemma L; Lennard, Martin S; Tucker, Geoffrey T; Rostami-Hodjegan, Amin

    2007-01-01

    What is already known about this subject Many studies have shown that genetic polymorphisms of the CYP2C9 gene contribute to some of the variability (around 20%) in warfarin dose requirements and therapeutic response to the drug. It is also clear that this effect must be elicited through differences in the plasma (S)-warfarin concentration between individuals of different genotypes, although assessing the effects of any single genotype of CYP2C9 on the kinetics of (S)-warfarin has generally failed. What this study adds The study aims to simulate the impact of genetic polymorphism in CYP2C9 on both the pharmacokinetics (PK) and pharmacodynamics (PD) of (S)-warfarin using a mechanistic, population approach to modelling. The outcomes with respect to the design of studies and their statistical power are compared against those of actual reported studies. The exercise with warfarin is offered as an example of how prior information on the in vitro PK and PD of new drugs might be used in association with knowledge of relevant genetic polymorphisms and their frequencies to carry out virtual clinical studies as an aid to the design, optimization and powering of subsequent real clinical trials assessing the impact of specific genetic differences. Aim To assess the power of in vivo studies needed to discern the effect of genotype on pharmacokinetics (PK) and pharmacodynamics (PD) using CYP2C9 and (S)-warfarin as an example. Methods Information on the in vitro metabolism of (S)-warfarin and genetic variation in CYP2C9 was incorporated into a mechanistic population-based PK–PD model. The influence of study design on the ability to detect significant differences in PK (AUC0−12 h) and PD (AUEC0−12 h INR) between CYP2C9 genotypes was investigated. Results A study size of 90 (based on the natural abundance of genotypes and uniform dosage) was required to achieve 80% power to discriminate the PK of (S)-warfarin between wild type (*1/*1) and the combination of all other

  17. Pharmacokinetic interactions between glimepiride and rosuvastatin in healthy Korean subjects: does the SLCO1B1 or CYP2C9 genetic polymorphism affect these drug interactions?

    PubMed Central

    Kim, Choon Ok; Oh, Eun Sil; Kim, Hohyun; Park, Min Soo

    2017-01-01

    To improve cardiovascular outcomes, dyslipidemia in patients with diabetes needs to be treated. Thus, these patients are likely to take glimepiride and rosuvastatin concomitantly. Therefore, this study aimed to evaluate the pharmacokinetic (PK) interactions between these two drugs in healthy males and to explore the effect of SLCO1B1 and CYP2C9 polymorphisms on their interactions in two randomized, open-label crossover studies. Glimepiride was studied in part 1 and rosuvastatin in part 2. Twenty-four participants were randomly assigned to each part. All subjects (n=24) completed part 1, and 22 subjects completed part 2. A total of 38 subjects among the participants of the PK interaction studies were enrolled in the genotype study to analyze their SLCO1B1 and CYP2C9 polymorphisms retrospectively (n=22 in part 1, n=16 in part 2). Comparison of the PK and safety of each drug alone with those of the drugs in combination showed that both glimepiride and rosuvastatin did not interact with each other and had tolerable safety profiles in all subjects. However, with regard to glimepiride PK, the SLCO1B1 521TC group had a significantly higher maximum plasma concentration (Cmax,ss) and area under the plasma concentration–time curve during the dose interval at steady state (AUCτ,ss) for glimepiride in combination with rosuvastatin than those for glimepiride alone. However, other significant effects of the SLCO1B1 or CYP2C9 polymorphism on the interaction between the two drugs were not observed. In conclusion, there were no significant PK interactions between the two drugs; however, the exposure to glimepiride could be affected by rosuvastatin in the presence of the SLCO1B1 polymorphism. PMID:28260863

  18. The G⁵¹⁶T CYP2B6 germline polymorphism affects the risk of acute myeloid leukemia and is associated with specific chromosomal abnormalities.

    PubMed

    Daraki, Aggeliki; Zachaki, Sophia; Koromila, Theodora; Diamantopoulou, Paraskevi; Pantelias, Gabriel E; Sambani, Constantina; Aleporou, Vasiliki; Kollia, Panagoula; Manola, Kalliopi N

    2014-01-01

    The etiology of acute myeloid leukemia (AML) underlies the influence of genetic variants in candidate genes. The CYP2B6 enzyme detoxifies many genotoxic xenobiotics, protecting cells from oxidative damage. The CYP2B6 gene is subjected to a single-nucleotide polymorphism (G⁵¹⁶T) with heterozygotes (GT) and homozygotes (TT) presenting decreased enzymatic activity. This case-control study aimed to investigate the association of CYP2B6 G⁵¹⁶T polymorphism with the susceptibility of AML and its cytogenetic and clinical characteristics. Genotyping was performed on 619 AML patients and 430 healthy individuals using RCR-RFLP and a novel LightSNip assay. The major finding was a statistically higher frequency of the variant genotypes (GT and TT) in patients compared to the controls (GT:38.8% vs 29.8% and TT:9.3% vs 5.3% respectively) (p<0.001). More specifically, a significantly higher frequency of GT+TT genotypes in de novo AML patients (46.6%) and an immensely high frequency of TT in secondary AML (s-AML) (20.5%) were observed. The statistical analysis showed that the variant T allele was approximately 1.5-fold and 2.4-fold higher in de novo and s-AML respectively than controls. Concerning FAB subtypes, the T allele presented an almost 2-fold increased in AML-M2. Interestingly, a higher incidence of the TT genotype was observed in patients with abnormal karyotypes. In particular, positive correlations of the mutant allele were found in patients carrying specific chromosomal aberrations [-7/del(7q), -5/del(5q), +8, +21 or t(8;21)], complex or monosomal karyotypes. Finally, a strikingly higher frequency of TT genotype was also observed in patients stratified to the poor risk group. In conclusion, our results provide evidence for the involvement of the CYP2B6 polymorphism in AML susceptibility and suggest a possible role of the CYP2B6 genetic background on the development of specific chromosomal aberrations.

  19. The G516T CYP2B6 Germline Polymorphism Affects the Risk of Acute Myeloid Leukemia and Is Associated with Specific Chromosomal Abnormalities

    PubMed Central

    Daraki, Aggeliki; Zachaki, Sophia; Koromila, Theodora; Diamantopoulou, Paraskevi; Pantelias, Gabriel E.; Sambani, Constantina; Aleporou, Vasiliki; Kollia, Panagoula; Manola, Kalliopi N.

    2014-01-01

    The etiology of acute myeloid leukemia (AML) underlies the influence of genetic variants in candidate genes. The CYP2B6 enzyme detoxifies many genotoxic xenobiotics, protecting cells from oxidative damage. The CYP2B6 gene is subjected to a single-nucleotide polymorphism (G516T) with heterozygotes (GT) and homozygotes (TT) presenting decreased enzymatic activity. This case-control study aimed to investigate the association of CYP2B6 G516T polymorphism with the susceptibility of AML and its cytogenetic and clinical characteristics. Genotyping was performed on 619 AML patients and 430 healthy individuals using RCR-RFLP and a novel LightSNip assay. The major finding was a statistically higher frequency of the variant genotypes (GT and TT) in patients compared to the controls (GT:38.8% vs 29.8% and TT:9.3% vs 5.3% respectively) (p<0.001). More specifically, a significantly higher frequency of GT+TT genotypes in de novo AML patients (46.6%) and an immensely high frequency of TT in secondary AML (s-AML) (20.5%) were observed. The statistical analysis showed that the variant T allele was approximately 1.5-fold and 2.4-fold higher in de novo and s-AML respectively than controls. Concerning FAB subtypes, the T allele presented an almost 2-fold increased in AML-M2. Interestingly, a higher incidence of the TT genotype was observed in patients with abnormal karyotypes. In particular, positive correlations of the mutant allele were found in patients carrying specific chromosomal aberrations [-7/del(7q), -5/del(5q), +8, +21 or t(8;21)], complex or monosomal karyotypes. Finally, a strikingly higher frequency of TT genotype was also observed in patients stratified to the poor risk group. In conclusion, our results provide evidence for the involvement of the CYP2B6 polymorphism in AML susceptibility and suggest a possible role of the CYP2B6 genetic background on the development of specific chromosomal aberrations. PMID:24586425

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

    PubMed

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

    2016-01-01

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

  1. CYP2B6 haplotype and biological factors responsible for hepatotoxicity in HIV-infected patients receiving efavirenz-based antiretroviral therapy.

    PubMed

    Manosuthi, Weerawat; Sukasem, Chonlaphat; Lueangniyomkul, Aroon; Mankatitham, Wiroj; Thongyen, Supeda; Nilkamhang, Samruay; Manosuthi, Sukanya; Sungkanuparph, Somnuek

    2014-03-01

    Data on the pharmacogenetic markers of CYP2B6 and biological factors associated with hepatotoxicity in HIV-infected patients receiving an efavirenz-based antiretroviral therapy (ART) regimen are very limited. A total of 134 HIV-infected Thai adults were prospectively enrolled to receive a once-daily regimen of efavirenz 600 mg/tenofovir/lamivudine. Seven single nucleotide polymorphisms (SNPs) within CYP2B6 were genotyped using real-time PCR. At 12 weeks after ART, plasma efavirenz concentrations at 12h after dosing were measured. The mean ± standard deviation patient age was 37 ± 8 years, and 77.6% were male. The median (IQR) CD4 count was 43 cells/mm(3) (17-105 cells/mm(3)). Eighteen patients (13.4%) had positive anti-HCV and 5 patients (3.7%) had positive HBsAg. The frequencies of heterozygous/homozygous mutants of each SNP were 64C>T (11%), 499C>G (0%), 516G>T (55%), 785A>G (63%), 1375A>G (0%), 1459C>T (3%) and 21563C>T (62%). The three most frequent haplotypes identified included *1/*6 (40.3%), *1/*1 (34.3%) and *6/*6 (8.2%). The median (IQR) plasma efavirenz concentration was 2.3mg/L (1.4-3.7 mg/L). At 24 weeks, median (IQR) serum ALP was 98 mg/dL (73-133 mg/dL) and direct bilirubin was 0.11 mg/dL (0.10-0.19 mg/dL). The proportion of grade 1 and grade 2 elevated serum ALP was 12.7% and 1.5%, respectively. By multivariate analysis, factors associated with high ALP, total bilirubin and direct bilirubin included CYP2B6 haplotype *6/*6, high serum ALP at Week 0 and positive anti-HCV (all P<0.05). In summary, HIV-infected patients with the pharmacogenetic marker 'CYP2B6 haplotype *6/*6' may have increased susceptibility to hepatotoxicity with efavirenz-based ART.

  2. Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1, CYP1A2, and CYP2C9.

    PubMed

    Otake, Yoko; Walle, Thomas

    2002-02-01

    There is very limited information on cytochrome P450 (P450)-mediated oxidative metabolism of dietary flavonoids in humans. In this study, we used human liver microsomes and recombinant P450 isoforms to examine the metabolism of two flavonols, galangin and kaempferide, and one flavone, chrysin. Both galangin and kaempferide, but not chrysin, were oxidized by human liver microsomes to kaempferol, with K(m) values of 9.5 and 17.8 microM, respectively. These oxidations were catalyzed mainly by CYP1A2 but also by CYP2C9. Consistent with these observations, the human liver microsomal metabolism of galangin and kaempferide were inhibited by the P450 inhibitors furafylline and sulfaphenazole. In addition, CYP1A1, although less efficient, was also able to oxidize the two flavonols. Thus, dietary flavonols are likely to undergo oxidative metabolism mainly in the liver but also extrahepatically.

  3. Construction of Metabolism Prediction Models for CYP450 3A4, 2D6, and 2C9 Based on Microsomal Metabolic Reaction System

    PubMed Central

    He, Shuai-Bing; Li, Man-Man; Zhang, Bai-Xia; Ye, Xiao-Tong; Du, Ran-Feng; Wang, Yun; Qiao, Yan-Jiang

    2016-01-01

    During the past decades, there have been continuous attempts in the prediction of metabolism mediated by cytochrome P450s (CYP450s) 3A4, 2D6, and 2C9. However, it has indeed remained a huge challenge to accurately predict the metabolism of xenobiotics mediated by these enzymes. To address this issue, microsomal metabolic reaction system (MMRS)—a novel concept, which integrates information about site of metabolism (SOM) and enzyme—was introduced. By incorporating the use of multiple feature selection (FS) techniques (ChiSquared (CHI), InfoGain (IG), GainRatio (GR), Relief) and hybrid classification procedures (Kstar, Bayes (BN), K-nearest neighbours (IBK), C4.5 decision tree (J48), RandomForest (RF), Support vector machines (SVM), AdaBoostM1, Bagging), metabolism prediction models were established based on metabolism data released by Sheridan et al. Four major biotransformations, including aliphatic C-hydroxylation, aromatic C-hydroxylation, N-dealkylation and O-dealkylation, were involved. For validation, the overall accuracies of all four biotransformations exceeded 0.95. For receiver operating characteristic (ROC) analysis, each of these models gave a significant area under curve (AUC) value >0.98. In addition, an external test was performed based on dataset published previously. As a result, 87.7% of the potential SOMs were correctly identified by our four models. In summary, four MMRS-based models were established, which can be used to predict the metabolism mediated by CYP3A4, 2D6, and 2C9 with high accuracy. PMID:27735849

  4. Novel drug metabolism indices for pharmacogenetic functional status based on combinatory genotyping of CYP2C9, CYP2C19 and CYP2D6 genes

    PubMed Central

    Villagra, David; Goethe, John; Schwartz, Harold I; Szarek, Bonnie; Kocherla, Mohan; Gorowski, Krystyna; Windemuth, Andreas; Ruaño, Gualberto

    2011-01-01

    Aims We aim to demonstrate clinical relevance and utility of four novel drug-metabolism indices derived from a combinatory (multigene) approach to CYP2C9, CYP2C19 and CYP2D6 allele scoring. Each index considers all three genes as complementary components of a liver enzyme drug metabolism system and uniquely benchmarks innate hepatic drug metabolism reserve or alteration through CYP450 combinatory genotype scores. Methods A total of 1199 psychiatric referrals were genotyped for polymorphisms in the CYP2C9, CYP2C19 and CYP2D6 gene loci and were scored on each of the four indices. The data were used to create distributions and rankings of innate drug metabolism capacity to which individuals can be compared. Drug-specific indices are a combination of the drug metabolism indices with substrate-specific coefficients. Results The combinatory drug metabolism indices proved useful in positioning individuals relative to a population with regard to innate drug metabolism capacity prior to pharmacotherapy. Drug-specific indices generate pharmacogenetic guidance of immediate clinical relevance, and can be further modified to incorporate covariates in particular clinical cases. Conclusions We believe that this combinatory approach represents an improvement over the current gene-by-gene reporting by providing greater scope while still allowing for the resolution of a single-gene index when needed. This method will result in novel clinical and research applications, facilitating the translation from pharmacogenomics to personalized medicine, particularly in psychiatry where many drugs are metabolized or activated by multiple CYP450 isoenzymes. PMID:21861665

  5. CCAAT/enhancer-binding protein alpha (C/EBPalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha) synergistically cooperate with constitutive androstane receptor to transactivate the human cytochrome P450 2B6 (CYP2B6) gene: application to the development of a metabolically competent human hepatic cell model.

    PubMed

    Benet, Marta; Lahoz, Agustín; Guzmán, Carla; Castell, José V; Jover, Ramiro

    2010-09-10

    The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/enhancer-binding protein alpha (C/EBPalpha), hepatocyte nuclear factor 4alpha (HNF4alpha), and constitutive androstane receptor, the CYP2B6 expression and inducibility by CITCO are restored in human hepatoma HepG2 cells at levels similar to those in cultured human hepatocytes. Moreover, several other phase I and II genes are simultaneously activated, which suggests that this is an effective approach to endow dedifferentiated human hepatoma cells with a particular metabolic competence and response to inducers. In order to gain insight into the molecular mechanism, we examined the cooperation of these three transcription factors on the CYP2B6 5'-flanking region. We show new CYP2B6-responsive sequences for C/EBPalpha and HNF4alpha and a novel synergistic regulatory mechanism whereby C/EBPalpha, HNF4alpha, and constitutive androstane receptor bind and cooperate through proximal and distal response elements to confer a maximal level of expression. The results obtained from human liver also suggest that important differences in the expression and binding of C/EBPalpha and HNF4alpha could account for the large interindividual variability of the hepatic CYP2B6 enzyme, which metabolizes commonly used drugs.

  6. High density H2 associative absorption on Titanium alpha-borozene (Ti2B6H6): An ab-initio case study

    NASA Astrophysics Data System (ADS)

    Akbarzadeh, Alireza; Tymzcak, C. J.

    2011-03-01

    Hydrogen is considered as a clean energy carrier that could be a future replacement for our addiction to fossil fuels. However, in order to have hydrogen economy at its highest efficiently we need to store hydrogen at high volumetric and gravimetric density. Using the all electron hybrid density functional theory, we have designed a benzene-like-molecule, Ti2B6H6, which has the promise of achieving this goal. Our results show that the molecule can associatively absorb the hydrogen up to ten percent by weight of hydrogen, which exceeds the 2015 US department of energy target. In this presentation we will discuss the mechanisms of H2 absorption and possible applications of this novel molecule. This research is funded by the Welch Foundation under Grant J. 1675 and the Texas Southern University High Performance Computing Center.

  7. The prognostic values of CYP2B6 genetic polymorphisms and metastatic sites for advanced breast cancer patients treated with docetaxel and thiotepa.

    PubMed

    Song, Qingkun; Zhou, Xinna; Yu, Jing; Dong, Ningning; Wang, Xiaoli; Yang, Huabing; Ren, Jun; Lyerly, H Kim

    2015-11-25

    This study investigated interactive effects of CYP2B6 genotypes and liver metastasis on the prognosis of metastatic breast cancer patients who received combined chemotherapy of docetaxel and thiotepa. Totally 153 patients were retrospectively genotyped rs8192719 (c.1294 + 53C > T) and rs2279343 (c.785A > G). Kaplan-Meier method and Cox Proportional Hazard Regression model were used to estimate the survival. Patients with liver metastasis had worsen prognosis, conferring a 2.26-fold high risk of progression and 1.93-fold high risk of death (p < 0.05). Both CT/TT genotype of rs8192719 (c.1294 +  3C > T) and AG genotype of rs2279343 (c.785A > G) prolonged survival (p < 0.05). Furthermore, among liver metastatic patients, AG genotype of rs2279343 (c.785A > G) was associated with a 47% reduced risk of death and a 6-month-longer overall survival (p < 0.05). Among non-liver metastatic patients, hazard ratios of CT/TT genotype of rs8192719 (c.1294 + 53C > T) were 0.45 for progression and 0.40 for death; and the corresponding survival was improved by 6 months and 16 months, respectively (p < 0.05). Genotypes of CYP2B6 had an interaction with clinical efficacy of docetaxel and thiotepa on metastatic breast cancer patients; and metastatic sites also affected clinical responses. Further therapies should take into account of chemotherapy regimen, genotypes of metabolizing enzymes and metastatic sites for the particular subpopulation.

  8. Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers.

    PubMed

    Huang, Guoxiang; Guo, Huan; Wu, Tangchun

    2012-06-20

    Polycyclic aromatic hydrocarbons (PAHs), the main components of coke oven emissions, can induce activation of cytochrome P450 (CYP) enzymes, which metabolize PAHs and result in DNA damage by forming adducts. This study was designed to know whether genetic variants of CYP genes are associated with plasma benzo[a]pyrene-7,8-diol-9,10-epoxide-albumin (BPDE-Alb) adducts and DNA damage in coke oven workers. In this study, 298 workers were divided into four groups according to the environmental PAHs exposure levels. The concentrations of plasma BPDE-Alb adducts were detected by reverse-phase high-performance liquid chromatography and the DNA damage levels were measured using comet assay. Twelve tag single nucleotide polymorphisms (tagSNPs) of 4 CYP genes were selected and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the top group, workers with CYP2B6 rs3760657GA genotype have lower BPDE-Alb adducts and DNA damage levels than those with rs3760657GG genotype (P<0.05). In the control group, the DNA damage levels of subjects with CYP1A1 rs4646421AA or GA+AA genotypes were lower than those with GG genotype (P<0.05). However, no such effects were shown for the other tagSNPs. These results suggested that genetic variations of CYP2B6 might be associated with low BPDE-Alb adducts and DNA damage levels in worker with high exposure to PAHs.

  9. In Vitro Inhibition of Human CYP450s 1A2, 2C9, 3A4/5, 2D6 and 2E1 by Grandisin.

    PubMed

    Habenschus, Maísa Daniela; Moreira, Fernanda de Lima; Lopes, Norberto Peporine; de Oliveira, Anderson R M

    2017-01-10

    Grandisin, a lignan isolated from many species of plants, such as Virola surinamensis, is a potential drug candidate due to its biological properties, highlighted by its antitumor and trypanocidal activities. In this study, the inhibitory effects of grandisin on the activities of human cytochrome P450 enzymes were investigated by using human liver microsomes. Results showed that grandisin is a competitive inhibitor of CYP2C9 and a competitive and mechanism-based inhibitor of CYP3A4/5. The apparent Ki value for CYP2C9 was 50.60 µM and those for CYP3A4/5 were 48.71 µM and 31.25 µM using two different probe substrates, nifedipine and midazolam, respectively. The apparent KI, kinact, and kinact/KI ratio for the mechanism-based inhibition of CYP3A4/5 were 6.40 µM, 0.037 min(-1), and 5.78 mL · min(-1) µmol(-1), respectively, by examining nifedipine oxidation, and 31.53 µM, 0.049 min(-1), and 1.55 mL · min(-1) µmol(-1), respectively, by examining midazolam 1'-hydroxylation. These apparent kinact/KI values were comparable to or even higher than those for several therapeutic drugs that act as mechanism-based inhibitors of CYP3A4/5. CYP1A2 and CYP2D6 activities, in turn, were not substantially inhibited by grandisin (IC50 > 200 µM and 100 µM, respectively). In contrast, from a concentration of 4 µM, grandisin significantly stimulated CYP2E1 activity. These results improve the prediction of grandisin-drug interactions, suggesting that the risk of interactions with drugs metabolized by CYP3A4/5 and CYP2E1 cannot be overlooked.

  10. Harnessing Knowledge on Very Important Pharmacogenes CYP2C9 and CYP2C19 Variation for Precision Medicine in Resource-Limited Global Conflict Zones.

    PubMed

    Barlas, İbrahim Ömer; Sezgin, Orhan; Dandara, Collet; Türköz, Gözde; Yengel, Emre; Cindi, Zinhle; Ankaralı, Handan; Şardaş, Semra

    2016-10-01

    Pharmacogenomics harnesses the utility of a patient's genome (n = 1) in decisions on which therapeutic drugs and in what amounts should be administered. Often, patients with shared ancestry present with comparable genetic profiles that predict drug response. However, populations are not static, thus, often, population mobility through migration, especially enmasse as is seen for refugees, changes the pharmacogenetic profiles of resultant populations and therefore observed responses to commonly used therapeutic drugs. For example, in the aftermath of the Syrian civil war since 2011, millions have fled their homes to neighboring countries in the Middle East. The growing permanence of refugees and mass migrations is a call to shift our focus in the life sciences community from old models of pharmaceutical innovation. These seismic social changes demand faster decisions for "population-to-population bridging," whereby novel drugs developed in or for particular regions/countries can meet with rational regulatory decisions/approval in world regions impacted by migrant/refugee populations whose profiles are dynamic, such as in the Eastern Mediterranean region at present. Thus, it is important to characterize and report on the prevalence of pharmacogenes that affect commonly used medications and predict if population changes may call for attention to particular differences that may impact health of patients. Thus, we report here on four single-nucleotide polymorphism (SNP) variations in CYP2C9 and CYP2C19 genes among Mersin-Turkish healthy volunteers in the Mersin Province in the Eastern Mediterranean region that is currently hosting a vast number of migrant populations from Syria. Both CYP2C9 and CYP2C19 are very important pharmacogene molecular targets. We compare and report here on the observed SNP genetic variation in our sample with data on 12 world populations from dbSNP and discuss the feasibility of forecasting the pharmacokinetics of drugs utilized by migrant

  11. Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid.

    PubMed

    Melet, Armelle; Assrir, Nadine; Jean, Pascale; Pilar Lopez-Garcia, Maria; Marques-Soares, Cristina; Jaouen, Maryse; Dansette, Patrick M; Sari, Marie Agnès; Mansuy, Daniel

    2003-01-01

    A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate

  12. Interaction of new sulfaphenazole derivatives with human liver cytochrome p450 2Cs: structural determinants required for selective recognition by CYP 2C9 and for inhibition of human CYP 2Cs.

    PubMed

    Ha-Duong, N T; Marques-Soares, C; Dijols, S; Sari, M A; Dansette, P M; Mansuy, D

    2001-10-15

    A series of new derivatives of sulfaphenazole (SPA), in which the NH(2) and phenyl substituents of SPA are replaced by various groups or in which the sulfonamide function of SPA is N-alkylated, were synthesized in order to further explore CYP 2C9 active site and to determine the structural factors explaining the selectivity of SPA for CYP 2C9 within the human P450 2C subfamily. Compounds in which the NH(2) group of SPA was replaced with R(1) = CH(3), Br, CH = CH(2), CH(2)CH = CH(2), and CH(2)CH(2)OH exhibited a high affinity for CYP 2C9, as shown by the dissociation constant of their CYP 2C9 complexes, K(s), which was determined by difference visible spectroscopy (K(s) between 0.1 and 0.4 microM) and their constant of CYP 2C9 inhibition (K(i) between 0.3 and 0.6 microM). This indicates that the CYP 2C9-iron(III)-NH(2)R bond previously described to exist in the CYP 2C9-SPA complex does not play a key role in the high affinity of SPA for CYP 2C9. Compounds in which the phenyl group of SPA was replaced with various aryl or alkyl R(2) substituents only exhibited a high affinity for CYP 2C9 if R(2) is a freely rotating and sufficiently electron-rich aryl substituent. Finally, compounds resulting from a N-alkylation of the SPA sulfonamide function (R(3) = CH(3), C(2)H(5), or C(3)H(7)) did not retain the selective inhibitory properties of SPA toward CYP 2C9. However, they are reasonably good inhibitors of CYP 2C8 and CYP 2C18 (IC(50) approximately 20 microM). These data allow one to better understand the structural factors that are important for selective binding in the CYP 2C9 active site. They also provide us with clues towards new selective inhibitors of CYP 2C8 and CYP 2C18.

  13. Characterizing the effect of cytochrome P450 (CYP) 2C8, CYP2C9, and CYP2D6 genetic polymorphisms on stereoselective N-demethylation of fluoxetine.

    PubMed

    Wang, Zhangting; Wang, Shengjia; Huang, Minmin; Hu, Haihong; Yu, Lushan; Zeng, Su

    2014-03-01

    Fluoxetine (FLX) is one of the most widely prescribed selective serotonin reuptake inhibitors. Although FLX is used as racemate in the clinic, the clinical pharmacokinetics of FLX and its N-demethylation metabolite norfluoxetine (NFLX) show obvious cytochrome P450 (CYP) polymorphism dependency and exhibit marked stereoselectivity. However, the kinetic profiles of CYP variants to FLX remain unclear. In the present study, some variants of human CYP2C8, CYP2C9, and CYP2D6 were first expressed in insect cells, and their catalytic roles with respect to FLX enantiomers were then investigated. CYP2C8.4 and CYP2C9.10 showed significantly lower activity and CYP2C8.3 showed significantly higher activity toward both R- and S-FLX compared with the wildtype, while CYP2C9.3, CYP2C9.13, and CYP2C9.16 showed significantly lower activity only toward R-FLX. Five CYP2C9 variants and CYP2D6.1 exhibited significantly stereoselective kinetic profiles prior to R-FLX, and CYP2C8.3 showed a slight stereoselectivity. Interestingly, obvious substrate inhibition was observed in the CYP2C9 wildtype and its three variants only in the case of R-FLX. Together, these findings suggest that CYP2C9 and CYP2D6 polymorphism may play an important role in the clearance of FLX and also in the stereoselective kinetic profiles of FLX enantiomers.

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

    PubMed

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

    2015-02-10

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

  15. Efavirenz and Metabolites in Cerebrospinal Fluid: Relationship with CYP2B6 c.516G→T Genotype and Perturbed Blood-Brain Barrier Due to Tuberculous Meningitis

    PubMed Central

    Chau, Tran Thi Hong; Fisher, Martin; Nelson, Mark; Winston, Alan; Else, Laura; Carr, Daniel F.; Taylor, Steven; Ustianowski, Andrew; Back, David; Pirmohamed, Munir; Solomon, Tom; Farrar, Jeremy; Törok, M. Estée; Khoo, Saye

    2016-01-01

    Efavirenz (EFZ) has been associated with neuropsychiatric side effects. Recently, the 8-hydroxy-EFZ (8OH-EFZ) metabolite has been shown to be a potent neurotoxin in vitro, inducing neuronal damage at concentrations of 3.3 ng/ml. EFZ induced similar neuronal damage at concentrations of 31.6 ng/ml. We investigated the effect of genotype and blood-brain barrier integrity on EFZ metabolite concentrations in cerebrospinal fluid (CSF). We measured CSF drug concentrations in subjects from two separate study populations: 47 subjects with tuberculous meningitis (TBM) coinfection in Vietnam receiving 800 mg EFZ with standard antituberculous treatment and 25 subjects from the PARTITION study in the United Kingdom without central nervous system infection receiving 600 mg EFZ. EFZ and metabolite concentrations in CSF and plasma were measured and compared with estimates of effectiveness and neurotoxicity from available published in vitro and in vivo data. The effect of the CYP2B6 c.516G→T genotype (GG genotype, fast EFV metabolizer status; GT genotype, intermediate EFV metabolizer status; TT genotype, slow EFV metabolizer status) was examined. The mean CSF concentrations of EFZ and 8OH-EFZ in the TBM group were 60.3 and 39.3 ng/ml, respectively, and those in the no-TBM group were 15.0 and 5.9 ng/ml, respectively. Plasma EFZ and 8OH-EFZ concentrations were similar between the two groups. CSF EFZ concentrations were above the in vitro toxic concentration in 76% of samples (GG genotype, 61%; GT genotype, 90%; TT genotype, 100%) in the TBM group and 13% of samples (GG genotype, 0%; GT genotype, 18%; TT genotype, 50%) in the no-TBM group. CSF 8OH-EFZ concentrations were above the in vitro toxic concentration in 98% of the TBM group and 87% of the no-TBM group; levels were independent of genotype but correlated with the CSF/plasma albumin ratio. Potentially neurotoxic concentrations of 8OH-EFZ are frequently observed in CSF independently of the CYP2B6 genotype, particularly in those

  16. Genetic variation in the CYP2B6 Gene is related to circulating 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) concentrations: an observational population-based study

    PubMed Central

    2014-01-01

    Background Since human CYP2B6 has been identified as the major CYP enzyme involved in the metabolism of 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47) and that human 2B6 is a highly polymorphic CYP, with known functional variants, we evaluated if circulating concentrations of a major brominated flame retardant, BDE-47, were related to genetic variation in the CYP2B6 gene in a population sample. Methods In the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (men and women all aged 70), 25 single nucleotide polymorphisms (SNPs) in the CYP2B6 gene were genotyped. Circulating concentrations of BDE-47 were analyzed by high-resolution gas chromatography coupled to high-resolution mass spectrometry (HRGC/ HRMS). Results Several SNPs in the CYP2B6 gene were associated with circulating concentrations of BDE-47 (P = 10-4 to 10-9). The investigated SNPs came primarily from two haplotypes, although the correlation between the haplotypes was rather high. Conditional analyses adjusting for the SNP with the strongest association with the exposure (rs2014141) did not provide evidence for independent signals. Conclusion Circulating concentrations of BDE-47 were related to genetic variation in the CYP2B6 gene in an elderly population. PMID:24885815

  17. Phytoremediation of the herbicides atrazine and metolachlor by transgenic rice plants expressing human CYP1A1, CYP2B6, and CYP2C19.

    PubMed

    Kawahigashi, Hiroyuki; Hirose, Sakiko; Ohkawa, Hideo; Ohkawa, Yasunobu

    2006-04-19

    This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.

  18. A Quantitative High-Throughput 96-well plate Fluorescence Assay for Mechanism-Based Inactivators of Cytochromes P450 Exemplified using CYP2B6

    PubMed Central

    Kenaan, Cesar; Zhang, Haoming; Hollenberg, Paul F.

    2010-01-01

    Mechanism-based inactivators such as bergamottin are useful chemical tools for identifying the roles of specific active-site amino acid residues in the reactions catalyzed by the cytochromes P450 (CYPs or P450s) that are responsible for the metabolism of a wide variety of drugs and endogenous substrates. In clinical settings mechanism-based inactivation of P450s involved in xenobiotic metabolism has the potential to lead to adverse drug-drug interactions and assays to identify and characterize drug candidates as P450 inactivators are important in drug discovery and development. Here we present a quantitative high-throughput protocol for investigating cytochrome P450 mechanism-based inactivators using the example of CYP2B6 and bergamottin to illustrate the finer points of this protocol. This protocol details the adaptation of a 7-ethoxytrifluoromethyl coumarin (7-EFC) O-deethylation fluorescence activity assay to a 96-well microtiter plate format and uses a plate-reader to detect the fluorescence of the product. Compared to previous methods, this protocol requires less P450 and takes significantly less time while greatly increasing throughput. The protocol as written takes approximately two hours to complete. The principles and procedures outlined in this protocol can be easily adapted to other inactivators, P450 isoforms, substrates and plate-readers. PMID:20885377

  19. Chemical mass balance receptor model applied to ambient C 2-C 9 VOC concentration in Seoul, Korea: Effect of chemical reaction losses

    NASA Astrophysics Data System (ADS)

    Na, Kwangsam; Pyo Kim, Yong

    A chemical mass balance (CMB) receptor model was used for estimating the diurnal contributions of VOC emission sources to the ambient C 2-C 9 VOC concentration in Seoul, Korea. For this purpose, the VOC concentrations were measured in the morning, the afternoon, and the evening. The samples were collected using a 2-h integrated SUMMA canister. The source profiles were developed for the CMB calculation in the Seoul area. To investigate the effect of the chemical reaction loss of VOCs on the CMB calculation, the modified model employing a decay factor and the standard model that considers no loss were compared. The modified model estimated that the vehicle exhaust (52%) was the largest leading source of VOCs in the Seoul atmosphere, followed by the use of solvents (26%), gasoline evaporation (15%), the use of liquefied petroleum gas (LPG) (5%), and the use of liquefied natural gas (LNG) (2%). Relative source contribution for vehicle exhaust showed a clear diurnal variation with a high in the morning and evening and a low in the afternoon, while the contribution of evaporative emissions (gasoline evaporation and solvent usage) showed a different diurnal pattern from that of the vehicle exhaust, exhibiting a high in the afternoon and evening and a low in the morning. It was found that the difference between the total source contribution (μg m -3) estimated from these two models was not statistically significant. However, when the paired-sample t-test is applied to the individual sources, a significant difference was found for the vehicle exhaust and the solvent use. In addition, the modified model brought forth a better performance with high R2 and low χ2 as compared to those obtained from the standard model in the CMB calculation. The vehicle exhaust and solvent use were estimated to be the largest and the second largest contributors to ambient benzene as well as ozone formation potential (OFP), respectively. Based on above results we believe that incorporating the

  20. Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0-Å Resolution

    PubMed Central

    Shah, Manish B.; Talakad, Jyothi C.; Maekawa, Keiko; Roberts, Arthur G.; Wilderman, P. Ross; Sun, Ling; Yang, Jane Y.; Huelga, Stephanie C.; Hong, Wen-Xu; Zhang, Qinghai; Stout, C. David; Halpert, James R.

    2010-01-01

    The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures. PMID:20061448

  1. Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

    PubMed

    Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

    2010-04-01

    The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

  2. Vitamins B2, B6 and B12 and Risk of New Colorectal Adenomas in a Randomized Trial of Aspirin Use and Folic Acid Supplementation

    PubMed Central

    Figueiredo, Jane C.; Levine, A. Joan; Grau, Maria V.; Midttun, Øivind; Ueland, Per M.; Ahnen, Dennis J.; Barry, Elizabeth L.; Tsang, Shirley; Munroe, David; Ali, Iqbal; Haile, Robert W.; Sandler, Robert S.; Baron, John A.

    2008-01-01

    Background Folate, other vitamin B cofactors, and genes involved in folate-mediated one-carbon metabolism (FOCM) all may play important roles in colorectal neoplasia. In this study, we examined the associations between dietary and circulating plasma levels of vitamins B2, B6 and B12 and risk colorectal adenomas. Methods The Aspirin/Folate Polyp Prevention Study is a randomized clinical trial of folic acid supplementation and incidence of new colorectal adenomas in individuals with a history of adenomas (n=1,084). Diet and supplement use were ascertained through a food-frequency questionnaire administered at baseline. Blood collected at baseline was used to determine plasma B-vitamin levels. We used generalized linear regression to estimate risk ratios (RR) and 95% confidence limits (CI) as measures of association. Results We found a borderline significant inverse association with plasma B6 (pyridoxal 5′phosphate, PLP) and adenoma risk (adjusted RR Q4 vs. Q1=0.78, 95% CI=0.61-1.00, p-trend=0.08). This association was not modified by folic acid supplementation or plasma folate. However, the protective association of PLP with adenoma risk was observed only among subjects who did not drink alcohol (p-interaction=0.03). Plasma B2 (riboflavin) was inversely associated with risk of advanced lesions (adjusted RR Q4 vs. Q1=0.51; 95% CI=0.26-0.99, p-trend=0.12). No significant associations were observed between adenoma risk and plasma vitamin B12 or dietary intake of vitamin B2 and B6. When we examined specific gene-B-vitamin interactions, we observed a possible interaction between MTHFR-C677T and plasma B2 on risk of all adenomas. Conclusion Our results suggest that high levels of PLP and B2 may protect against colorectal adenomas. PMID:18708408

  3. Multiplex pyrosequencing method to determine CYP2C9*3, VKORC1*2, and CYP4F2*3 polymorphisms simultaneously: its application to a Korean population and comparisons with other ethnic groups.

    PubMed

    Kim, Kyoung-Ah; Song, Wan-Geun; Lee, Hae-Mi; Joo, Hyun-Jin; Park, Ji-Young

    2014-11-01

    Warfarin is an anticoagulant that is difficult to administer because of the wide variation in dose requirements to achieve a therapeutic effect. CYP2C9, VKROC1, and CYP4F2 play important roles in warfarin metabolism, and their genetic polymorphisms are related to the variability in dose determination. In this study we describe a new multiplex pyrosequencing method to identify CYP2C9*3 (rs1057910), VKORC1*2 (rs9923231), and CYP4F2*3 (rs2108661) simultaneously. A multiplex pyrosequencing method to simultaneously detect CYP2C9*3, VKORC1*2, and CYP4F2*3 alleles was designed. We assessed the allele frequencies of the polymorphisms in 250 Korean subjects using the multiplex pyrosequencing method. The results showed 100 % concordance between single and multiplex pyrosequencing methods, and the polymorphisms identified by pyrosequencing were also validated with the direct sequencing method. The allele frequencies of these polymorphisms in this population were as follows: 0.040 for CYP2C9*3, 0.918 for VKORC1*2, and 0.416 for CYP4F2*3. Although the allele frequencies of the CYP2C9*3 and VKROC1*2 were comparable to those in Japanese and Chinese populations, their frequencies in this Korean population differed from those in other ethnic groups; the CYP4F2*3 frequency was the highest among other ethnic populations including Chinese and Japanese populations. The pyrosequencing methods developed were rapid and reliable for detecting CYP2C9*3, VKORC1*2, and CYP4F2*3. Large ethnic differences in the frequency of these genetic polymorphisms were noted among ethnic groups. CYP4F2*3 exhibited its highest allele frequency among other ethnic populations compared to that in a Korean population.

  4. Microdose pharmacogenetic study of ¹⁴C-tolbutamide in healthy subjects with accelerator mass spectrometry to examine the effects of CYP2C9∗3 on its pharmacokinetics and metabolism.

    PubMed

    Ikeda, Toshihiko; Aoyama, Shinsuke; Tozuka, Zenzaburo; Nozawa, Kohei; Hamabe, Yoshimi; Matsui, Takao; Kainuma, Michiko; Hasegawa, Setsuo; Maeda, Kazuya; Sugiyama, Yuichi

    2013-07-16

    Microdose study enables us to understand the pharmacokinetic profiles of drugs in humans prior to the conventional clinical trials. The advantage of microdose study is that the unexpected pharmacological/toxicological effects of drugs caused by drug interactions or genetic polymorphisms of metabolic enzymes/transporters can be avoided due to the limited dose. With a combination use of accelerator mass spectrometry (AMS) and (14)C-labaled compounds, the pharmacokinetics of both parent drug and its metabolites can be sensitively monitored. Thus, to demonstrate the usability of microdose study with AMS for the prediction of the impact of genetic polymorphisms of CYP enzyme on the pharmacokinetics of unchanged drugs and metabolites, we performed microdose pharmacogenetic study using tolbutamide as a CYP2C9 probe drug. A microdose of (14)C-tolbutamide (100 μg) was administered orally to healthy volunteers with the CYP2C9(∗)1/(∗)1 or CYP2C9(∗)1/(∗)3 diplotype. Area under the plasma concentration-time curve for the (14)C-radioactivity, determined by AMS, or that for the parent drug, determined by liquid chromatography/mass spectrometry, was about 1.6 times or 1.7 times greater in the CYP2C9(∗)1/(∗)3 than in the CYP2C9(∗)1/(∗)1 group, which was comparable to the previous reports at therapeutic dose. In the plasma and urine, tolbutamide, carboxytolbutamide, and 4-hydroxytolbutamide were detected and practically no other metabolites could be found in both diplotype groups. The fraction of metabolites in plasma radioactivity was slightly lower in the CYP2C9(∗)1/(∗)3 group. Microdose study can be used for the prediction of the effects of genetic polymorphisms of enzymes on the pharmacokinetics and metabolic profiles of drugs with minimal care of their pharmacological/toxicological effects.

  5. Variation in Genes Controlling Warfarin Disposition and Response in American Indian and Alaska Native People: CYP2C9, VKORC1, CYP4F2, CYP4F11, GGCX

    PubMed Central

    Yracheta, Joseph; Dillard, Denise A.; Schilling, Brian; Khan, Burhan; Hopkins, Scarlett; Boyer, Bert; Black, Jynene; Wiener, Howard; Tiwari, Hemant K.; Gordon, Adam; Nickerson, Deborah; Tsai, Jesse M.; Farin, Federico M.; Thornton, Timothy A.; Rettie, Allan E.; Thummel, Kenneth E.

    2015-01-01

    Objectives Pharmacogenetic testing is projected to improve health outcomes and reduce the cost of care by increasing therapeutic efficacy and minimizing drug toxicity. American Indian and Alaska Native (AI/AN) people historically have been excluded from pharmacogenetic research and its potential benefits, a deficiency we sought to address. The vitamin K antagonist warfarin is prescribed for prevention of thromboembolic events, although its narrow therapeutic index and wide inter-individual variability necessitate close monitoring of drug response. Therefore, we were interested in variation in CYP2C9, VKORC1, CYP4F2, CYP4F11, and GGCX, which encode enzymes important for the activity of warfarin and synthesis of vitamin K dependent blood clotting factors. Methods We resequenced these genes in 188 AI/AN people in partnership with Southcentral Foundation (SCF) in Anchorage, AK and 94 Yup'ik people living in the Yukon-Kuskokwim Delta of southwest Alaska to identify known or novel function-disrupting variation. We conducted genotyping for specific SNPs in larger cohorts of each study population (380 and 350, respectively). Results We identified high frequencies of the lower-warfarin dose VKORC1 haplotype (−1639G>A and 1173C>T) and the higher-warfarin dose CYP4F2*3 variant. We also identified two relatively common, novel, and potentially function-disrupting variants in CYP2C9 (M1L and N218I), which, along with CYP2C9*3, CYP2C9*2 and CYP2C9*29, predict that a significant proportion of AI/AN people will have decreased CYP2C9 activity. Conclusions Overall, we predict a lower average warfarin dose requirement in AI/AN populations in Alaska than that seen in non-AI/AN populations of the US, a finding consistent with clinical experience in Alaska. PMID:25946405

  6. 42 CFR 2a.6 - Issuance of Confidentiality Certificates; single project limitation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Issuance of Confidentiality Certificates; single project limitation. 2a.6 Section 2a.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.6 Issuance of...

  7. 42 CFR 2a.6 - Issuance of Confidentiality Certificates; single project limitation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Issuance of Confidentiality Certificates; single project limitation. 2a.6 Section 2a.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.6 Issuance of...

  8. 42 CFR 2a.6 - Issuance of Confidentiality Certificates; single project limitation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Issuance of Confidentiality Certificates; single project limitation. 2a.6 Section 2a.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.6 Issuance of...

  9. 42 CFR 2a.6 - Issuance of Confidentiality Certificates; single project limitation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Issuance of Confidentiality Certificates; single project limitation. 2a.6 Section 2a.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.6 Issuance of...

  10. 42 CFR 2a.6 - Issuance of Confidentiality Certificates; single project limitation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Issuance of Confidentiality Certificates; single project limitation. 2a.6 Section 2a.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL PROVISIONS PROTECTION OF IDENTITY-RESEARCH SUBJECTS § 2a.6 Issuance of...

  11. Structures of Cytochrome P450 2B6 Bound to 4-Benzylpyridine and 4-(4-Nitrobenzyl)pyridine: Insight into Inhibitor Binding and Rearrangement of Active Site Side Chains

    PubMed Central

    Pascual, Jaime; Zhang, Qinghai; Stout, C. David; Halpert, James R.

    2011-01-01

    The biochemical, biophysical, and structural analysis of the cytochrome P450 2B subfamily of enzymes has provided a wealth of information regarding conformational plasticity and substrate recognition. The recent X-ray crystal structure of the drug-metabolizing P450 2B6 in complex with 4-(4-chlorophenyl)imidazole (4-CPI) yielded the first atomic view of this human enzyme. However, knowledge of the structural basis of P450 2B6 specificity and inhibition has remained limited. In this study, structures of P450 2B6 were determined in complex with the potent inhibitors 4-benzylpyridine (4-BP) and 4-(4-nitrobenzyl)pyridine (4-NBP). Comparison of the present structures with the previous P450 2B6-4-CPI complex showed that reorientation of side chains of the active site residue Phe206 on the F-helix and Phe297 on the I-helix was necessary to accommodate the inhibitors. However, P450 2B6 does not require any major side chain rearrangement to bind 4-NBP compared with 4-BP, and the enzyme provides no hydrogen-bonding partners for the polar nitro group of 4-NBP within the hydrophobic active site. In addition, on the basis of these new structures, substitution of residue 172 with histidine as observed in the single nucleotide polymorphism Q172H and in P450 2B4 may contribute to a hydrogen bonding network connecting the E- and I-helices, thereby stabilizing active site residues on the I-helix. These results provide insight into the role of active site side chains upon inhibitor binding and indicate that the recognition of the benzylpyridines in the closed conformation structure of P450 2B6 is based solely on hydrophobicity, size, and shape. PMID:21875942

  12. Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction.

    PubMed

    Nirogi, Ramakrishna; Palacharla, Raghava Choudary; Mohammed, Abdul Rasheed; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Bhyrapuneni, Gopinadh

    2015-03-25

    The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug-drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5 ± 0.3 and k(inact) value of 0.045 ± 0.001 min(-1). Phenelzine inactivated CYP2B6 with KI and k(inact) values of 44.9 ± 6.9 μM and 0.085 ± 0.003 min(-1) respectively. Inactivation of selegiline was characterized with KI and k(inact) values of 22.0 ± 3.3 and 0.074 ± 0.002 min(-1) respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index.

  13. Syntheses, structure, and luminescent properties of novel hydrated rare earth borates Ln2B6O10OH4•H2O (Ln = Pr, Nd, Sm, Eu, Gd, Dy, Ho, and Y).

    PubMed

    Cong, Rihong; Yang, Tao; Wang, Zheming; Sun, Junliang; Liao, Fuhui; Wang, Yingxia; Lin, Jianhua

    2011-03-07

    Ln(2)B(6)O(10)(OH)(4)•H(2)O (Ln = Pr, Nd, Sm-Gd, Dy, Ho, and Y), a new series of hydrated rare earth borates, have been synthesized under hydrothermal conditions. A single crystal of Nd analogue was used for the structure determination by X-ray diffraction. It crystallizes in the monoclinic space group C2/c with lattice constants a = 21.756(4), b = 4.3671(9), c = 12.192(2) Å, and β = 108.29(3)°. The other compounds are isostructural to Nd(2)B(6)O(10)(OH)(4)•H(2)O. The fundamental building block (FBB) of the polyborate anion in this structure is a three-membered ring [B(3)O(6)(OH)(2)](5-). The FBBs are connected by sharing oxygen atoms forming an infinite [B(3)O(5)(OH)(2)](3-) chain, and the chains are linked by hydrogen bonds, establishing a two-dimensional (2-D) [B(6)O(10)(OH)(4)•H(2)O](6-) layer. The 2-D borate layers are thus interconnected by Ln(3+) ions to form the complex three-dimensional structure. Ln(2)B(6)O(10)(OH)(4)•H(2)O dehydrates stepwise, giving rise to two new intermediate compounds Ln(2)B(6)O(10)(OH)(4) and Ln(2)B(6)O(11)(OH)(2). The investigation on the luminescent properties of Gd(2-2x)Eu(2x)B(6)O(10)(OH)(4)•H(2)O (x = 0.01-1.00) shows a high efficiency of Eu(3+) f-f transitions and the existence of the energy transfer process from Gd(3+) to Eu(3+). Eu(2)B(6)O(10)(OH)(4)•H(2)O and its two dehydrated products, Eu(2)B(6)O(10)(OH)(4) and Eu(2)B(6)O(11)(OH)(2), present the strongest emission peak at 620 nm ((5)D(0) → (7)F(2) transition), which may be potential red phosphors.

  14. Genetic polymorphisms of drug-metabolizing phase I enzymes CYP3A4, CYP2C9, CYP2C19 and CYP2D6 in Han, Uighur, Hui and Mongolian Chinese populations.

    PubMed

    Zuo, Jinliang; Xia, Dongya; Jia, Lihui; Guo, Tao

    2012-07-01

    We randomly evaluated 672 unrelated, healthy Chinese volunteers (136 Han, 214 Uighur, 164 Hui and 158 Mongolian) to compare CYP3A4, CYP2C9, CYP2C19 and CYP2D6 allele frequencies. Genomic DNA was extracted from peripheral leukocytes and genotyped for CYP3A4*5, CYP3A4*18, CYP2C9*2, CYP2C9*13, CYP2C19*2, CYP2C19*3 and CYP2D6*10 by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Our results showed that there is no significant difference in the distribution of CYP2C19*3 and CYP3A4*18 genotypes in the Han, Uighur, Hui and Mongolian Chinese populations. The CYP2C9*13/*13 and CYP3A4*5 genotypes were not observed in any of the four Chinese populations. We found a higher incidence of the CYP2C9*2 allele in Uighur populations, compared to the Han, Hui and Mongolian populations. The incidence of the CYP2C19*2 allele in the Han population was not significantly different from that in the Uighur, Hui or Mongolian populations; however, the Uighur population showed significantly lower rates of this allele than the Hui and Mongolian populations, and the Mongolian population had a significantly lower incidence of this allele than the Hui population. There was no significant difference in the presence of the CYP2D6*10 allele in the Mongolian, Han or Hui populations. However, the Uighur population showed significantly lower rates of this allele than the other three populations. These findings provide basic genetic information for further pharmacogenomic investigations in the Chinese population.

  15. Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings.

    PubMed

    Thomford, Nicholas E; Awortwe, Charles; Dzobo, Kevin; Adu, Faustina; Chopera, Denis; Wonkam, Ambroise; Skelton, Michelle; Blackhurst, Dee; Dandara, Collet

    2016-02-16

    Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts

  16. Effects of green tea catechins on cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A activities in human liver and intestinal microsomes.

    PubMed

    Misaka, Shingen; Kawabe, Keisuke; Onoue, Satomi; Werba, José Pablo; Giroli, Monica; Tamaki, Sekihiro; Kan, Toshiyuki; Kimura, Junko; Watanabe, Hiroshi; Yamada, Shizuo

    2013-01-01

    The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

  17. Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture.

    PubMed

    Badolo, Lassina; Jensen, Bente; Säll, Carolina; Norinder, Ulf; Kallunki, Pekka; Montanari, Dino

    2015-02-01

    1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2). 2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)>BCRP (9)>OCT1 (8)>OATP1B1 (5)>MRP3 (2). 3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2. 4. Structure-activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.

  18. Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

    PubMed

    Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

    2011-09-01

    The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

  19. 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

  20. Metabolism of bilirubin by human cytochrome P450 2A6

    SciTech Connect

    Abu-Bakar, A'edah; Arthur, Dionne M.; Wikman, Anna S.; Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu; Ng, Jack C.; Lang, Matti A.

    2012-05-15

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2

  1. Effects of CYP2B6 and CYP1A2 Genetic Variation on Nevirapine Plasma Concentration and Pharmacodynamics as Measured by CD4 Cell Count in Zimbabwean HIV-Infected Patients.

    PubMed

    Mhandire, Doreen; Lacerda, Miguel; Castel, Sandra; Mhandire, Kudakwashe; Zhou, Danai; Swart, Marelize; Shamu, Tinei; Smith, Peter; Musingwini, Tutsirai; Wiesner, Lubbe; Stray-Pedersen, Babill; Dandara, Collet

    2015-09-01

    The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to other

  2. Effects of CYP2B6 and CYP1A2 Genetic Variation on Nevirapine Plasma Concentration and Pharmacodynamics as Measured by CD4 Cell Count in Zimbabwean HIV-Infected Patients

    PubMed Central

    Mhandire, Doreen; Lacerda, Miguel; Castel, Sandra; Mhandire, Kudakwashe; Zhou, Danai; Swart, Marelize; Shamu, Tinei; Smith, Peter; Musingwini, Tutsirai; Wiesner, Lubbe; Stray-Pedersen, Babill

    2015-01-01

    Abstract The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to

  3. CYP2B6*6 genotype and high efavirenz plasma concentration but not nevirapine are associated with low lumefantrine plasma exposure and poor treatment response in HIV-malaria-coinfected patients.

    PubMed

    Maganda, B A; Minzi, O M S; Ngaimisi, E; Kamuhabwa, A A R; Aklillu, E

    2016-02-01

    We investigated the influence of efavirenz (EFV)- or nevirapine (NVP)-based antiretroviral therapy (ART) on lumefantrine plasma exposure in HIV-malaria-coinfected patients and implication of pharmacogenetic variations. A total of 269 HIV patients with uncomplicated falciparum malaria on NVP-based ART (NVP-arm), EFV-based ART (EFV-arm) or not receiving ART (control-arm) were enrolled and treated with artemether-lumefantrine. Day-7 lumefantrine, baseline EFV and NVP plasma concentrations, and CYP2B6*6,*18, CYP3A4*1B, CYP3A5*3,*6,*7, ABCB1 c.3435C>T and ABCB1 c.4036A>G genotypes were determined. The median day-7 lumefantrine plasma concentration was significantly lower in the EFV-arm compared with that in NVP- and control-arm. High EFV plasma concentrations and CYP2B6*6/*6 genotype significantly correlated with low lumefantrine plasma concentrations and high rate of recurrent parasitemia. No significant effect of NVP-based ART on lumefantrine exposure was observed. In conclusion, owing to long-term CYP3A induction, EFV-based ART cotreatment significantly reduces lumefantrine plasma exposure leading to poor malaria treatment response, which is more pronounced in CYP2B6 slow metabolizers.

  4. CYP2A6 polymorphisms and risk for tobacco-related cancers.

    PubMed

    Rossini, Ana; de Almeida Simão, Tatiana; Albano, Rodolpho Mattos; Pinto, Luis Felipe Ribeiro

    2008-11-01

    Tobacco consumption is the main identifiable risk to cancer, contributing to the majority of tumors in upper aerodigestive tissues. The psychoactive compound responsible for tobacco addiction, nicotine and the potent carcinogens present at high concentrations either in cigarette mainstream smoke or in smokeless tobacco products, 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) and N-nitrosonornicotine (NNN) can be metabolized by CYP2A6. CYP2A6 is expressed in many aerodigestive tissues with high interindividual variability. The CYP2A6 gene is highly polymorphic and CYP2A6 alleles coding for enzymes with altered expression or metabolic capacity produce alterations in nicotine metabolism in vivo and seem to influence smoking behavior. These polymorphisms may change the rate of NNK and NNN activation and, therefore, may influence cancer risk associated with tobacco consumption. However, to date only a few and inconclusive studies have addressed the risk that a given CYP2A6 polymorphism presents for the development of tobacco-related tumors. Most, but not all, show a reduced risk associated with alleles that result in decreased enzyme activity. The overlapping substrate specificity and tissue expression between CYP2A6 and the highly similar CYP2A13 may add to the conflicting results observed. The intricate regulation of CYP2A6 and the variation of structurally different chemical compounds capable of inhibiting CYP2A enzymes also add to the complexity. Finally, the interaction between polymorphisms of genes that code for CYP2A6, CYP2A13 and other potent carcinogen-metabolizing CYP enzymes may help to determine individuals that are at higher risk of developing tumors associated with tobacco consumption.

  5. Nicotine Metabolism and Smoking: Ethnic Differences in the Role of P450 2A6.

    PubMed

    Murphy, Sharon E

    2017-01-17

    Nicotine is the primary addictive agent in tobacco, and P450 2A6 (gene name: CYP2A6) is the primary catalyst of nicotine metabolism. It was proposed more than 20 years ago that individuals who metabolize nicotine poorly would smoke less, either fewer cigarettes per day or less intensely per cigarette, compared to smokers who metabolize nicotine more efficiently. These poor metabolizers would then be less likely to develop lung cancer due to their lower exposure to the many carcinogens delivered with nicotine in each puff of smoke. Numerous studies have reported that smokers who carry reduced activity or null CYP2A6 alleles do smoke less. Yet only in Asian populations, both Japanese and Chinese, which have a high prevalence of genetic variants, has a link between CYP2A6, smoking dose, and lung cancer been established. In other ethnic groups, it has been challenging to confirm a direct link between P450 2A6-mediated nicotine metabolism and the risk of lung cancer. This challenge is due in part to the difficulty in accurately quantifying smoking dose and accurately predicting or measuring P450 2A6-mediated nicotine metabolism. Biomarkers of nicotine metabolism and smoking exposure, including the ratio of trans-3-hydroxycotine to cotinine, a measure of P450 2A6 activity and plasma cotinine, or urinary total nicotine equivalents (the sum of nicotine and six metabolites) as measures of exposure are useful for addressing this challenge. However, to take full advantage of these biomarkers in the study of ethnic/racial differences in the risk of lung cancer requires the complete characterization of nicotine metabolism across ethnic/racial groups. Variation in metabolism pathways, other than those catalyzed by P450 2A6, can impact biomarkers of both nicotine metabolism and dose. This is clearly important for smokers with low levels of UGT2B10-catalyzed nicotine and cotinine glucuronidation because the UGT2B10 genotype influences plasma cotinine levels. Cotinine is not

  6. High plasma efavirenz level and CYP2B6*6 are associated with efavirenz-based HAART-induced liver injury in the treatment of naïve HIV patients from Ethiopia: a prospective cohort study.

    PubMed

    Yimer, G; Amogne, W; Habtewold, A; Makonnen, E; Ueda, N; Suda, A; Worku, A; Haefeli, W E; Burhenne, J; Aderaye, G; Lindquist, L; Aklillu, E

    2012-12-01

    The objective of this study was to assess the incidence, timing and identify pharmacogenetic, efavirenz (EFV) pharmacokinetic and biochemical predictors of EFV-based antiretroviral therapy (ART) drug-induced liver injury (DILI). ART-naïve HIV patients (n = 285) were prospectively enrolled. Pretreatment laboratory evaluations included hepatitis B surface antigen and C antibody, CD4 count and viral load. Liver tests were done at baseline, 1st, 2nd, 4th, 8th, 12th, 24th and 48th weeks during ART. Plasma EFV and 8-hydroxyefvairenz concentration was determined at week 4 using liquid chromatography-mass spectrometry. CYP2B6, CYP3A5, ABCB1 3435C/T and UGT2B7*2 genotyping was done using Taqman genotyping assay. Data were analyzed using survival analysis and Cox proportional hazards model. The incidence of DILI was 15.7% or 27.9 per 100 person-years and that of severe injury was 3.4% or 6.13 per 100 person-years. The median time for the development of DILI and severe injury was 2 and 4 weeks after initiation of ART, respectively. There was significant association of DILI with lower baseline platelet, albumin, log plasma viral load and CD4 count (P = 0.031, 0.037, 0.06 and 0.019, respectively). Elevated baseline alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, plasma EFV level and CYP2B6*6 were good predictors for the development of DILI (P = 0.03, 0.01, 0.016, 0.017 and 0.04, respectively). We report for the first time CYP2B6*6 as a putative genetic marker and high plasma EFV concentration as intermediate biomarker for vulnerability to EFV-induced liver injury in HIV patients. CYP2B6 genotyping and/or regular monitoring of EFV and lever enzymes level during early therapy is advised for early diagnosis and management of DILI.

  7. Structure-function relationships of inhibition of human cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives.

    PubMed

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V; Foroozesh, Maryam K; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2010-12-20

    Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

  8. 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

    2016-03-02

    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

  9. Structure-Function Relationships of Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 Flavonoid Derivatives

    PubMed Central

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Murayama, Norie; Martin, Martha V.; Foroozesh, Maryam K.; Yamazaki, Hiroshi; Guengerich, F. Peter; Komori, Masayuki

    2010-01-01

    Structure-function relationships for inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced Reverse Type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, e.g. 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 µM to 0.09, 0.21, 0.25, and 0.27 µM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 µM. The introduction of a 4’-methoxy- or 3’,4’-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 µM, respectively. The above hydroxyl- and/or methoxy-groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always towards P450 1A2, where 3-, 5-, or 7-hydroxyflavone, and 4’-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-,5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3-and 5-hydroxyflavone. Flavone and several other flavonoids produced Type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities towards P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids towards these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids. PMID

  10. Investigation of binding features: effects on the interaction between CYP2A6 and inhibitors.

    PubMed

    Ai, Chunzhi; Li, Yan; Wang, Yonghua; Li, Wei; Dong, Peipei; Ge, Guangbo; Yang, Ling

    2010-07-15

    A computational investigation has been carried out on CYP2A6 and its naphthalene inhibitors to explore the crucial molecular features contributing to binding specificity. The molecular bioactive orientations were obtained by docking (FlexX) these compounds into the active site of the enzyme. And the density functional theory method was further used to optimize the molecular structures with the subsequent analysis of molecular lipophilic potential (MLP) and molecular electrostatic potential (MEP). The minimal MLPs, minimal MEPs, and the band gap energies (the energy difference between the highest occupied molecular orbital and lowest unoccupied molecular orbital) showed high correlations with the inhibition activities (pIC(50)s), illustrating their significant roles in driving the inhibitor to adopt an appropriate bioactive conformation oriented in the active site of CYP2A6 enzyme. The differences in MLPs, MEPs, and the orbital energies have been identified as key features in determining the binding specificity of this series of compounds to CYP2A6 and the consequent inhibitory effects. In addition, the combinational use of the docking, MLP and MEP analysis is also demonstrated as a good attempt to gain an insight into the interaction between CYP2A6 and its inhibitors.

  11. Structural Insight into the Altered Substrate Specificity of Human Cytochrome P450 2A6 Mutants

    PubMed Central

    Sansen, Stefaan; Hsu, Mei-Hui; Stout, C. David; Johnson, Eric F.

    2009-01-01

    Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M. and Guengerich, F.P. (2005) J.Biol.Chem. 49, 41090-41100). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B′-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility. PMID:17540336

  12. Gene-gene interactions between DRD3, MRP4 and CYP2B6 polymorphisms and its influence on the pharmacokinetic parameters of efavirenz in HIV infected patients.

    PubMed

    Sánchez-Martín, Almudena; Cabrera Figueroa, Salvador; Cruz, Raquel; Porras-Hurtado, Liliana; Calvo-Boyero, Fernando; Rasool, Mahmood; Domínguez-Gil Hurlé, Alfonso; Carracedo, Angel

    2016-10-01

    Genetic factors have a significant impact on the PK variability of EFV, much higher than other non-genetic factors, such as demography. In this work we have performed a comprehensive PG analysis of genes encoding the major metabolizing enzymes and transporters of EFV, establishing a clear relationship between the PK parameters and genetic factors, which explain 50% of the variability in EFV PK parameters. The most relevant associations for metabolizing enzymes were found in CYP2B6 (rs3745274), in agreement with previous studies. The influence of transporters on the kinetics of EFV was also proved with significant correlations between the PK parameters of EFV and MRP4 (rs1751034, rs2274407). Analysis of gene-gene interactions with CYP2B6 was particularly useful to reinforce the role of MRP4 and to reveal unknown associations, such as that of DRD3. However, the role of DRD3 cannot be a direct effect but an indirect one due to physical proximity of NAT and the DRD3 locus in the genome.

  13. Genetic markers in CYP2C19 and CYP2B6 for prediction of cyclophosphamide's 4‐hydroxylation, efficacy and side effects in Chinese patients with systemic lupus erythematosus

    PubMed Central

    Shu, Wenying; Guan, Su; Yang, Xiuyan; Liang, Liuqin; Li, Jiali; Chen, Zhuojia; Zhang, Yu; Chen, Lingyan

    2015-01-01

    Aims The aim of the study was to investigate the combined impact of genetic polymorphisms in key pharmacokinetic genes on plasma concentrations and clinical outcomes of cyclophosphamide (CPA) in Chinese patients with systemic lupus erythematosus (SLE). Methods One hundred and eighty nine Chinese SLE patients treated with CPA induction therapy (200 mg, every other day) were recruited and adverse reactions were recorded. After 4 weeks induction therapy, 128 lupus nephritis (LN) patients continued to CPA maintenance therapy (200–600 mg week–1) for 6 months, and their clinical outcomes were recorded. Blood samples were collected for CYP2C19, CYP2B6, GST and PXR polymorphism analysis, as well as CPA and its active metabolite (4‐hydroxycyclophosphamide (4‐OH‐CPA)) plasma concentration determination. Results Multiple linear regression analysis revealed that CYP2B6 ‐750 T > C (P < 0.001), −2320 T > C (P < 0.001), 15582C > T (P = 0.017), CYP2C19*2 (P < 0.001) and PXR 66034 T > C (P = 0.028) accounted for 47% of the variation in 4‐OH‐CPA plasma concentration. Among these variants, CYP2B6 ‐750 T > C and CYP2C19*2 were selected as the combination genetic marker because these two SNPs contributed the most to the inter‐individual variability in 4‐OH‐CPA concentration, accounting for 23.6% and 21.5% of the variation, respectively. Extensive metabolizers (EMs) (CYP2B6 ‐750TT, CYP2C19*1*1) had significantly higher median 4‐OH‐CPA plasma concentrations (34.8, 11.0 and 6.6 ng ml‐1 for EMs, intermediate metabolizers (IMs) and poor metabolizers (PMs), P < 0.0001), higher risks of leukocytopenia (OR = 7.538, 95% CI 2.951, 19.256, P < 0.0001) and gastrointestinal toxicity (OR = 7.579, 95% CI 2.934, 19.578, P < 0.0001), as well as shorter median time to achieve complete remission (13.2, 18.3 and 23.3 weeks for EMs, IMs and PMs, respectively, P = 0.026) in LN patients than PMs (CYP2B6 ‐750CC, CYP2C19*2*2) and

  14. Correlations of CYP2C9∗3/CYP2D6∗10/CYP3A5∗3 gene polymorphisms with efficacy of etanercept treatment for patients with ankylosing spondylitis

    PubMed Central

    Chen, Yuan-Yuan

    2017-01-01

    Abstract Background: The tumor necrosis factor alpha (TNF-α) inhibitor etanercept has been proven to be effective in the treatment of ankylosing spondylitis (AS), while genetic polymorphism may affect drug metabolism or drug receptor, resulting in interindividual variability in drug disposition and efficacy. The purpose of this study is to investigate the correlations between CYP2C9∗3/CYP2D6∗10/CYP3A5∗3 gene polymorphisms and the efficacy of etanercept treatment for patients with AS. Methods: From March 2012 to June 2015, 312 AS patients (174 males and 138 females, mean age: 35.2 ± 5.83 years) from 18 to 56 years old were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism was applied to detect the allele and genotype frequencies of CYP2C9∗3, CYP2D6∗10, and CYP3A5∗3 gene polymorphisms. The joint swelling score, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level of AS patients were compared before and after 24-week etanercept treatment. Assessment in Ankylosing Spondylitis (ASAS) and bath ankylosing spondylitis disease activity index (BASDAI) scores were recorded to assess the efficacy of etanercept treatment. Results: The AS patients with wild-type ∗1/∗1 and heterozygous ∗1/∗3 genotypes of CYP2C9∗3 polymorphism accounted for 93.59% and 6.41%, respectively, without ∗3/∗3 genotype. The AS patients with wild-type CC, heterozygous CT, and mutation homozygous TT genotypes of CYP2D6∗10 polymorphism accounted for 19.23%, 39.10%, and 41.67%, respectively. The AS patients with wild-type ∗1/∗1, heterozygous ∗1/∗3, and mutation homozygous ∗3/∗3 genotypes of CYP3A5∗3 polymorphism accounted for 7.69%, 36.22%, and 56.09%, respectively. After 24-week treatment, AS patients with wild-type ∗1/∗1 genotype of CYP2C9∗3, CC genotype of CYP2D6∗10, and ∗3/∗3 genotype of CYP3A5∗3 polymorphisms had lower joint swelling score, ESR, and CRP level. The joint swelling

  15. Anti-CD28 monoclonal antibody-stimulated cytokines released from blood suppress CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes in vitro.

    PubMed

    Czerwiński, Maciej; Kazmi, Faraz; Parkinson, Andrew; Buckley, David B

    2015-01-01

    Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood-mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism-namely, the release of cytokines from PBMCs.

  16. CYP2C19 but not CYP2B6, CYP3A4, CYP3A5, ABCB1, PON1 or P2Y12 genetic polymorphism impacts antiplatelet response after clopidogrel in Koreans.

    PubMed

    Zhang, Hong-Zhe; Kim, Moo Hyun; Guo, Long-Zhe; Serebruany, Victor

    2017-01-01

    Clopidogrel response variability (CRV) is well documented, and may affect clinical outcomes. Impact of genetic polymorphisms is important for assessing and predicting CRV. The extensive evidence indicates the importance of CYP2C19 variants in reducing efficacy of clopidogrel. This study defined the impact of numerous genetic polymorphisms on CRV before and after percutaneous coronary interventions (PCI) exclusively in a Korean cohort assuming less genetic variability noise. One hundred and thirty-six patients of Korean origin undergoing PCI were included. Platelet reactivity was measured by VerifyNow assay before and after PCI. Genetic polymorphism of seven single nucleotides of CYP2B6, CYP2C19, CYP3A4, CYP3A5, ABCB1, PON1, and P2Y12 were evaluated and matched with platelet reactivity. Carriers of at least one CYP2C19*2 or *3 allele uniformly exhibited higher platelet reactivity compared to 0-carrier pre-PCI (odds ratio 3.1, 95% confidence interval 1.4-6.9, P < 0.01) and post-PCI (odds ratio 3.4, 95% confidence interval 1.7-6.8, P < 0.001). The carriers of other gene allele variants lack uniformed impact on CRV. The Korean carriers of CYP2C19*2 or *3 allele are linked to CRV, whereas CYP2B6, CYP3A4, CYP3A5, ABCB1, PON1, and P2Y12 failed to predict CRV. The exact clinical utility of these findings is uncertain, and requires a large randomized national trial for proof of concept.

  17. Genetic variations in the xenobiotic-metabolizing enzymes CYP1A1, CYP1A2, CYP2C9, CYP2C19 and susceptibility to colorectal cancer among Turkish people.

    PubMed

    Özhan, Gül; Mutur, Mine; Ercan, Gulcin; Alpertunga, Buket

    2014-04-01

    Cytochrome P450 (CYP) enzymes are genetically polymorphic and play key roles in the metabolism of xenobiotics. Colorectal cancer (CRC) is one of the most common malignant tumors in Turkey as well as in the world. In this study, it was aimed both to evaluate the effects of CYP variants on the susceptibility to CRC and to predict the individual response of the Turkish people to xenobiotics metabolized by CYP enzymes. For that, we assessed the association of CYP1A1, CYP1A2, CYP2C9, and CYP2C19 polymorphisms in patients with CRC in the Turkish population through a case-control study. Distributions of the variants were determined in 104 patients with CRC and 183 healthy volunteers. As results, CYP1A1 6235T/C was significantly associated with CRC risk (odds ratio [OR]=2.53; 95% confidence interval [CI]=0.99-6.45; p=0.046). In a haplotype-based analysis, CYP1A1 haplotype C6235-A2455 might be associated with the development of CRC (OR=2.70; 95% CI=0.58-5.90; p=0.046). We believe that the findings are the first results of CYP allele distributions in the Turkish population and provide an understanding of the epidemiological studies that correlate therapeutic approaches and etiology of CRC especially in Turkish patients.

  18. CYP2D6 and CYP2A6 biotransform dietary tyrosol into hydroxytyrosol.

    PubMed

    Rodríguez-Morató, Jose; Robledo, Patricia; Tanner, Julie-Anne; Boronat, Anna; Pérez-Mañá, Clara; Oliver Chen, C-Y; Tyndale, Rachel F; de la Torre, Rafael

    2017-02-15

    The dietary phenol tyrosol has been reported to be endogenously transformed into hydroxytyrosol, a potent antioxidant with multiple health benefits. In this work, we evaluated whether tyrosine hydroxylase (TH) and cytochrome P450s (CYPs) catalyzed this process. To assess TH involvement, Wistar rats were treated with α-methyl-L-tyrosine and tyrosol. Tyrosol was converted into hydroxytyrosol whilst α-methyl-L-tyrosine did not inhibit the biotransformation. The role of CYP was assessed in human liver microsomes (HLM) and tyrosol-to-hydroxytyrosol conversion was observed. Screening with selective enzymatic CYP inhibitors identified CYP2A6 as the major isoform involved in this process. Studies with baculosomes further demonstrated that CYP2D6 and CYP3A4 could transform tyrosol into hydroxytyrosol. Experiments using human genotyped livers showed an interindividual variability in hydroxytyrosol formation and supported findings that CYP2D6 and CYP2A6 mediated this reaction. The dietary health benefits of tyrosol-containing foods remain to be evaluated in light of CYP pharmacogenetics.

  19. Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6.

    PubMed

    Zhang, Qing-Yu; Gu, Jun; Su, Ting; Cui, Huadong; Zhang, Xiuling; D'Agostino, Jaime; Zhuo, Xiaoliang; Yang, Weizhu; Swiatek, Pamela J; Ding, Xinxin

    2005-12-09

    The aim of this study was to prepare and characterize a transgenic mouse model in which CYP2A6, a human P450 enzyme, is expressed specifically in the liver. CYP2A6, which is mainly expressed in human liver, is active toward many xenobiotics. Our transgene construct contained the mouse transthyretin promoter/enhancer, a full-length CYP2A6 cDNA, and a downstream neomycin-resistance gene for positive selection in embryonic stem cells. Hepatic expression of the CYP2A6 transgene was demonstrated by immunoblotting, whereas tissue specificity of CYP2A6 expression was confirmed by RNA-PCR. The transgenic mouse was further characterized after being backcrossed to the B6 strain for six generations. Hepatic microsomes from homozygous transgenic mice had activities significantly higher than those of B6 mice toward coumarin. The in vivo activity of transgenic CYP2A6 was also determined. Systemic clearance of coumarin was significantly higher in the transgenic mice than in B6 controls, consistent with the predicted role of CYP2A6 as the major coumarin hydroxylase in human liver. The CYP2A6-transgenic mouse model should be valuable for studying the in vivo function of this polymorphic human enzyme in drug metabolism and chemical toxicity.

  20. 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.

  1. A comparative molecular field analysis of cytochrome P450 2A5 and 2A6 inhibitors.

    PubMed

    Poso, A; Gynther, J; Juvonen, R

    2001-03-01

    Structure-activity relationships of 23 P450 2A5 and 2A6 inhibitors were analysed using the CoMFA and GOLPE/GRID with smart region definition (SRD). The predictive power of the resulting models was validated using five compounds not belonging to the model set. All models have high internal and external predictive power and resulting 3D-QSAR models are supporting each other. Both Sybyl and GOLPE highlight properties near lactone moiety to be important for 2A5 and 2A6 inhibition. Another important feature for pIC50 was the size of the substituent in the 7-positon of coumarin. The models suggest that the 2A5 binding site is larger that that of 2A6 due to larger steric regions in the CoMFA coefficient maps and corresponding GOLPE maps. In addition, the maps reveal that 2A6 disfavours negative charge near the lactone moiety of coumarin.

  2. Celery extract inhibits mouse CYP2A5 and human CYP2A6 activities via different mechanisms.

    PubMed

    Deng, Xiao; Pu, Qianghong; Wang, Erhao; Yu, Chao

    2016-12-01

    Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7-hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose-dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time- and nicotinamide adenine dinucleotide phosphate (NADPH)-independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH-dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5-methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism-based inhibition for CYP2A6.

  3. Celery extract inhibits mouse CYP2A5 and human CYP2A6 activities via different mechanisms

    PubMed Central

    Deng, Xiao; Pu, Qianghong; Wang, Erhao; Yu, Chao

    2016-01-01

    Human cytochrome P450 (CYP) 2A6 participates in the metabolism of nicotine and precarcinogens, thus the deliberate inhibition of CYP2A6 may reduce cigarette consumption and therefore reduce the risk of developing the types of cancer associated with smoking. The inhibitory effects and mechanisms of celery (Apium graveolens) extract on mouse CYP2A5 and human CYP2A6 activity remain unclear. These effects were investigated in mouse and human liver microsomes using coumarin 7-hydroxylation in a probe reaction. Celery extract reduced CYP2A5 and CYP2A6 activities in vitro in a dose-dependent manner. In vivo experiments also showed that celery extract markedly decreased CYP2A5 activity. The inhibition of celery extract on CYP2A5 was time- and nicotinamide adenine dinucleotide phosphate (NADPH)-independent, and was markedly reduced by ultracentrifugation. Additionally, the inhibition of celery extract on CYP2A6 was time and NADPH-dependent. Levels of inhibition were characterized by a Ki, the measure of the tightness of bonds between the enzyme and its inhibitor, of 266.4 µg/ml for CYP2A5, and a Ki of 1,018 µg/ml and Kinact of 0.3/min for CYP2A6. Kinact is the maximal rate of enzyme inactivation at a saturating concentration of inhibitor. The coumarin derivative 5-methoxypsoralen present in celery extract did not solely to the inhibition of CYP2A5/6 activity. In conclusion, celery extract inhibited the levels of mouse CYP2A5 and human CYP2A6 activity via different mechanisms: Mixed competitive inhibition for CYP2A5 and mechanism-based inhibition for CYP2A6. PMID:28101244

  4. A comparative molecular field analysis of cytochrome P450 2A5 and 2A6 inhibitors

    NASA Astrophysics Data System (ADS)

    Poso, Antti; Gynther, Jukka; Juvonen, Risto

    2001-03-01

    Structure-activity relationships of 23 P450 2A5 and 2A6 inhibitors were analysed using the CoMFA [1] and GOLPE/GRID with smart region definition (SRD) [2]. The predictive power of the resulting models was validated using five compounds not belonging to the model set. All models have high internal and external predictive power and resulting 3D-QSAR models are supporting each other. Both Sybyl and GOLPE highlight properties near lactone moiety to be important for 2A5 and 2A6 inhibition. Another important feature for pIC50 was the size of the substituent in the 7-positon of coumarin. The models suggest that the 2A5 binding site is larger that that of 2A6 due to larger steric regions in the CoMFA coefficient maps and corresponding GOLPE maps. In addition, the maps reveal that 2A6 disfavours negative charge near the lactone moiety of coumarin.

  5. Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

    SciTech Connect

    DeVore, Natasha M.; Meneely, Kathleen M.; Bart, Aaron G.; Stephens, Eva S.; Battaile, Kevin P.; Scott, Emily E.

    2013-11-20

    Human xenobiotic-metabolizing cytochrome P450 (CYP) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally, a single ligand can interact with multiple CYP enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the CYP superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human CYP enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key residues involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 {angstrom}), CYP2A13 (3.0 {angstrom}), CYP2E1 (2.35 {angstrom}), and the CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 {angstrom}) have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual residues lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine.

  6. CYP2A6- and CYP2A13-catalyzed metabolism of the nicotine Δ5'(1')iminium ion.

    PubMed

    von Weymarn, Linda B; Retzlaff, Cassandra; Murphy, Sharon E

    2012-11-01

    Nicotine, the major addictive agent in tobacco, is metabolized primarily by CYP2A6-catalyzed oxidation. The product of this reaction, 5'-hydroxynicotine, is in equilibrium with the nicotine Δ5'(1')iminium ion and is further metabolized to cotinine. We reported previously that both CYP2A6 and the closely related extrahepatic enzyme CYP2A13 were inactivated during nicotine metabolism; however, inactivation occurred after metabolism was complete. This led to the hypothesis that oxidation of a nicotine metabolite, possibly the nicotine Δ5'(1')iminium ion, was responsible for generating the inactivating species. In the studies presented here, we confirm that the nicotine Δ5'(1')iminium ion is an inactivator of both CYP2A6 and CYP2A13, and inactivation depends on time, concentration, and the presence of NADPH. Inactivation was not reversible and was accompanied by a parallel loss in spectrally active protein, as measured by reduced CO spectra. These data are consistent with the characterization of the nicotine Δ5'(1')iminium ion as a mechanism-based inactivator of both CYP2A13 and CYP2A6. We also confirm that both CYP2A6 and CYP2A13 catalyze the metabolism of the nicotine Δ5'(1')iminium ion to cotinine and provide evidence that both enzymes catalyze the sequential metabolism of the nicotine Δ5'(1')iminium ion. That is, a fraction of the cotinine formed may not be released from the enzyme before further oxidation to 3'-hydroxycotinine.

  7. A genetic polymorphism in coumarin 7-hydroxylation: Sequence of the human CYP2A genes and identification of variant CYP2A6 alleles

    SciTech Connect

    Fernandez-Salguero, P.; Hoffman, S.M.G.; Mohrenweiser, H.

    1995-09-01

    A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes - CYP2A6, CYP2A7, and CYP2A13 - plus two pseudogenes truncated after exon 5 were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6{nu}2. Sequence differences in the CY-P2A6{nu}2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6{nu}2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6{nu}2 allele, and a variant - designated CYP2A6{nu}1 - that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6{nu}1 and CYP2A6{nu}2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism. 30 refs., 4 figs., 2 tabs.

  8. Predictors of Variation in CYP2A6 mRNA, Protein, and Enzyme Activity in a Human Liver Bank: Influence of Genetic and Nongenetic Factors.

    PubMed

    Tanner, Julie-Anne; Prasad, Bhagwat; Claw, Katrina G; Stapleton, Patricia; Chaudhry, Amarjit; Schuetz, Erin G; Thummel, Kenneth E; Tyndale, Rachel F

    2017-01-01

    Cytochrome P450 2A6 CYP2A6: metabolizes several clinically relevant substrates, including nicotine, the primary psychoactive component in cigarette smoke. Smokers vary widely in their rate of inactivation and clearance of nicotine, altering numerous smoking phenotypes. We aimed to characterize independent and shared impact of genetic and nongenetic sources of variation in CYP2A6 mRNA, protein, and enzyme activity in a human liver bank (n = 360). For the assessment of genetic factors, we quantified levels of CYP2A6, cytochrome P450 oxidoreductase (POR), and aldo-keto reductase 1D1 (AKR1D1) mRNA, and CYP2A6 and POR proteins. CYP2A6 enzyme activity was determined through measurement of cotinine formation from nicotine and 7-hydroxycoumarin formation from coumarin. Donor DNA was genotyped for CYP2A6, POR, and AKR1D1 genetic variants. Nongenetic factors assessed included gender, age, and liver disease. CYP2A6 phenotype measures were positively correlated to each other (r values ranging from 0.47-0.88, P < 0.001). Female donors exhibited higher CYP2A6 mRNA expression relative to males (P < 0.05). Donor age was weakly positively correlated with CYP2A6 protein (r = 0.12, P < 0.05) and activity (r = 0.20, P < 0.001). CYP2A6 reduced-function genotypes, but not POR or AKR1D1 genotypes, were associated with lower CYP2A6 protein (P < 0.001) and activity (P < 0.01). AKR1D1 mRNA was correlated with CYP2A6 mRNA (r = 0.57, P < 0.001), protein (r = 0.30, P < 0.001), and activity (r = 0.34, P < 0.001). POR protein was correlated with CYP2A6 activity (r = 0.45, P < 0.001). Through regression analyses, we accounted for 17% (P < 0.001), 37% (P < 0.001), and 77% (P < 0.001) of the variation in CYP2A6 mRNA, protein, and activity, respectively. Overall, several independent and shared sources of variation in CYP2A6 activity in vitro have been identified, which could translate to variable hepatic clearance of nicotine.

  9. Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

    PubMed Central

    Ueng, Yune-Fang; Chen, Chien-Chih; Chung, Yu-Ting; Liu, Tsung-Yun; Chang, Yu-Ping; Lo, Wei-Sheng; Murayama, Norie; Yamazaki, Hiroshi; Souček, Pavel; Chau, Gar-Yang; Chi, Chin-Wen; Chen, Ruei-Ming; Li, Ding-Tzai

    2011-01-01

    BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min−1; KI 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice. PMID:21418183

  10. Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers, nicotine, coumarin, N-nitrosodiethylamine, and N-nitrosobenzylmethylamine.

    PubMed

    Le Gal, Annabelle; Dréano, Yvonne; Lucas, Danièle; Berthou, François

    2003-09-15

    Cytochrome P450 2A6 constitutes 5-10% of the total microsomal CYPs of human liver. Although CYP2A6 is the major coumarin 7-hydroxylase, other known substrates of CYP2A6 include many toxicants and precarcinogens. The chemical structure diversity of these substrates raises the question of their selectivity. Thus, kinetic parameters were determined for the hydroxylation of five substrates of diverse chemical structures known to be selective for cytochrome P450 2A6: methyl tert-butyl ether (MTBE), nicotine, coumarin, N-nitrosobenzylmethylamine (NBzMA), and N-nitrosodiethylamine (NDEA). Sources of enzymes were either human liver microsomes or heterologously expressed CYPs. Coumarin was shown to be the substrate with the highest affinity, followed by NDEA, nicotine, NBzMA, and MTBE. Variability of CYP2A6 catalytic activities in human liver was between 24-fold for MTBE to sevenfold for coumarin, while CYP2A6 content varied 68-fold in human liver microsomes. These five catalytic activities were highly significantly correlated between them and with hepatic CYP2A6 content. The most selective chemical inhibitor of these five substrates was shown to be 8-methoxypsoralen. Based upon chemical inhibition of the enzymatic activities of pure recombinant human CYPs, it cannot be totally excluded that P450s other than CYP2A6, especially CYP2E1, are involved, although to a lesser extent, in NDEA and NBzMA metabolism. In conclusion, the prototype probes for CYP2A6 phenotyping are coumarin and nicotine.

  11. Evidence for Concurrent Effects of Exposure to Environmental Cadmium and Lead on Hepatic CYP2A6 Phenotype and Renal Function Biomarkers in Nonsmokers

    PubMed Central

    Satarug, Soisungwan; Nishijo, Muneko; Ujjin, Pailin; Vanavanitkun, Yuvaree; Baker, Jason R.; Moore, Michael R.

    2004-01-01

    We examined the interrelationships between phenotype of hepatic cytochrome P450 2A6 (CYP2A6), nephropathy, and exposure to cadmium and lead in a group of 118 healthy Thai men and women who had never smoked. Their urinary Cd excretion ranged from 0.05 to 2.36 μg/g creatinine, whereas their urinary Pb excretion ranged from 0.1 to 12 μg/g creatinine. Average age and Cd burden of women and men did not differ. Women, however, on average showed a 46% higher urinary Pb excretion (p < 0.001) and lower zinc status, suggested by lower average serum Zn and urinary Zn excretion compared with those in men. Cd-linked nephropathy was detected in both men and women. However, Pb-linked nephropathy was seen only in women, possibly because of higher Pb burden coupled with lower protective factors, notably of Zn (p < 0.001), in women compared with men. In men, Pb burden showed a negative association with CYP2A6 activity (adjusted β= −0.29, p = 0.003), whereas Cd burden showed a positive association with CYP2A6 activity (adjusted β= 0.38, p = 0.001), suggesting opposing effects of Cd and Pb on hepatic CYP2A6 phenotype. The weaker correlation between Cd burden CYP2A6 activity in women despite similarity in Cd burden between men and women is consistent with opposing effects of Pb and Cd on hepatic CYP2A6 phenotypic expression. A positive correlation between Cd-linked nephropathy (urinary N-acetyl-β-d-glucosaminidase excretion) and CYP2A6 activity in men (r = 0.39, p = 0.002) and women (r = 0.37, p = 0.001) suggests that Cd induction of hepatic CYP2A6 expression and Cd-linked nephropathy occurred simultaneously. PMID:15531436

  12. Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity.

    PubMed

    Park, Sungshim L; Tiirikainen, Maarit I; Patel, Yesha M; Wilkens, Lynne R; Stram, Daniel O; Le Marchand, Loic; Murphy, Sharon E

    2016-03-01

    Genetic variation in cytochrome P450 2A6 (CYP2A6) gene is the primary contributor to the intraindividual and interindividual differences in nicotine metabolism and has been found to influence smoking intensity. However, no study has evaluated the relationship between CYP2A6 genetic variants and the CYP2A6 activity ratio (total 3-hydroxycotinine/cotinine) and their influence on smoking intensity [total nicotine equivalents (TNE)], across five racial/ethnic groups found to have disparate rates of lung cancer. This study genotyped 10 known functional CYP2A6 genetic or copy number variants in 2115 current smokers from the multiethnic cohort study [African Americans (AA) = 350, Native Hawaiians (NH) = 288, Whites = 413, Latinos (LA) = 437 and Japanese Americans (JA) = 627] to conduct such an investigation. Here, we found that LA had the highest CYP2A6 activity followed by Whites, AA, NH and JA, who had the lowest levels. Adjusting for age, sex, race/ethnicity and body mass index, we found that CYP2A6 diplotypes were predictive of TNE levels, particularly in AA and JA (P trend < 0.0001). However, only in JA did the association remain after accounting for cigarettes per day. Also, it is only in this population that the lower activity ratio supports lower TNE levels, carcinogen exposure and thereby lower risk of lung cancer. Despite the association between nicotine metabolism (CYP2A6 activity phenotype and diplotypes) and smoking intensity (TNE), CYP2A6 levels did not correlate with the higher TNE levels found in AA nor the lower TNE levels found in LA, suggesting that other factors may influence smoking dose in these populations. Therefore, further study in these populations is recommended.

  13. A Global Health Diagnostic for Personalized Medicine in Resource-Constrained World Settings: A Simple PCR-RFLP Method for Genotyping CYP2B6 g.15582C>T and Science and Policy Relevance for Optimal Use of Antiretroviral Drug Efavirenz.

    PubMed

    Evans, Jonathan; Swart, Marelize; Soko, Nyarai; Wonkam, Ambroise; Huzair, Farah; Dandara, Collet

    2015-06-01

    The use of pharmacogenomics (PGx) knowledge in treatment of individual patients is becoming a common phenomenon in the developed world. However, poorly resourced countries have thus far been constrained for three main reasons. First, the cost of whole genome sequencing is still considerably high in comparison to other (non-genomics) diagnostics in the developing world where both science and social dynamics create a dynamic and fragile healthcare ecosystem. Second, studies correlating genomic differences with drug pharmacokinetics and pharmacodynamics have not been consistent, and more importantly, often not indexed to impact on societal end-points, beyond clinical practice. Third, ethics regulatory frames over PGx testing require improvements based on nested accountability systems and in ways that address the user community needs. Thus, CYP2B6 is a crucial enzyme in the metabolism of antiretroviral drugs, efavirenz and nevirapine. More than 40 genetic variants have been reported, but only a few contribute to differences in plasma EFV and NVP concentrations. The most widely reported CYP2B6 variants affecting plasma drug levels include c.516G>T, c.983T>C, and to a lesser extent, g.15582C>T, which should be considered in future PGx tests. While the first two variants are easily characterized, the g.15582C>T detection has been performed primarily by sequencing, which is costly, labor intensive, and requires access to barely available expertise in the developing world. We report here on a simple, practical PCR-RFLP method with vast potentials for use in resource-constrained world regions to detect the g.15582C>T variation among South African and Cameroonian persons. The effects of CYP2B6 g.15582C>T on plasma EFV concentration were further evaluated among HIV/AIDS patients. We report no differences in the frequency of the g.15582T variant between the South African (0.08) and Cameroonian (0.06) groups, which are significantly lower than reported in Asians (0.39) and

  14. The role of human cytochrome P450 enzymes in the formation of 2-hydroxymetronidazole: CYP2A6 is the high affinity (low Km) catalyst.

    PubMed

    Pearce, Robin E; Cohen-Wolkowiez, Michael; Sampson, Mario R; Kearns, Gregory L

    2013-09-01

    Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

  15. The impact of Cytochrome P450 CYP1A2, CYP2C9, CYP2C19 and CYP2D6 genes on suicide attempt and suicide risk-a European multicentre study on treatment-resistant major depressive disorder.

    PubMed

    Höfer, Peter; Schosser, Alexandra; Calati, Raffaella; Serretti, Alessandro; Massat, Isabelle; Kocabas, Neslihan Aygun; Konstantinidis, Anastasios; Linotte, Sylvie; Mendlewicz, Julien; Souery, Daniel; Zohar, Joseph; Juven-Wetzler, Alzbeta; Montgomery, Stuart; Kasper, Siegfried

    2013-08-01

    Recently published data have reported associations between cytochrome P450 metabolizer status and suicidality. The aim of our study was to investigate the role of genetic polymorphisms of the cytochrome P450 genes on suicide risk and/or a personal history of suicide attempts. Two hundred forty-three major depressive disorder patients were collected in the context of a European multicentre resistant depression study and treated with antidepressants at adequate doses for at least 4 weeks. Suicidality was assessed using the Mini International Neuropsychiatric Interview and the Hamilton Rating Scale for Depression (HAM-D). Treatment response was defined as HAM-D ≤ 17 and remission as HAM-D ≤ 7 after 4 weeks of treatment with antidepressants at adequate dose. Genotyping was performed for all relevant variations of the CYP1A2 gene (*1A, *1F, *1C, *1 J, *1 K), the CYP2C9 gene (*2, *3), the CYP2C19 gene (*2, *17) and the CYP2D6 gene (*3, *4, *5, *6, *9, *19, *XN). No association between both suicide risk and personal history of suicide attempts, and the above mentioned metabolic profiles were found after multiple testing corrections. In conclusion, the investigated cytochrome gene polymorphisms do not seem to be associated with suicide risk and/or a personal history of suicide attempts, though methodological and sample size limitations do not allow definitive conclusions.

  16. Curcumin Successfully Inhibited the Computationally Identified CYP2A6 Enzyme-Mediated Bioactivation of Aflatoxin B1 in Arbor Acres broiler

    PubMed Central

    Muhammad, Ishfaq; Sun, Xiaoqi; Wang, He; Li, Wei; Wang, Xinghe; Cheng, Ping; Li, Sihong; Zhang, Xiuying; Hamid, Sattar

    2017-01-01

    Cytochrome P450 enzymes are often responsible for the toxic and carcinogenic effects of toxicants, such as aflatoxin B1 (AFB1). The human hepatic CYP2A6 enzyme mediates the oxidative metabolism of several procarcinogens. In this study, we characterized a partial sequence of CYP2A6 gene from Arbor Acres (AA) broiler and studied its role in AFB1 bioactivation. Moreover, the effect of curcumin on CYP2A6 is illustrated. Six groups of AA broiler were treated for 28 days including the control group (fed only basal diet), curcumin alone-treated group (450 mg/kg feed), the group fed AFB1-contaminated feed (5 mg/kg feed) plus the low (150 mg), medium (300 mg) or high (450 mg) of curcumin, and the group fed AFB1-contaminated diet alone (5 mg/kg feed). After the end of treatment period, liver samples were collected for different analyses. The results revealed that the histopathological examination showed clear signs of liver toxicity in AA broliers in AFB1-fed group, but curcumin-supplementation in feed prevented partially AFB1-induced liver toxicity. Liver and body weights were recorded to study the AFB1 harmful effects. We noted an obvious increase in liver weight and decrease in body weight in AFB1-fed group. But, the administration of curcumin partially ameliorated the increase in liver weight and decrease in body weight in a dose-dependent manner. The results (RT-PCR and Elisa) revealed that mRNA and protein expression level enhanced in AFB1-fed group. Consistently, CYP2A6 enzyme activity also increased in AFB1-fed group, suggesting that AA broiler CYP2A6 actively involved in bioactivation of AFB1. However, curcumin treatment inhibited CYP2A6 at mRNA and protein levels in AFB1 treated AA broiler in a dose-dependent manner. Maximum inhibition of liver CYP2A6 enzyme activity in AA broiler has been achieved at a dose of 450 mg/kg curcumin. This is the first study identifying and confirming the role of CYP2A6 enzyme in AFB1 bioactivation in AA broiler liver (in vivo), and

  17. Curcumin Successfully Inhibited the Computationally Identified CYP2A6 Enzyme-Mediated Bioactivation of Aflatoxin B1 in Arbor Acres broiler.

    PubMed

    Muhammad, Ishfaq; Sun, Xiaoqi; Wang, He; Li, Wei; Wang, Xinghe; Cheng, Ping; Li, Sihong; Zhang, Xiuying; Hamid, Sattar

    2017-01-01

    Cytochrome P450 enzymes are often responsible for the toxic and carcinogenic effects of toxicants, such as aflatoxin B1 (AFB1). The human hepatic CYP2A6 enzyme mediates the oxidative metabolism of several procarcinogens. In this study, we characterized a partial sequence of CYP2A6 gene from Arbor Acres (AA) broiler and studied its role in AFB1 bioactivation. Moreover, the effect of curcumin on CYP2A6 is illustrated. Six groups of AA broiler were treated for 28 days including the control group (fed only basal diet), curcumin alone-treated group (450 mg/kg feed), the group fed AFB1-contaminated feed (5 mg/kg feed) plus the low (150 mg), medium (300 mg) or high (450 mg) of curcumin, and the group fed AFB1-contaminated diet alone (5 mg/kg feed). After the end of treatment period, liver samples were collected for different analyses. The results revealed that the histopathological examination showed clear signs of liver toxicity in AA broliers in AFB1-fed group, but curcumin-supplementation in feed prevented partially AFB1-induced liver toxicity. Liver and body weights were recorded to study the AFB1 harmful effects. We noted an obvious increase in liver weight and decrease in body weight in AFB1-fed group. But, the administration of curcumin partially ameliorated the increase in liver weight and decrease in body weight in a dose-dependent manner. The results (RT-PCR and Elisa) revealed that mRNA and protein expression level enhanced in AFB1-fed group. Consistently, CYP2A6 enzyme activity also increased in AFB1-fed group, suggesting that AA broiler CYP2A6 actively involved in bioactivation of AFB1. However, curcumin treatment inhibited CYP2A6 at mRNA and protein levels in AFB1 treated AA broiler in a dose-dependent manner. Maximum inhibition of liver CYP2A6 enzyme activity in AA broiler has been achieved at a dose of 450 mg/kg curcumin. This is the first study identifying and confirming the role of CYP2A6 enzyme in AFB1 bioactivation in AA broiler liver (in vivo), and

  18. More potent inhibition of human CYP2A6 than mouse CYP2A5 enzyme activities by derivatives of phenylethylamine and benzaldehyde.

    PubMed

    Rahnasto, M; Raunio, H; Poso, A; Juvonen, R O

    2003-05-01

    1. A rapid 96-well plate assay method was developed and validated to measure liver microsomal coumarin 7-hydroxylation in vitro. 2. The method was used to test inhibition of human and mouse CYP2A enzymes by three phenylethylamine derivatives 2-(p-tolyl)-ethylamine, amphetamine, 2-phenylethylamine and benzaldehyde, and two of its derivatives, 4-methylbenzaldehyde and 4-methoxybenzaldehyde. 3. The benzaldehyde derivatives were more potent inhibitors of CYP2A5 than the phenylethylamines. The K(ic) value of 4-methylbenzaldehyde was 3.4 micro M and for 4-methoxybenzaldehyde it was 0.86 micro M for CYP2A5. 4. Amphetamine is a weak inhibitor of CYP2A6, whereas benzaldehyde is a suicide inhibitor with K(inact) = 0.16 min(-1) and K(I) = 18 micro M. The K(ic) values of 2-phenylethylamine, 2-(p-tolyl)-ethylamine, 4-methylbenzaldehyde and 4-methoxybenzaldehyde were 1.13, 0.23, 0.36 and 0.73 micro M for CYP2A6, respectively. 5. Novel potent inhibitors were found for CYP2A6 and, except for 4-methoxybenzaldehyde, all the compounds inhibited CYP2A5 and CYP2A6 enzymes differentially. These data add to the refinement of CYP2A enzyme active sites and provide chemical leads for developing novel chemical inhibitors of the CYP2A6 enzyme.

  19. Genome-Wide Pharmacogenomic Study on Methadone Maintenance Treatment Identifies SNP rs17180299 and Multiple Haplotypes on CYP2B6, SPON1, and GSG1L Associated with Plasma Concentrations of Methadone R- and S-enantiomers in Heroin-Dependent Patients.

    PubMed

    Yang, Hsin-Chou; Chu, Shih-Kai; Huang, Chieh-Liang; Kuo, Hsiang-Wei; Wang, Sheng-Chang; Liu, Sheng-Wen; Ho, Ing-Kang; Liu, Yu-Li

    2016-03-01

    Methadone maintenance treatment (MMT) is commonly used for controlling opioid dependence, preventing withdrawal symptoms, and improving the quality of life of heroin-dependent patients. A steady-state plasma concentration of methadone enantiomers, a measure of methadone metabolism, is an index of treatment response and efficacy of MMT. Although the methadone metabolism pathway has been partially revealed, no genome-wide pharmacogenomic study has been performed to identify genetic determinants and characterize genetic mechanisms for the plasma concentrations of methadone R- and S-enantiomers. This study was the first genome-wide pharmacogenomic study to identify genes associated with the plasma concentrations of methadone R- and S-enantiomers and their respective metabolites in a methadone maintenance cohort. After data quality control was ensured, a dataset of 344 heroin-dependent patients in the Han Chinese population of Taiwan who underwent MMT was analyzed. Genome-wide single-locus and haplotype-based association tests were performed to analyze four quantitative traits: the plasma concentrations of methadone R- and S-enantiomers and their respective metabolites. A significant single nucleotide polymorphism (SNP), rs17180299 (raw p = 2.24 × 10(-8)), was identified, accounting for 9.541% of the variation in the plasma concentration of the methadone R-enantiomer. In addition, 17 haplotypes were identified on SPON1, GSG1L, and CYP450 genes associated with the plasma concentration of methadone S-enantiomer. These haplotypes accounted for approximately one-fourth of the variation of the overall S-methadone plasma concentration. The association between the S-methadone plasma concentration and CYP2B6, SPON1, and GSG1L were replicated in another independent study. A gene expression experiment revealed that CYP2B6, SPON1, and GSG1L can be activated concomitantly through a constitutive androstane receptor (CAR) activation pathway. In conclusion, this study revealed new

  20. Genome-Wide Pharmacogenomic Study on Methadone Maintenance Treatment Identifies SNP rs17180299 and Multiple Haplotypes on CYP2B6, SPON1, and GSG1L Associated with Plasma Concentrations of Methadone R- and S-enantiomers in Heroin-Dependent Patients

    PubMed Central

    Yang, Hsin-Chou; Chu, Shih-Kai; Huang, Chieh-Liang; Kuo, Hsiang-Wei; Wang, Sheng-Chang; Liu, Sheng-Wen; Ho, Ing-Kang; Liu, Yu-Li

    2016-01-01

    Methadone maintenance treatment (MMT) is commonly used for controlling opioid dependence, preventing withdrawal symptoms, and improving the quality of life of heroin-dependent patients. A steady-state plasma concentration of methadone enantiomers, a measure of methadone metabolism, is an index of treatment response and efficacy of MMT. Although the methadone metabolism pathway has been partially revealed, no genome-wide pharmacogenomic study has been performed to identify genetic determinants and characterize genetic mechanisms for the plasma concentrations of methadone R- and S-enantiomers. This study was the first genome-wide pharmacogenomic study to identify genes associated with the plasma concentrations of methadone R- and S-enantiomers and their respective metabolites in a methadone maintenance cohort. After data quality control was ensured, a dataset of 344 heroin-dependent patients in the Han Chinese population of Taiwan who underwent MMT was analyzed. Genome-wide single-locus and haplotype-based association tests were performed to analyze four quantitative traits: the plasma concentrations of methadone R- and S-enantiomers and their respective metabolites. A significant single nucleotide polymorphism (SNP), rs17180299 (raw p = 2.24 × 10−8), was identified, accounting for 9.541% of the variation in the plasma concentration of the methadone R-enantiomer. In addition, 17 haplotypes were identified on SPON1, GSG1L, and CYP450 genes associated with the plasma concentration of methadone S-enantiomer. These haplotypes accounted for approximately one-fourth of the variation of the overall S-methadone plasma concentration. The association between the S-methadone plasma concentration and CYP2B6, SPON1, and GSG1L were replicated in another independent study. A gene expression experiment revealed that CYP2B6, SPON1, and GSG1L can be activated concomitantly through a constitutive androstane receptor (CAR) activation pathway. In conclusion, this study revealed new

  1. Identification of N-(hydroxymethyl) norcotinine as a major product of cytochrome P450 2A6, but not cytochrome P450 2A13-catalyzed cotinine metabolism.

    PubMed

    Brown, Kathryn M; von Weymarn, Linda B; Murphy, Sharon E

    2005-12-01

    Cotinine formation is the major pathway of nicotine metabolism in smokers, and the primary pathway of cotinine metabolism is trans-3'-hydroxylation. trans-3'-Hydroxycotinine and its glucuronide conjugate account for up to 50% of the nicotine metabolites excreted by smokers. Minor metabolites of cotinine excreted by smokers include norcotinine and cotinine N-oxide, each of which account for <5% of the nicotine dose. It has been reported that P450 2A6 is the catalyst of cotinine metabolism. However, we report here that the major product of P450 2A6-catalyzed cotinine metabolism is N-(hydroxymethyl)norcotinine, a previously unknown human metabolite of cotinine. N-(Hydroxymethyl)norcotinine was chemically synthesized, and its stability under the conditions of the enzyme reactions was confirmed. The products of P450 2A6-catalyzed [5-3H]cotinine metabolism were quantified by radioflow HPLC. The identification of N-(hydroxymethyl)norcotinine as the major metabolite was based on HPLC analysis on three unique systems and coelution with N-(hydroxymethyl)norcotinine standard. 5'-Hydroxycotinine and trans-3'-hydroxycotinine were minor products of P450 2A6-catalyzed cotinine metabolism, accounting for 14 and 8% of the total cotinine metabolites, respectively. N-(Hydroxymethyl)norcotinine was a product of cotinine metabolism by the extrahepatic P450, 2A13, but it was a minor one. The major product of P450 2A13-catalyzed cotinine metabolism was 5'-hydroxycotinine, which was formed at twice the rate of trans-3'-hydroxycotinine. The identification of all cotinine metabolites formed by both enzymes was confirmed by LC/MS/MS analysis. Kinetic parameters for cotinine metabolism were determined for P450 2A6 and P450 2A13. This work has confirmed that the major metabolite of cotinine in smokers, trans-3'-hydroxycotinine, is only a minor metabolite of P450 2A6-catalyzed cotinine metabolism.

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

    NASA Astrophysics Data System (ADS)

    Jett, John Edward, Jr.

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

  3. Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

    PubMed

    Prasopthum, Aruna; Pouyfung, Phisit; Sarapusit, Songklod; Srisook, Ekaruth; Rongnoparut, Pornpimol

    2015-04-01

    The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

  4. Influence of synthetic and natural food dyes on activities of CYP2A6, UGT1A6, and UGT2B7.

    PubMed

    Kuno, Nayumi; Mizutani, Takaharu

    2005-08-27

    Synthetic or natural food dyes are typical xenobiotics, as are drugs and pollutants. After ingestion, part of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes and excreted by transporters of phase III enzymes. However, there is little information regarding the metabolism of these dyes. It was investigated whether these dyes are substrates for CYP2A6 and UDP-glucuronosyltransferase (UGT). The in vitro inhibition of drug-metabolizing enzymes by these dyes was also examined. The synthetic food dyes studied were amaranth (food red no. 2), erythrosine B (food red no. 3), allura red (food red no. 40), new coccine (food red no. 102), acid red (food red no. 106), tartrazine (food Yellow no. 4), sunset yellow FCF (food yellow no. 5), brilliant blue FCF (food blue no. 1), and indigo carmine (food blue no. 2). The natural additive dyes studied were extracts from purple sweet potato, purple corn, cochineal, monascus, grape skin, elderberry, red beet, gardenia, and curthamus. Data confirmed that these dyes were not substrates for CYP2A6, UGT1A6, and UGT2B7. Only indigo carmine inhibited CYP2A6 in a noncompetitive manner, while erythrosine B inhibited UGT1A6 (glucuronidation of p-nitrophenol) and UGT2B7 (glucuronidation of androsterone). In the natural additive dyes just listed, only monascus inhibited UGT1A6 and UGT2B7.

  5. Oxidation of N-Nitrosoalkylamines by human cytochrome P450 2A6: sequential oxidation to aldehydes and carboxylic acids and analysis of reaction steps.

    PubMed

    Chowdhury, Goutam; Calcutt, M Wade; Guengerich, F Peter

    2010-03-12

    Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH(3)CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect ((D)k(app) approximately 10), which was highly expressed in a variety of competitive and non-competitive experiments. The (D)k(app) for DEN was approximately 3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO(2)H and CH(3)CO(2)H, respectively. In neither case was a lag observed, which was unexpected considering the k(cat) and K(m) parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).

  6. Exploring QSAR and QAAR for inhibitors of cytochrome P450 2A6 and 2A5 enzymes using GFA and G/PLS techniques.

    PubMed

    Roy, Kunal; Roy, Partha Pratim

    2009-05-01

    A series of naphthalene and non-naphthalene derivatives (n=42) having cytochrome P450 2A6 and 2A5 inhibitory activities, reported by Rahnasto et al., were subjected to QSAR and QAAR studies. The analyses were performed using electronic, spatial, shape and thermodynamic descriptors to develop quantitative models for prediction of the inhibitory activities and to explore importance of different descriptors for the responses. The data set was divided into training and test sets (with test set size being approximately 25% of the full data set size) based on K-means clustering applied on the standardized descriptor matrix. Genetic function approximation (GFA) and genetic partial least-squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical and prediction (both internal and external) qualities although different equations varied in quality in a wide range (R(2): 0.561-0.898, R(a)(2): 0.508-0.870, Q(2): 0.495-0.814, R(pred)(2): 0.615-0.914, r(2): 0.679-0.905, r(0)(2): 0.639-0.904, r(m)(2): 0.494-0.876). In the case of CYP2A5 inhibition, the GFA derived QSAR model is better than the G/PLS derived model considering both internal and external validations. In the case of CYP2A6 inhibitory potency data, the GFA derived QSAR model is better than the G/PLS model considering internal validation whereas the latter is better in external validation (which is more important) than the former. The model development process was subjected to randomization test at 90% confidence level by taking into account the whole pool of descriptors, while the developed models were also subjected to randomization test (99% confidence level) for validation. Based on the randomization test results, GFA models are found to be superior to the G/PLS models. Among the parameters, which were found important in modeling both the responses, were different Jurs descriptors, electronic descriptors (like Sr, Apol), steric descriptors (like shadow

  7. Water pipe (Shisha, Hookah, Arghile) Smoking and Secondhand Tobacco Smoke Effects on CYP1A2 and CYP2A6 Phenotypes as Measured by Caffeine Urine Test.

    PubMed

    Yılmaz, Şenay Görücü; Llerena, Adrián; De Andrés, Fernando; Karakaş, Ümit; Gündoğar, Hasan; Erciyas, Kamile; Kimyon, Sabit; Mete, Alper; Güngör, Kıvanç; Özdemir, Vural

    2017-03-01

    Public policies to stop or reduce cigarette smoking and exposure to secondhand smoke and associated diseases have yielded successful results over the past decade. Yet, the growing worldwide popularity of another form of tobacco consumption, water pipe smoking, has received relatively less attention. To the best of our knowledge, no study to date has evaluated the effects of water pipe smoking on cytochrome P450 (CYP450) activities and drug interaction potential in humans, whereas only limited information is available on the impact of secondhand smoke on drug metabolism. In a sample of 99 healthy volunteers (28 water pipe smokers, 30 secondhand tobacco smoke exposed persons, and 41 controls), we systematically compared CYP1A2 and CYP2A6 enzyme activities in vivo using caffeine urine test. The median self-reported duration of water pipe smoking was 7.5 h/week and 3 years of exposure in total. The secondhand smoke group had a median of 14 h of self-reported weekly exposure to tobacco smoke indoor where a minimum of five cigarettes were smoked/hour for a total of 3.5 years (median). Analysis of variance did not find a significant difference in CYP1A2 and CYP2A6 activities among the three study groups (p > 0.05). Nor was there a significant association between the extent of water pipe or secondhand smoke exposure and the CYP1A2 and CYP2A6 activities (p > 0.05). Further analysis in a subsample with smoke exposure more than the median values also did not reveal a significant difference from the controls. Although we do not rule out an appreciable possible impact of water pipe smoke and secondhand smoke on in vivo activities of these two drug metabolism pathways, variability in smoke constituents from different tobacco consumption methods (e.g., water pipe) might affect drug metabolism in ways that might differ from that of cigarette smoke. Further studies in larger prospective samples are recommended to evaluate water pipe and secondhand tobacco smoke effects

  8. Alaska Native smokers and smokeless tobacco users with slower CYP2A6 activity have lower tobacco consumption, lower tobacco-specific nitrosamine exposure and lower tobacco-specific nitrosamine bioactivation.

    PubMed

    Zhu, Andy Z X; Binnington, Matthew J; Renner, Caroline C; Lanier, Anne P; Hatsukami, Dorothy K; Stepanov, Irina; Watson, Clifford H; Sosnoff, Connie S; Benowitz, Neal L; Tyndale, Rachel F

    2013-01-01

    Nicotine, the psychoactive ingredient in tobacco, is metabolically inactivated by CYP2A6 to cotinine. CYP2A6 also activates procarcinogenic tobacco-specific nitrosamines (TSNA). Genetic variation in CYP2A6 is known to alter smoking quantity and lung cancer risk in heavy smokers. Our objective was to investigate how CYP2A6 activity influences tobacco consumption and procarcinogen levels in light smokers and smokeless tobacco users. Cigarette smokers (n = 141), commercial smokeless tobacco users (n = 73) and iqmik users (n = 20) were recruited in a cross-sectional study of Alaska Native people. The participants' CYP2A6 activity was measured by both endophenotype and genotype, and their tobacco and procarcinogen exposure biomarker levels were also measured. Smokers, smokeless tobacco users and iqmik users with lower CYP2A6 activity had lower urinary total nicotine equivalents (TNE) and (methylnitrosamino)-1-(3)pyridyl-1-butanol (NNAL) levels (a biomarker of TSNA exposure). Levels of N-nitrosonornicotine (NNN), a TSNA metabolically bioactivated by CYP2A6, were higher in smokers with lower CYP2A6 activities. Light smokers and smokeless tobacco users with lower CYP2A6 activity reduce their tobacco consumption in ways (e.g. inhaling less deeply) that are not reflected by self-report indicators. Tobacco users with lower CYP2A6 activity are exposed to lower procarcinogen levels (lower NNAL levels) and have lower procarcinogen bioactivation (as indicated by the higher urinary NNN levels suggesting reduced clearance), which is consistent with a lower risk of developing smoking-related cancers. This study demonstrates the importance of CYP2A6 in the regulation of tobacco consumption behaviors, procarcinogen exposure and metabolism in both light smokers and smokeless tobacco users.

  9. Binding of diverse environmental chemicals with human cytochromes P450 2A13, 2A6, and 1B1 and enzyme inhibition.

    PubMed

    Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D; Folkman, Lindsay M; Foroozesh, Maryam K; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F Peter

    2013-04-15

    A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e., the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450

  10. Binding of Diverse Environmental Chemicals with Human Cytochromes P450 2A13, 2A6, and 1B1 and Enzyme Inhibition

    PubMed Central

    Shimada, Tsutomu; Kim, Donghak; Murayama, Norie; Tanaka, Katsuhiro; Takenaka, Shigeo; Nagy, Leslie D.; Folkman, Lindsay M.; Foroozesh, Maryam K.; Komori, Masayuki; Yamazaki, Hiroshi; Guengerich, F. Peter

    2014-01-01

    A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2’,5’-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2’-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2’-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family

  11. Metabolic effects of CYP2A6 and CYP2A13 on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced gene mutation-A mammalian cell-based mutagenesis approach

    SciTech Connect

    Chiang, Huai-chih; Wang, Chin-Ying; Lee, Hui-Ling; Tsou, Tsui-Chun

    2011-06-01

    Both cytochrome P450 2A6 (CYP2A6) and cytochrome P450 2A13 (CYP2A13) are involved in metabolic activation of tobacco-specific nitrosamines and may play important roles in cigarette smoking-induced lung cancer. Unlike CYP2A6, effects of CYP2A13 on the tobacco-specific nitrosamine-induced mutagenesis in lung cells remain unclear. This study uses a supF mutagenesis assay to examine the relative effects of CYP2A6 and CYP2A13 on metabolic activation of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its resulting mutagenesis in human lung cells. A recombinant adenovirus-mediated CYP2A6/CYP2A13 expression system was established to specifically address the relative effects of these two CYPs. Mutagenesis results revealed that both CYP2A6 and CYP2A13 significantly enhanced the NNK-induced supF mutation and that the mutagenic effect of CYP2A13 was markedly higher than that of CYP2A6. Analysis of NNK metabolism indicated that {>=} 70% of NNK was detoxified to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), either with or without CYP2A6/CYP2A13 expression. Both CYP2A6 and CYP2A13 significantly enhanced the {alpha}-hydroxylation of NNK; and the {alpha}-hydroxylation activity of CYP2A13 was significantly higher than that of CYP2A6. Analysis of the NNK-related DNA adduct formation indicated that, in the presence of CYP2A13, NNK treatments caused marked increases in O{sup 6}-methylguanine (O{sup 6}-MeG). The present results provide the first direct in vitro evidence demonstrating the predominant roles of CYP2A13 in NNK-induced mutagenesis, possibly via metabolic activation of NNK {alpha}-hydroxylation.

  12. A LC-MS/MS method for concurrent determination of nicotine metabolites and role of CYP2A6 in nicotine metabolism in U937 macrophages: implications in oxidative stress in HIV + smokers.

    PubMed

    Jin, Mengyao; Earla, Ravinder; Shah, Ankit; Earla, Rajya L; Gupte, Raeesa; Mitra, Ashim K; Kumar, Anil; Kumar, Santosh

    2012-03-01

    Nicotine, the major constituent of tobacco, is predominantly metabolized by liver CYP2A6 into cotinine and many other compounds, including nicotine-derived nitrosamine ketone (NNK), which is known to cause oxidative stress. We have recently shown that CYP2A6 is highly expressed in U937 monocyte-derived macrophages. In this study we investigated the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages. To study nicotine metabolism, we developed a highly sensitive LC-MS/MS method for simultaneous quantitative determination of nicotine, cotinine, and NNK. The LC-MS/MS analysis was carried out by multiple reaction monitoring mass transitions with m/z of 163.2/130.1, 177.4/98.3, and 208.4/122.1 for nicotine, cotinine, and NNK, respectively. The calibration curves were linear within 3.3-1028.1 ng/ml for nicotine and 0.3-652.6 ng/ml for cotinine and NNK. This novel method was then applied to quantify nicotine metabolites, cotinine and NNK, in nicotine-treated U937 macrophages. Cotinine and NNK initially formed at 30 min, followed by a peak at 2-3 h. The role of CYP2A6 in nicotine metabolism in U937 macrophages was further confirmed by using CYP2A6-selective inhibitor, tryptamine, which significantly decreased cotinine (70%) and completely inhibited NNK formations. Finally, we showed that nicotine-treated macrophages increase the formation of oxidant at 30-60 min, which is consistent with the initial formation of cotinine and NNK. In conclusion, we have developed a new LCMS/MS method for concurrent determination of nicotine metabolites and analyzed the role of CYP2A6 in nicotine metabolism and oxidative stress in U937 macrophages, which may have implications in viral replication among HIV + smokers.

  13. Piezoelectric crystal microbalance measurements of enthalpy of sublimation of C2-C9 dicarboxylic acids

    NASA Astrophysics Data System (ADS)

    Dirri, F.; Palomba, E.; Longobardo, A.; Zampetti, E.

    2016-02-01

    We present here a novel experimental set-up that is able to measure the enthalpy of sublimation of a given compound by means of piezoelectric crystal microbalances (PCMs). The PCM sensors have already been used for space measurements, such as for the detection of organic and non-organic volatile species and refractory materials in planetary environments. In Earth atmospherics applications, PCMs can be also used to obtain some physical-chemical processes concerning the volatile organic compounds (VOCs) present in atmospheric environments. The experimental set-up has been developed and tested on dicarboxylic acids. In this work, a temperature-controlled effusion cell was used to sublimate VOC, creating a molecular flux that was collimated onto a cold PCM. The VOC recondensed onto the PCM quartz crystal, allowing the determination of the deposition rate. From the measurements of deposition rates, it has been possible to infer the enthalpy of sublimation of adipic acid, i.e. ΔHsub : 141.6 ± 0.8 kJ mol-1, succinic acid, i.e. 113.3 ± 1.3 kJ mol-1, oxalic acid, i.e. 62.5 ± 3.1 kJ mol-1, and azelaic acid, i.e. 124.2 ± 1.2 kJ mol-1. The results obtained show an accuracy of 1 % for succinic, adipic, and azelaic acid and within 5 % for oxalic acid and are in very good agreement with previous works (within 6 % for adipic, succinic, and oxalic acid and within 11 % or larger for azelaic acid).

  14. Piezoelectric crystal microbalance measurements of enthalpy of sublimation of C2-C9 dicarboxylic acids

    NASA Astrophysics Data System (ADS)

    Dirri, F.; Palomba, E.; Longobardo, A.; Zampetti, E.

    2015-07-01

    We present here a novel experimental setup able to measure the enthalpy of sublimation of a given compound by means of Piezoelectric Crystal Microbalances (PCM). This experiment was performed in the TG-Lab facility in IAPS-INAF, dedicated to the development of TGA sensors for space measurements, such as detection of organic and non-organic volatile species and refractory materials in planetary environments. In order to study physical-chemical processes concerning the Volatile Organic Compounds (VOC) present in atmospheric environments, the setup has been tested on Dicarboxylic acids. Acids with low molecular weight are among the components of organic fraction of particulate matter in the atmosphere, coming from different sources (biogenic and anthropogenic). Considering their relative abundance, it is useful to consider Dicarboxylic acid as "markers" to define the biogenic or anthropogenic origin of the aerosol, thus obtaining some information of the emission sources. In this work, a temperature controlled effusion cell was used to sublimate VOC, creating a molecular flux that was collimated onto a cold PCM. The VOC re-condensed onto the PCM quartz crystal allowing the determination of the deposition rate. From the measurements of deposition rates, it was possible to infer the enthalpy of sublimation of Adipic acid, i.e. Δ Hsub: 141.6 ± 0.8 kJ mol-1, Succinic acid, i.e. 113.3 ± 1.3 kJ mol-1, Oxalic acid, i.e. 62.5 ± 3.1 kJ mol-1 and Azelaic acid, i.e. 124.2 ± 1.2 kJ mol-1 (weight average values). The results obtained are in very good agreement with literature within 10 % for the Adipic, Succinic and Oxalic acid.

  15. VKORC1 and CYP2C9 Gene Polymorphisms and Warfarin Management

    ClinicalTrials.gov

    2009-09-02

    Atrial Fibrillation; Cardiac Thrombus; Deep Vein Thrombosis; Pulmonary Embolism; Heart Valve Replacement (Mechanical or Biological With AF); Cardiomyopathy (Ischemic or Dilated); Peripheral Vascular Disease

  16. Genomic Landscape of Experimental Bladder Cancer in Rodents and Its Application to Human Bladder Cancer: Gene Amplification and Potential Overexpression of Cyp2a5/CYP2A6 Are Associated with the Invasive Phenotype

    PubMed Central

    Kanemoto, Kazuhiro; Fukuta, Katsuhiro; Kawai, Noriyasu; Tozawa, Keiichi; Ochiai, Masako; Okamoto, Koji; Ohnami, Sumiko; Sakamoto, Hiromi; Yoshida, Teruhiko; Kanai, Yae; Katoh, Masaru; Yasui, Takahiro; Kohri, Kenjiro; Kakizoe, Tadao; Nakagama, Hitoshi

    2016-01-01

    Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with

  17. Materials Data on Gd(Co2B)6 (SG:166) by Materials Project

    SciTech Connect

    Kristin Persson

    2014-07-09

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  18. Materials Data on K2B6H5NO2 (SG:63) by Materials Project

    SciTech Connect

    Kristin Persson

    2016-02-04

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  19. Materials Data on Sr(Ni2B)6 (SG:166) by Materials Project

    DOE Data Explorer

    Kristin Persson

    2016-02-10

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  20. Materials Data on Ba(Ni2B)6 (SG:166) by Materials Project

    DOE Data Explorer

    Kristin Persson

    2016-02-10

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  1. Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

    PubMed

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Lee, M; Yang, C S; Hu, W Y; Pan, J

    1999-10-01

    To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the

  2. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

  3. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  4. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover, applications of immobilized P450 enzyme constructs will also be used for monitoring protein-protein interaction and metabolite production with the use of immobilized-P450 bioreactor constructs. This work provides insight into the effect on catalytic activity caused by both P450 aggregation as well as isoform-specific protein-protein interactions and provides insight in the production of biosynthetically produced drug metabolites

  5. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

    AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  6. Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

    PubMed

    Kopečná-Zapletalová, Michaela; Krasulová, Kristýna; Anzenbacher, Pavel; Hodek, Petr; Anzenbacherová, Eva

    2017-04-01

    1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

  7. RS-WebPredictor: a server for predicting CYP-mediated sites of metabolism on drug-like molecules

    PubMed Central

    Zaretzki, Jed; Bergeron, Charles; Huang, Tao-wei; Rydberg, Patrik; Swamidass, S. Joshua; Breneman, Curt M.

    2013-01-01

    Summary: Regioselectivity-WebPredictor (RS-WebPredictor) is a server that predicts isozyme-specific cytochrome P450 (CYP)-mediated sites of metabolism (SOMs) on drug-like molecules. Predictions may be made for the promiscuous 2C9, 2D6 and 3A4 CYP isozymes, as well as CYPs 1A2, 2A6, 2B6, 2C8, 2C19 and 2E1. RS-WebPredictor is the first freely accessible server that predicts the regioselectivity of the last six isozymes. Server execution time is fast, taking on average 2s to encode a submitted molecule and 1s to apply a given model, allowing for high-throughput use in lead optimization projects. Availability: RS-WebPredictor is accessible for free use at http://reccr.chem.rpi.edu/Software/RS-WebPredictor/ Contact: brenec@rpi.edu PMID:23242264

  8. An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s

    PubMed Central

    Chen, Zhong-hua; Zhang, Su-xing; Long, Na; Lin, Li-shan; Chen, Tao; Zhang, Fei-peng; Lv, Xue-qin; Ye, Pei-zhen; Li, Ning; Zhang, Ke-zhi

    2016-01-01

    Aim: The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. Methods: In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT). Results: The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay. Conclusion: The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates. PMID:27063220

  9. In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

    PubMed

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

  10. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes.

    PubMed

    Kariya, S; Isozaki, S; Uchino, K; Suzuki, T; Narimatsu, S

    1996-11-01

    The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 microM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 microM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 microM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.

  11. 2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) revisited: comparative cytochrome P450 inhibition in human liver microsomes by SKF525A, its metabolites, and SKF-acid and SKF-alcohol.

    PubMed

    Franklin, Michael R; Hathaway, Laura B

    2008-12-01

    When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

  12. Urinary excretion of vitamin B1, B2, B6, niacin, pantothenic acid, folate, and vitamin C correlates with dietary intakes of free-living elderly, female Japanese.

    PubMed

    Tsuji, Tomiko; Fukuwatari, Tsutomu; Sasaki, Satoshi; Shibata, Katsumi

    2010-03-01

    We hypothesized that 24-hour urinary excretion of water-soluble vitamins might correlate with their intake in free-living Japanese elderly females aged 70 to 84 years. We performed a cross-sectional study composed of 37 healthy, elderly, Japanese females living freely. All foods and the corresponding weights consumed for 4 consecutive days were recorded accurately. A 24-hour urine sample was collected on the fourth day, and the urinary content of water-soluble vitamins was measured. The urinary levels of all vitamins, except for B(12) (r = 0.01; P = .936), were correlated positively with the mean intake over the recent 4 days (vitamin B1: r = 0.62; P < .001; vitamin B2: r = 0.57; P < .001; vitamin B6: r = 0.37; P < .005; niacin: r = 0.54; P < .001; niacin equivalents: r = 0.54; P < .001; pantothenic acid: r = 0.59; P < .001; folate: r = 0.55; P = .001; and vitamin C: r = 0.53; P < .001). Mean estimated intakes of water-soluble vitamins calculated using urinary concentrations and recovery rates showed 96% to 107% of their 3-day mean intake, except for vitamin B12 (65%). We conclude that urinary levels of water-soluble vitamins, except for B12, reflected their recent intake in free-living Japanese elderly females and could be used as a measure of their intake during the previous few days both for group means and for individual rankings within a group.

  13. Metabolism of Endosulfan-Alpha by Human Liver Microsomes and its Utility as a Simultaneous In Vitro Probe for CYP2B6 and CYP3A4

    DTIC Science & Technology

    2006-03-30

    cyclophosphamide and ifosfamide (Huang et al., 2000), S-mephenytoin (Heyn et al., 1996; Ko et al., 1998), bupropion (Faucette et al., 2000; Hesse et al...cyclophosphamide and ifosfamide . Biochem Pharmacol 59:961-972. Khanna RN, Misra D, Anand M and Sharma HK (1979) Distribution of endosulfan in cat

  14. In vitro cytochrome P450 inhibition potential of methylenedioxy-derived designer drugs studied with a two-cocktail approach.

    PubMed

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2016-02-01

    In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta-keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

  15. RS-Predictor models augmented with SMARTCyp reactivities: Robust metabolic regioselectivity predictions for nine CYP isozymes

    PubMed Central

    Zaretzki, Jed; Rydberg, Patrik; Bergeron, Charles; Bennett, Kristin P.; Olsen, Lars

    2012-01-01

    RS-Predictor is a tool for creating pathway-independent, isozyme-specific site of metabolism (SOM) prediction models using any set of known cytochrome P450 substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1 and 3A4, which we believe is the largest publicly accessible collection of P450 ligands and metabolites ever released. A comprehensive investigation into the importance of different descriptor classes for predicting the regioselectivity of each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a DFT reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by StarDrop and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2(83.0%), 2A6(85.7%), 2B6(82.1%), 2C19(86.2%), 2C8(83.8%), 2C9(84.5%), 2D6(85.9%), 2E1(82.8%), 3A4(82.3%) and merged(86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs. PMID:22524152

  16. RS-Predictor models augmented with SMARTCyp reactivities: robust metabolic regioselectivity predictions for nine CYP isozymes.

    PubMed

    Zaretzki, Jed; Rydberg, Patrik; Bergeron, Charles; Bennett, Kristin P; Olsen, Lars; Breneman, Curt M

    2012-06-25

    RS-Predictor is a tool for creating pathway-independent, isozyme-specific, site of metabolism (SOM) prediction models using any set of known cytochrome P450 (CYP) substrates and metabolites. Until now, the RS-Predictor method was only trained and validated on CYP 3A4 data, but in the present study, we report on the versatility the RS-Predictor modeling paradigm by creating and testing regioselectivity models for substrates of the nine most important CYP isozymes. Through curation of source literature, we have assembled 680 substrates distributed among CYPs 1A2, 2A6, 2B6, 2C19, 2C8, 2C9, 2D6, 2E1, and 3A4, the largest publicly accessible collection of P450 ligands and metabolites released to date. A comprehensive investigation into the importance of different descriptor classes for identifying the regioselectivity mediated by each isozyme is made through the generation of multiple independent RS-Predictor models for each set of isozyme substrates. Two of these models include a density functional theory (DFT) reactivity descriptor derived from SMARTCyp. Optimal combinations of RS-Predictor and SMARTCyp are shown to have stronger performance than either method alone, while also exceeding the accuracy of the commercial regioselectivity prediction methods distributed by Optibrium and Schrödinger, correctly identifying a large proportion of the metabolites in each substrate set within the top two rank-positions: 1A2 (83.0%), 2A6 (85.7%), 2B6 (82.1%), 2C19 (86.2%), 2C8 (83.8%), 2C9 (84.5%), 2D6 (85.9%), 2E1 (82.8%), 3A4 (82.3%), and merged (86.0%). Comprehensive datamining of each substrate set and careful statistical analyses of the predictions made by the different models revealed new insights into molecular features that control metabolic regioselectivity and enable accurate prospective prediction of likely SOMs.

  17. Main contribution of the cytochrome P450 isoenzyme 1A2 (CYP1A2) to N-demethylation and 5-sulfoxidation of the phenothiazine neuroleptic chlorpromazine in human liver--A comparison with other phenothiazines.

    PubMed

    Wójcikowski, Jacek; Boksa, Jan; Daniel, Władysława A

    2010-10-15

    The aim of the present study was to identify cytochrome P450 (CYP) isoenzymes involved in the 5-sulfoxidation, mono-N-demethylation and di-N-demethylation of the aliphatic-type phenothiazine neuroleptic chlorpromazine in human liver. Experiments were performed in vitro using cDNA-expressed human CYP isoforms (Supersomes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4), liver microsomes from different donors and CYP-selective inhibitors. The obtained results indicate that CYP1A2 is the only CYP isoform that catalyzes the mono-N-demethylation and di-N-demethylation of chlorpromazine (100%) and is the main isoform responsible for chlorpromazine 5-sulfoxidation (64%) at a therapeutic concentration of the drug (10 microM). CYP3A4 contributes to a lesser degree to chlorpromazine 5-sulfoxidation (34%). The role of CYP2B6, CYP2C19 and CYP2D6 in catalyzing of the latter reaction is negligible (0.1-2%). Similar results were obtained at a higher, non-therapeutic concentration of the drug (100 microM); however, the contribution of CYP1A2 to chlorpromazine mono-N-demethylation was noticeably lower (75%), mostly in favour of CYP2C19 and CYP3A4 (about 12% each). The obtained results indicate that the catalysis of chlorpromazine N-demethylation and 5-sulfoxidation in humans exhibits a stricter CYP1A2 preference compared to the previously tested phenothiazines (promazine, perazine, and thioridazine). Hence pharmacokinetic interactions involving chlorpromazine and CYP1A2 substrates and inhibitors are likely to occur. Considering strong dopaminergic D(2), noradrenergic alpha(1) and cholinergic M(1) receptor blocking properties of chlorpromazine and some of its metabolites, as well as their serious side effects, the obtained results may be of pharmacological and clinical importance.

  18. Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

    PubMed

    Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Padala, Nagasurya Prakash; Boggavarapu, Rajesh Kumar; Bhyrapuneni, Gopinadh; Ajjala, Devender Reddy; Nirogi, Ramakrishna

    2017-04-01

    The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values.

  19. Insights on Cytochrome P450 Enzymes and Inhibitors Obtained Through QSAR Studies

    PubMed Central

    Sridhar, Jayalakshmi; Liu, Jiawang; Foroozesh, Maryam; Stevens, Cheryl L. Klein

    2013-01-01

    The cytochrome P450 (CYP) superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR), and three-dimensional quantitative structure activity relationships (3D-QSAR) represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions. PMID:22864238

  20. Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts.

    PubMed

    Ding, Xinxin; Kaminsky, Laurence S

    2003-01-01

    Cytochrome P450 (CYP) enzymes in extrahepatic tissues often play a dominant role in target tissue metabolic activation of xenobiotic compounds. They may also determine drug efficacy and influence the tissue burden of foreign chemicals or bioavailability of therapeutic agents. This review focuses on xenobiotic-metabolizing CYPs of the human respiratory and gastrointestinal tracts, including the lung, trachea, nasal respiratory and olfactory mucosa, esophagus, stomach, small intestine, and colon. Many CYPs are expressed in one or more of these organs, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2S1, CYP3A4, CYP3A5, and CYP4B1. Of particular interest are the preferential expression of certain CYPs in the respiratory tract and the regional differences in CYP expression profile in different parts of the gastrointestinal tract. Current research activities on the characterization of CYP expression, function, and regulation in these tissues, as well as future research needs, are discussed.

  1. High-throughput screening of inhibitory effects of Bo-yang-hwan-o-tang on human cytochrome P450 isoforms in vitro using UPLC/MS/MS.

    PubMed

    Lee, Miran; Park, Jeonghyeon; Lim, Mi-sun; Seong, Sook Jin; Lee, Joomi; Seo, Jeong Ju; Park, Yong-Ki; Lee, Hae Won; Yoon, Young-Ran

    2012-01-01

    Bo-yang-hwan-o-tang (BHT) is an oriental herbal medicine for treating brain disorders such as cerebral ischemia. The objective of this study was to develop an economically feasible and time-saving high-throughput screening method to monitor the potential inhibitory effects of BHT on human cytochrome P450 (CYP) enzymes in vitro. Two cocktail sets were used for incubation of human liver microsomes: Cocktail A: 6 probe substrates for CYP1A2, CYP2A6, CYP2C8, CYP2C19, CYP2D6, CYP3A4; Cocktail B: 3 for CYP2B6, CYP2C9, CYP2E1. The concentrations of the substrate metabolites were simultaneously analyzed using UPLC/MS/MS. The BHT extract had almost negligible inhibitory effects on the nine human CYP isoforms tested, with the half-maximal inhibitory concentration value ranged from 3624.99 to 45412.44 μg/ml. The results suggest that BHT extract has no inhibitory effects on CYP isoforms within the clinically recommended dosage range. We conclude that BHT might be free of drug-herb interactions when co-administered with other medicines. However, more in vivo human studies are needed to confirm these results. The high-throughput screening method can be a useful tool for drug discovery and for understanding drug interactions.

  2. Cytochrome P450 inhibition potential of new psychoactive substances of the tryptamine class.

    PubMed

    Dinger, Julia; Woods, Campbell; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

    2016-01-22

    New psychoactive substances (NPS) are not tested for their cytochrome P450 (CYP) inhibition potential before consumption. Therefore, this potential was explored for tryptamine-derived NPS (TDNPS) including alpha-methyl tryptamines (AMTs), dimethyl tryptamines (DMTs), diallyl tryptamines (DALTs), and diisopropyl tryptamines (DiPTs) using test substrates preferred by the Food and Drug Administration in a cocktail assay. All tested TDNPS with the exception of DMT inhibited CYP2D6 activity with IC50 values below 100μM. DALTs inhibited CYP2D6 activity similar to paroxetine and quinidine and CYP1A2 activity comparable to fluvoxamine. 5-Methoxy-N,N-diallyltryptamine reduced in vivo the caffeine metabolism in rats consistent with in vitro results. Five of the AMTs also inhibited CYP1A2 activity comparable to amiodarone. AMT and 6-F-AMT inhibited CYP2A6 activity in the range of the test inhibitor tranylcypromine. CYP2B6 activity was inhibited by 19 tryptamines, but weakly compared to efavirenz. CYP2C8 activity was inhibited by five of the tested TDNPS and three showed values comparable to trimethoprim and gemfibrozil. Six tryptamines inhibited CYP2C9 and seven CYP2C19 activities comparable to fluconazole and chloramphenicol, respectively. Nineteen compounds showed inhibition of CYP2E1 and 18 of CYP3A activity, respectively. These results showed that the CYP inhibition by TDNPS might be clinically relevant, but clinical studies are needed to explore this further.

  3. A Comparison of the In Vitro Inhibitory Effects of Thelephoric Acid and SKF-525A on Human Cytochrome P450 Activity.

    PubMed

    Song, Min; Do, Hyunhee; Kwon, Oh Kwang; Yang, Eun-Ju; Bae, Jong-Sup; Jeong, Tae Cheon; Song, Kyung-Sik; Lee, Sangkyu

    2014-02-01

    Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2-33.7 μM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.

  4. In vitro metabolism studies of nomifensine monooxygenation pathways: metabolite identification, reaction phenotyping, and bioactivation mechanism.

    PubMed

    Yu, Jian; Brown, Dean G; Burdette, Doug

    2010-10-01

    Multiple GSH adducts of the oxidative products of nomifensine (M1-M9) in human hepatocytes and liver microsomes have been identified recently. The current study reports three new types of monooxygenated metabolites of nomifensine identified in human liver microsomes: C-linked hydroxylated metabolites with modifications at the A ring (H1 and H4), an N-hydroxylamine (H6), and nomifensine N-oxides (H7 and H8). GSH conjugate formation in incubates containing cDNA-expressed P450s and GSH suggests that nomifensine GSH-sulfinamides (M1 and M2) are formed through the reaction between GSH and the oxidative product of H6. C-linked GSH conjugates M3, M4, M5, and M6 are probably formed via nomifensine benzoquinone imine intermediates via H4 and/or nomifensine epoxides. C-linked GSH conjugates M7, M8, and M9 are probably formed through similar mechanisms via H1. Nomifensine N-oxides do not form reactive metabolites that react with GSH. In vitro metabolism studies using a panel of cDNA-expressed human P450 and flavin monooxygenase (FMO) isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, FMO1, FMO3, and FMO5) indicated that CYP3A4, CYP2C19, and CYP2B6 generate the largest quantities of H1, H4, and H6, respectively. H7 and H8 are formed almost exclusively by FMOs. The contribution of the individual P450s involved in the formation of H1, H4, and H6 in human liver microsomes was confirmed by the inhibition of product formation by monoclonal anti-cytochrome 450 antibodies. These results showed that CYP3A4 and CYP2B6 contributed primarily to the formation of H1 and H6, respectively. CYP2C19 and CYP1A2 seemed to contribute significantly to the formation of H4.

  5. Cytochrome P450-mediated metabolism of triclosan attenuates its cytotoxicity in hepatic cells.

    PubMed

    Wu, Yuanfeng; Chitranshi, Priyanka; Loukotková, Lucie; Gamboa da Costa, Gonçalo; Beland, Frederick A; Zhang, Jie; Fang, Jia-Long

    2016-11-28

    Triclosan is a widely used broad-spectrum anti-bacterial agent. The objectives of this study were to identify which cytochrome P450 (CYP) isoforms metabolize triclosan and to examine the effects of CYP-mediated metabolism on triclosan-induced cytotoxicity. A panel of HepG2-derived cell lines was established, each of which overexpressed a single CYP isoform, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1. The extent of triclosan metabolism by each CYP was assessed by reversed-phase high-performance liquid chromatography with online radiochemical detection. Seven isoforms were capable of metabolizing triclosan, with the order of activity being CYP1A2 > CYP2B6 > CYP2C19 > CYP2D6 ≈ CYP1B1 > CYP2C18 ≈ CYP1A1. The remaining 11 isoforms (CYP2A6, CYP2A7, CYP2A13, CYP2C8, CYP2C9, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11, and CYP4B1) had little or no activity toward triclosan. Three metabolites were detected: 2,4-dichlorophenol, 4-chlorocatechol, and 5'-hydroxytriclosan. Consistent with the in vitro screening data, triclosan was extensively metabolized in HepG2 cells overexpressing CYP1A2, CYP2B6, CYP2C19, CYP2D6, and CYP2C18, and these cells were much more resistant to triclosan-induced cytotoxicity compared to vector cells, suggesting that CYP-mediated metabolism of triclosan attenuated its cytotoxicity. In addition, 2,4-dichlorophenol and 4-chlorocatechol were less toxic than triclosan to HepG2/vector cells. Conjugation of triclosan, catalyzed by human glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), also occurred in HepG2/CYP-overexpressing cells and primary human hepatocytes, with a greater extent of conjugation being associated with higher cell viability. Co-administration of triclosan with UGT or SULT inhibitors led to greater cytotoxicity in HepG2 cells and primary human hepatocytes, indicating that glucuronidation and

  6. 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...

  7. Novel Marmoset Cytochrome P450 2C19 in Livers Efficiently Metabolizes Human P450 2C9 and 2C19 Substrates, S-Warfarin, Tolbutamide, Flurbiprofen, and Omeprazole.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Kawano, Mirai; Shimizu, Makiko; Toda, Akiko; Utoh, Masahiro; Sasaki, Erika; Yamazaki, Hiroshi

    2015-10-01

    The common marmoset (Callithrix jacchus), a small New World monkey, has the potential for use in human drug development due to its evolutionary closeness to humans. Four novel cDNAs, encoding cytochrome P450 (P450) 2C18, 2C19, 2C58, and 2C76, were cloned from marmoset livers to characterize P450 2C molecular properties, including previously reported P450 2C8. The deduced amino acid sequence showed high sequence identities (>86%) with those of human P450 2Cs, except for marmoset P450 2C76, which has a low sequence identity (∼70%) with any human P450 2Cs. Phylogenetic analysis showed that marmoset P450 2Cs were more closely clustered with those of humans and macaques than other species investigated. Quantitative polymerase chain reaction analysis showed that all of the marmoset P450 2C mRNAs were predominantly expressed in liver as opposed to the other tissues tested. Marmoset P450 2C proteins were detected in liver by immunoblotting using antibodies against human P450 2Cs. Among marmoset P450 2Cs heterologously expressed in Escherichia coli, marmoset P450 2C19 efficiently catalyzed human P450 2C substrates, S-warfarin, diclofenac, tolbutamide, flurbiprofen, and omeprazole. Marmoset P450 2C19 had high Vmax and low Km values for S-warfarin 7-hydroxylation that were comparable to those in human liver microsomes, indicating warfarin stereoselectivity similar to findings in humans. Faster in vivo S-warfarin clearance than R-warfarin after intravenous administration of racemic warfarin (0.2 mg/kg) to marmosets was consistent with the in vitro kinetic parameters. These results indicated that marmoset P450 2C enzymes had functional characteristics similar to those of humans, and that P450 2C-dependent metabolic properties are likewise similar between marmosets and humans.

  8. The possible role of gradual accumulation of copper, cadmium, lead and iron and gradual depletion of zinc, magnesium, selenium, vitamins B2, B6, D, and E and essential fatty acids in multiple sclerosis.

    PubMed

    Johnson, S

    2000-09-01

    Multiple sclerosis (MS) has a much higher incidence among caucasians that in any other race. Furthermore: females are much more susceptible than males and white females living in colder, wetter areas are much more susceptible than those living in warmer areas. On the other hand, menstruating women have increased copper (Cu) absorption and half-life, so they tend to accumulate more Cu than males. Moreover, rapidly growing girls have an increased demand for zinc (Zn), but their rapidly decreasing production of melatonin results in impaired Zn absorption, which is exacerbated by the high Cu levels. The low Zn levels result in deficient CuZnSuperoxide dismutase (CuZnSOD), which in turn leads to increased levels of superoxide. Menstruating females also often present with low magnesium (Mg) and vitamin B6 levels. Vitamin B6 moderates intracellular nitric oxide (NO) production and extracellular Mg is required for NO release from the cell, so that a deficiency of these nutrients results in increased NO production in the cell and reduced release from the cell. The trapped NO combines with superoxide to form peroxinitrite, an extremely powerful free radical that leads to the myelin damage of MS. Iron (Fe), molybdenum (Mo) and cadmium (Cd) accumulation also increase superoxide production. Which explains MS in males, who tend to accumulate Fe much faster and Cu much less rapidly than females. Since vitamin D is paramount for Mg absorption, the much reduced exposure to sunlight in the higher latitudes may account for the higher incidence in these areas. Moreover, vitamin B2 is a cofactor for xanthine oxidase, and its deficiency exacerbates the low levels of uric acid caused by high Cu levels, resulting in myelin degeneration. Finally Selenium (Se) and vitamin E prevent lipid peroxidation and EPA and DHA upregulate CuZnSOD. Therefore, supplementation with 100 mg MG, 25 mg vit B6, 10 mg vit B2, 15 mg Zn and 400 IU vit D and E, 100 microg Se, 180 mg EPA and 120 mg DHA per day between 14 and 16 years of age may prevent MS.

  9. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    PubMed

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined.

  10. Stereo-selective metabolism of methadone by human liver microsomes and cDNA-expressed cytochrome P450s: a reconciliation.

    PubMed

    Chang, Yan; Fang, Wenfang B; Lin, Shen-Nan; Moody, David E

    2011-01-01

    In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racaemic methadone was used for incubations; (R)- and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63-74, 12-32 and 1. 4-14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60-85%; inhibition of CYP2B6 in 2B6 high-activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers.

  11. Cytochrome P450 gene polymorphism and cancer.

    PubMed

    Agundez, Jose A G

    2004-06-01

    Human cytochrome P450 (CYP) enzymes play a key role in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. Phenotyping analyses revealed an association between CYP enzyme activity and the risk to develop several forms of cancer. Research carried out in the last decade demonstrated that several CYP enzymes are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures became available for most human CYP, an impressive number of association studies on CYP polymorphisms and cancer risk were conducted. Here we review the findings obtained in these studies regarding CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP8A1 and CYP21 gene polymorphisms. Consistent evidences for association between CYP polymorphisms and lung, head and neck, and liver cancer were reported. Controversial findings suggest that colorectal and prostate cancers may be associated to CYP polymorphisms, whereas no evidences for a relevant association with breast or bladder cancers were reported. We summarize the available information related to the association of CYP polymorphisms with leukaemia, lymphomas and diverse types of cancer that were investigated only for some CYP genes, including brain, esophagus, stomach, pancreas, pituitary, cervical epithelium, melanoma, ovarian, kidney, anal and vulvar cancers. This review discusses on causes of heterogeneity in the proposed associations, controversial findings on cancer risk, and identifies topics that require further investigation. In addition, some recommendations on study design, in order to obtain more conclusive findings in further studies, are provided.

  12. Development of HepG2-derived cells expressing cytochrome P450s for assessing metabolism-associated drug-induced liver toxicity.

    PubMed

    Xuan, Jiekun; Chen, Si; Ning, Baitang; Tolleson, William H; Guo, Lei

    2016-08-05

    The generation of reactive metabolites from therapeutic agents is one of the major mechanisms of drug-induced liver injury (DILI). In order to evaluate metabolism-related toxicity and improve drug efficacy and safety, we generated a battery of HepG2-derived cell lines that express 14 cytochrome P450s (CYPs) (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 3A7) individually using a lentiviral expression system. The expression/production of a specific CYP in each cell line was confirmed by an increased abundance of the CYP at both mRNA and protein levels. Moreover, the enzymatic activities of representative CYPs in the corresponding cell lines were also measured. Using our CYP-expressed HepG2 cells, the toxicity of three drugs that could induce DILI (amiodarone, chlorpromazine and primaquine) was assessed, and all of them showed altered (increased or decreased) toxicity compared to the toxicity in drug-treated wild-type HepG2 cells. CYP-mediated drug toxicity examined in our cell system is consistent with previous reports, demonstrating the potential of these cells for assessing metabolism-related drug toxicity. This cell system provides a practical in vitro approach for drug metabolism screening and for early detection of drug toxicity. It is also a surrogate enzyme source for the enzymatic characterization of a particular CYP that contributes to drug-induced liver toxicity.

  13. Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450.

    PubMed

    Bojić, Mirza; Sedgeman, Carl A; Nagy, Leslie D; Guengerich, F Peter

    2015-06-20

    Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

  14. Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes

    PubMed Central

    Pratt, Victoria M.; Everts, Robin E.; Aggarwal, Praful; Beyer, Brittany N.; Broeckel, Ulrich; Epstein-Baak, Ruth; Hujsak, Paul; Kornreich, Ruth; Liao, Jun; Lorier, Rachel; Scott, Stuart A.; Smith, Chingying Huang; Toji, Lorraine H.; Turner, Amy; Kalman, Lisa V.

    2017-01-01

    Pharmacogenetic testing is increasingly available from clinical laboratories. However, only a limited number of quality control and other reference materials are currently available to support clinical testing. To address this need, the Centers for Disease Control and Prevention–based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, has characterized 137 genomic DNA samples for 28 genes commonly genotyped by pharmacogenetic testing assays (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP4F2, DPYD, GSTM1, GSTP1, GSTT1, NAT1, NAT2, SLC15A2, SLC22A2, SLCO1B1, SLCO2B1, TPMT, UGT1A1, UGT2B7, UGT2B15, UGT2B17, and VKORC1). One hundred thirty-seven Coriell cell lines were selected based on ethnic diversity and partial genotype characterization from earlier testing. DNA samples were coded and distributed to volunteer testing laboratories for targeted genotyping using a number of commercially available and laboratory developed tests. Through consensus verification, we confirmed the presence of at least 108 variant pharmacogenetic alleles. These samples are also being characterized by other pharmacogenetic assays, including next-generation sequencing, which will be reported separately. Genotyping results were consistent among laboratories, with most differences in allele assignments attributed to assay design and variability in reported allele nomenclature, particularly for CYP2D6, UGT1A1, and VKORC1. These publicly available samples will help ensure the accuracy of pharmacogenetic testing. PMID:26621101

  15. Gene expression profiling of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR.

    PubMed

    Toselli, Francesca; de Waziers, Isabelle; Dutheil, Mary; Vincent, Marc; Wilce, Peter A; Dodd, Peter R; Beaune, Philippe; Loriot, Marie-Anne; Gillam, Elizabeth M J

    2015-01-01

    1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

  16. Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

    PubMed

    Lu, Ying-Yuan; Cheng, Hai-Xu; Wang, Xin; Wang, Xiao-Wei; Liu, Jun-Yi; Li, Pu; Lou, Ya-Qing; Li, Jun; Lu, Chuang; Zhang, Guo-Liang

    2017-01-12

    1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including Km, Vmax, and CLint were 2.3 μM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.

  17. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers.

    PubMed

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H; Frei, Eva; Arlt, Volker M; Stiborová, Marie

    2016-04-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5 , to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP-7,8-dihydrodiol and BaP-9-ol, which are intermediates in BaP-derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP-3-ol, a metabolite that is a 'detoxified' product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP-7,8-dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP-9-ol. BaP-3-ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active.

  18. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

    PubMed

    Wortham, Matthew; Czerwinski, Maciej; He, Lin; Parkinson, Andrew; Wan, Yu-Jui Yvonne

    2007-09-01

    Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

  19. Identification, Synthesis, and Biological Evaluation of the Major Human Metabolite of NLRP3 Inflammasome Inhibitor MCC950.

    PubMed

    Salla, Manohar; Butler, Mark S; Pelingon, Ruby; Kaeslin, Geraldine; Croker, Daniel E; Reid, Janet C; Baek, Jong Min; Bernhardt, Paul V; Gillam, Elizabeth M J; Cooper, Matthew A; Robertson, Avril A B

    2016-12-08

    MCC950 is an orally bioavailable small molecule inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that exhibits remarkable activity in multiple models of inflammatory disease. Incubation of MCC950 with human liver microsomes, and subsequent analysis by HPLC-MS/MS, revealed a major metabolite, where hydroxylation of MCC950 had occurred on the 1,2,3,5,6,7-hexahydro-s-indacene moiety. Three possible regioisomers were synthesized, and coelution using HPLC-MS/MS confirmed the structure of the metabolite. Further synthesis of individual enantiomers and coelution studies using a chiral column in HPLC-MS/MS showed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide (2a). Incubation of MCC950 with a panel of cytochrome P450 enzymes showed P450s 2A6, 2C9, 2C18, 2C19, 2J2, and 3A4 catalyze the formation of the major metabolite 2a, with a lower level of activity shown by P450s 1A2 and 2B6. All of the synthesized compounds were tested for inhibition of NLRP3-induced production of the pro-inflammatory cytokine IL-1β from human monocyte derived macrophages. The identified metabolite 2a was 170-fold less potent than MCC950, while one regioisomer had nanomolar inhibitory activity. These findings also give first insight into the SAR of the hexahydroindacene moiety.

  20. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  1. United States Air Force Training Extract AFSC 2A6X4 Aircraft Fuel Systems (ANG/AFRC)

    DTIC Science & Technology

    2001-02-01

    boost pumps 91 87 97 92 89 93 E0243 Remove or install butterfly -type shutoff valves ...8 19 19 19 F0323 Repair butterfly -type shutoff valves 14 13 14 25 28 23 F0324 Repair check...1073 A10.6.2.2. Fuel Jettison Butterfly Valve

  2. Evaluation of Pharmacokinetic Interactions Between Lesinurad, a New Selective Urate Reabsorption Inhibitor, and CYP Enzyme Substrates Sildenafil, Amlodipine, Tolbutamide, and Repaglinide.

    PubMed

    Gillen, Michael; Yang, Chun; Wilson, David; Valdez, Shakti; Lee, Caroline; Kerr, Bradley; Shen, Zancong

    2017-01-09

    Lesinurad is a selective uric acid reabsorption inhibitor approved for the treatment of hyperuricemia associated with gout in combination with xanthine oxidase inhibitors. In vitro assays indicate that lesinurad is an inducer of CYPs in the order CYP3A > CYP2C8 > CYP2C9 > CYP2C19 > CYP2B6 and an inhibitor of CYP2C8 and CYP2C9. To investigate the drug interaction potential of lesinurad, clinical drug interaction studies were conducted. Open-label studies in volunteers investigated the effects of single-/multiple-dose lesinurad on the pharmacokinetics of sildenafil and amlodipine (CYP3A4 induction), tolbutamide (CYP2C9 inhibition/induction), and repaglinide (CYP2C8 inhibition/induction). There was no apparent induction of CYP2C8 and CYP2C9 following repeated lesinurad administration, although no inhibition of CYP2C9 and modest inhibition of CYP2C8 were observed following single-dose lesinurad. Consistent with in vitro observations, lesinurad (200 mg once daily) was an inducer of CYP3A based on the effects on sildenafil exposure. Sildenafil exposure decreased by approximately 34% for Cmax and AUC when administered with multiple-dose lesinurad 200 mg and allopurinol 300 mg, relative to sildenafil alone. During lesinurad therapy, the possibility of reduced efficacy of concomitant drugs that are CYP3A substrates should be considered and their efficacy monitored because of induction of CYP3A by lesinurad.

  3. Simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma by UPLC-MS/MS and its application to cytochrome P450 activity study in rats.

    PubMed

    Ma, Jianshe; Wang, Shuanghu; Zhang, Meiling; Zhang, Qingwei; Zhou, Yunfang; Lin, Chongliang; Lin, Guanyang; Wang, Xianqin

    2015-08-01

    A specific ultra-performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C18 column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2-2000 ng/mL, with correlation coefficient (r(2) ) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats.

  4. Hydroxylation and N-dechloroethylation of Ifosfamide and deuterated Ifosfamide by the human cytochrome p450s and their commonly occurring polymorphisms.

    PubMed

    Calinski, Diane M; Zhang, Haoming; Ludeman, Susan; Dolan, M Eileen; Hollenberg, Paul F

    2015-07-01

    The hydroxylation and N-dechloroethylation of deuterated ifosfamide (d4IFO) and ifosfamide (IFO) by several human P450s have been determined and compared. d4IFO was synthesized with deuterium at the alpha and alpha' carbons to decrease the rate of N-dechloroethylation and thereby enhance hydroxylation of the drug at the 4' position. The purpose was to decrease the toxic and increase the efficacious metabolites of IFO. For all of the P450s tested, hydroxylation of d4IFO was improved and dechloroethylation was reduced as compared with nondeuterated IFO. Although the differences were not statistically significant, the trend favoring the 4'-hydroxylation pathway was noteworthy. CYP3A5 and CYP2C19 were the most efficient enzymes for catalyzing IFO hydroxylation. The importance of these enzymes in IFO metabolism has not been reported previously and warrants further investigation. The catalytic ability of the common polymorphisms of CYP2B6 and CYP2C9 for both reactions were tested with IFO and d4IFO. It was determined that the commonly expressed polymorphisms CYP2B6*4 and CYP2B6*6 had reduced catalytic ability for IFO compared with CYP2B6*1, whereas CYP2B6*7 and CYP2B6*9 had enhanced catalytic ability. As with the wild-type enzymes, d4IFO was more readily hydroxylated by the polymorphic variants than IFO, and d4IFO was not dechloroethylated by any of the polymorphic forms. We also assessed the use of specific inhibitors of P450 to favor hydroxylation in human liver microsomes. We were unable to separate the pathways with these experiments, suggesting that multiple P450s are responsible for catalyzing both metabolic pathways for IFO, which is not observed with the closely related drug cyclophosphamide.

  5. Applicability of second-generation upcyte® human hepatocytes for use in CYP inhibition and induction studies

    PubMed Central

    Ramachandran, Sarada D; Vivarès, Aurélie; Klieber, Sylvie; Hewitt, Nicola J; Muenst, Bernhard; Heinz, Stefan; Walles, Heike; Braspenning, Joris

    2015-01-01

    Human upcyte® hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte® hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC50 values for each compound correctly classified them as potent inhibitors. Upcyte® hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte® hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUCu/F2, and Cmax,u/Ind50. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte® hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte® hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations. PMID:26516577

  6. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions

    PubMed Central

    Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

    2016-01-01

    Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug’s impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and

  7. Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery.

    PubMed

    Pan, Shu-Ting; Xue, Danfeng; Li, Zhi-Ling; Zhou, Zhi-Wei; He, Zhi-Xu; Yang, Yinxue; Yang, Tianxin; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2016-06-28

    The human cytochrome P450 (CYP) superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA ("Orthologous MAtrix") Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

  8. The impact of individual cytochrome P450 enzymes on oxidative metabolism of benzo[a]pyrene in human livers

    PubMed Central

    Šulc, Miroslav; Indra, Radek; Moserová, Michaela; Schmeiser, Heinz H.; Frei, Eva; Arlt, Volker M.; White, P.

    2016-01-01

    Benzo[a]pyrene (BaP) is a human carcinogen that covalently binds to DNA after metabolic activation by cytochrome P450 (CYP) enzymes. In this study human recombinant CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5) were expressed in Supersomes™ together with their reductases, NADPH:CYP oxidoreductase, epoxide hydrolase and cytochrome b5, to investigate BaP metabolism. Human CYPs produced up to eight BaP metabolites. Among these, BaP‐7,8‐dihydrodiol and BaP‐9‐ol, which are intermediates in BaP‐derived DNA adduct formation, were mainly formed by CYP1A1 and 1B1, and to a lesser extent by CYP2C19 and 3A4. BaP‐3‐ol, a metabolite that is a ‘detoxified’ product of BaP, was formed by most human CYPs tested, although CYP1A1 and 1B1 produced it the most efficiently. Based on the amounts of the individual BaP metabolites formed by these CYPs and their expression levels in human liver, we determined their contributions to BaP metabolite formation in this organ. Our results indicate that hepatic CYP1A1 and CYP2C19 are most important in the activation of BaP to BaP‐7,8‐dihydrodiol, whereas CYP2C19, 3A4, and 1A1 are the major enzymes contributing to the formation of BaP‐9‐ol. BaP‐3‐ol is predominantly formed by hepatic CYP3A4, while CYP1A1 and 2C19 are less active. Environ. Mol. Mutagen. 57:229–235, 2016. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc. PMID:26919089

  9. Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery

    PubMed Central

    Pan, Shu-Ting; Xue, Danfeng; Li, Zhi-Ling; Zhou, Zhi-Wei; He, Zhi-Xu; Yang, Yinxue; Yang, Tianxin; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2016-01-01

    The human cytochrome P450 (CYP) superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA (“Orthologous MAtrix”) Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery. PMID:27367670

  10. Nuclear Receptors in Drug Metabolism, Drug Response and Drug Interactions.

    PubMed

    Prakash, Chandra; Zuniga, Baltazar; Song, Chung Seog; Jiang, Shoulei; Cropper, Jodie; Park, Sulgi; Chatterjee, Bandana

    Orally delivered small-molecule therapeutics are metabolized in the liver and intestine by phase I and phase II drug-metabolizing enzymes (DMEs), and transport proteins coordinate drug influx (phase 0) and drug/drug-metabolite efflux (phase III). Genes involved in drug metabolism and disposition are induced by xenobiotic-activated nuclear receptors (NRs), i.e. PXR (pregnane X receptor) and CAR (constitutive androstane receptor), and by the 1α, 25-dihydroxy vitamin D3-activated vitamin D receptor (VDR), due to transactivation of xenobiotic-response elements (XREs) present in phase 0-III genes. Additional NRs, like HNF4-α, FXR, LXR-α play important roles in drug metabolism in certain settings, such as in relation to cholesterol and bile acid metabolism. The phase I enzymes CYP3A4/A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, CYP1A2, CYP2C8, CYP2A6, CYP2J2, and CYP2E1 metabolize >90% of all prescription drugs, and phase II conjugation of hydrophilic functional groups (with/without phase I modification) facilitates drug clearance. The conjugation step is mediated by broad-specificity transferases like UGTs, SULTs, GSTs. This review delves into our current understanding of PXR/CAR/VDR-mediated regulation of DME and transporter expression, as well as effects of single nucleotide polymorphism (SNP) and epigenome (specified by promoter methylation, histone modification, microRNAs, long non coding RNAs) on the expression of PXR/CAR/VDR and phase 0-III mediators, and their impacts on variable drug response. Therapeutic agents that target epigenetic regulation and the molecular basis and consequences (overdosing, underdosing, or beneficial outcome) of drug-drug/drug-food/drug-herb interactions are also discussed. Precision medicine requires understanding of a drug's impact on DME and transporter activity and their NR-regulated expression in order to achieve optimal drug efficacy without adverse drug reactions. In future drug screening, new tools such as humanized mouse models and

  11. The effects of acute hydrogen sulfide poisoning on cytochrome P450 isoforms activity in rats.

    PubMed

    Wang, Xianqin; Chen, Mengchun; Chen, Xinxin; Ma, Jianshe; Wen, Congcong; Pan, Jianchun; Hu, Lufeng; Lin, Guanyang

    2014-01-01

    Hydrogen sulfide (H2S) is the second leading cause of toxin related death (after carbon monoxide) in the workplace. H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, respectively. The experimental rats were randomly divided into two groups, control group and acute H2S poisoning group (inhaling 300 ppm for 2 h). The mixture of six probes was given to rats by 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. The results for acute H2S poisoning and control groups were as follows: there was a statistically significant difference in the AUC and C max for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetic difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 in rats.

  12. Traditional Herbal Formulas to as Treatments for Musculoskeletal Disorders: Their Inhibitory Effects on the Activities of Human Microsomal Cytochrome P450s and UDP-glucuronosyltransferases

    PubMed Central

    Jin, Seong Eun; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Ha, Hyekyung

    2016-01-01

    Objective: The aim of this study was to assess the influence of traditional herbal formulas, including Bangpungtongseong-san (BPTSS; Fangfengtongsheng-san, Bofu-tsusho-san), Ojeok-san (OJS; Wuji-san, Goshaku-san), and Oyaksungi-san (OYSGS; Wuyaoshungi-san, Uyakujyunki-san), on the activities of the human cytochrome P450s (CYP450s) and UDP-glucuronosyltransferases (UGTs), which are drug-metabolizing enzymes. Materials and Methods: The activities of the major human CYP450 isozymes (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) and UGTs (UGT1A1, UGT1A4, and UGT2B7) were investigated using in vitro fluorescence-based and luminescence-based enzyme assays, respectively. The inhibitory effects of the herbal formulas were characterized, and their IC50 values were determined. Results: BPTSS inhibited the activities of CYP1A2, CYP2C19, CYP2E1, and UGT1A1 while it exerted relatively weak inhibition on CYP2B6, CYP2C9, CYP2D6, and CYP3A4. BPTSS also negligibly inhibited the activities of UGT1A4 and UGT2B7, with IC50 values in the excess of 1000 μg/mL. OJS and OYSGS inhibited the activity of CYP2D6, whereas they exhibited no inhibition of the UGT1A4 activity at doses <1000 μg/mL. In addition, OJS inhibited the CYP1A2 activity but exerted a relatively weak inhibition on the activities of CYP2C9, CYP2C19, CYP2E1, and CYP3A4. Conversely, OJS negligibly inhibited the activities of CYP2B6, UGT1A1, and UGT2B7 with IC50 values in excess of 1000 μg/mL. OYSGS weakly inhibited the activities of CYP1A2, CYP2C19, CYP2E1, CYP3A4, and UGT1A1, with a negligible inhibition on the activities of CYP2B6, CYP2C9, and UGT2B7, with IC50 values in excess of 1000 μg/mL. Conclusions: These results provide information regarding the safety and effectiveness of BPTSS, OJS, and OYSGS when combined with conventional drugs. SUMMARY Bangpungtongseong-san inhibited the activities of human microsomal CYP1A2, CYP2C19, CYP2E1, and UGT1A1, with a negligibly inhibition on the activities of CYP2B6

  13. Association of cytochrome P450 genetic polymorphisms with neoadjuvant chemotherapy efficacy in breast cancer patients

    PubMed Central

    2012-01-01

    Background The enzymes of the cytochrome P450 family (CYPs) play an important role in the metabolism of a great variety of anticancer agents; therefore, polymorphisms in genes encoding for metabolizing enzymes and drugs transporters can affect drug efficacy and toxicity. Methods The genetic polymorphisms of cytochrome P450 were studied in 395 patients with breast cancer by RLFP analysis. Results Here, we studied the association of functionally significant variant alleles of CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 with the clinical response to neoadjuvant chemotherapy in breast cancer patients. A significant correlation was observed between the CYP2C9*2 polymorphism and chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91), as well as between CYP2C9*2 heterozygotes and chemotherapy resistance in women with nodal forms of breast cancer and a cancer hereditary load (OR = 15.50; CI 95% = 1.08 – 826.12) when the potential combined effects were examined. No significant association between chemotherapy resistance and the other examined genotypes and the potential combined clinical and tumour-related parameters were discovered. Conclusion In conclusion, CYP2C9*2 was associated with neoadjuvant chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91) in the population of interest. PMID:22702493

  14. In vitro metabolism of a novel antithrombotic compound, S002-333, and its enantiomers: quantitative cytochrome P450 phenotyping, metabolic profiling and enzyme kinetic studies.

    PubMed

    Saxena, Amrita; Jain, Girish K; Siddiqui, Hefazat H; Bhunia, Shom S; Saxena, Anil K; Gayen, Jiaur R

    2014-04-01

    1. S002-333, (2-(4'-methoxy-benzenesulfonyl)-2,3,4,9-tetrahydro-1H-pyrido (3,4-b) indole-3-carboxylic acid amide) is a novel potent antithrombotic molecule currently under development phase. It is the racemic mixture of two enantiomers, namely S004-1032 (R-form) and S007-1558 (S-form). 2. The contribution of five major isoenzymes, namely CYP2B6, 2C9, 2C19, 2D6 and 3A4 was quantified using recombinant P450s in the phase-I metabolism through relative activity factor approach. CYP2C19 was found to be the major contributor for S002-333 and S007-1558, while CYP3A4 showed greater involvement in S004-1032 metabolism. Chemical inhibition and immunoinhibition studies reconfirmed the results in human liver microsomes (HLM). 3. Four major phase-I metabolites of S002-333; M-1 and M-3 (oxidative), M-2 (O-demethylated) and M-4 (dehydrogenated) were characterized in HLM. These metabolites constituted 11.2, 11.3 and 21.5% of the parent in comparison with the net phase-I metabolism of 29.9, 31.4 and 38.3% of S002-333, S004-1032 and S007-1558, respectively. 4. Among CYP2C9, 2C19 and 3A4, the relative contribution of CYP2C9 was found to be maximum during M-1 through M-4 formation. Enzyme kinetic analysis for detected metabolites indicated that M-1 to M-3 followed classical hyperbolic kinetics, whereas M-4 showed evidence of autoactivation. In conclusion, the results suggest prominent role of CYP2C9, 2C19 and 3A4 isoforms for enantioselective disposition of S002-333 in vitro.

  15. In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran.

    PubMed

    Paris, Brandy L; Ogilvie, Brian W; Scheinkoenig, Julie A; Ndikum-Moffor, Florence; Gibson, Remi; Parkinson, Andrew

    2009-10-01

    Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

  16. Potential herb-drug interaction of shexiang baoxin pill in vitro based on drug metabolism/transporter

    PubMed Central

    Shen, Zhijie; Wang, Yingjie; Guo, Wei; Yao, Yili; Wang, Xiaolong

    2016-01-01

    Many researches have proved functions of anti-oxidation, endothelial protection and pro-angiogenesis efficiency of Shexiang Baoxin Pill (SBP). This study aims to investigate potential for metabolism-based interaction on CYP450s and transporter based interaction on OATP1B1, BRCP and MDR1. Human primary hepatocytes were used in this study. Probe substrates of cytochrome P450 enzymes were incubated in human liver microsomes (HLMs) with or without SBP and IC50 values were estimated. Inhibitive potential of SBP on activities of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4 was evaluated. Inducible potential of SBP on activities of CYP1A2, 2B6 and 3A4 was accessed. Inhibitive potential of SBP on human OATP1B1 was evaluated using cell-based assay. Inhibitive potential of SBP on human MDR1 and BCRP was also evaluated using vesicles assay. MDR1 and BCRP vesicle kit were used to determine ATP dependent uptake activity when incubated with SBP. SBP was a competitive inhibitor of CYP2B6, 2C19, while neither inhibitory nor inductive potentials toward other CYP450s were detected. No significant MDR1 inhibitory potential was estimated, while only high concentration of SBP (500 μg/ml) could inhibit activity of BCRP. Probe substrates Estradiol-17 β-glucuronide was incubated in HEK293-OATP1B1 and HEK293-MOCK cell system with different concentration of SBP and estimated IC50 was 179 μg/mL, which demonstrated a moderate inhibition potential against OATP1B1. In conclusion, outcome of this study suggests that SBP plays an important role in inhibition of CYP450 isozymes (including CYP2B6 and 2C9) and transporter OATP1B1. Therefore, precautions should be taken when using SBP for CYP and OATP-related herb-drug interactions. PMID:28078025

  17. The Effect of Gene Variants on Levonorgestrel Pharmacokinetics when Combined with Antiretroviral Therapy containing Efavirenz or Nevirapine.

    PubMed

    Neary, M; Lamorde, M; Olagunju, A; Darin, K M; Merry, C; Byakika-Kibwika, P; Back, D J; Siccardi, M; Owen, A; Scarsi, K K

    2017-02-10

    Reduced levonorgestrel concentrations from the levonorgestrel contraceptive implant was previously seen when given concomitantly with efavirenz. We sought to assess whether single nucleotide polymorphisms (SNPs) in genes involved in efavirenz and nevirapine metabolism were linked to these changes in levonorgestrel concentration. SNPs in CYP2B6, CYP2A6, NR1I2 and NR1I3 were analysed. Associations of participant demographics and genotype with levonorgestrel pharmacokinetics were evaluated in HIV-positive women using the levonorgestrel implant plus efavirenz- or nevirapine-based ART, in comparison to ART-naïve women using multivariate linear regression. Efavirenz group: CYP2B6 516G>T was associated with lower levonorgestrel log10 Cmax and log10 AUC. CYP2B6 15582C>T was associated with lower log10 AUC. Nevirapine group: CYP2B6 516G>T was associated with higher log10 Cmax and lower log10 Cmin . Pharmacogenetic variations influenced subdermal levonorgestrel pharmacokinetics in HIV-positive women, indicating that the magnitude of the interaction with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is influenced by host genetics. This article is protected by copyright. All rights reserved.

  18. Synthesis, biological evaluation and molecular modelling studies of methyleneimidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17). Part I: Heterocyclic modifications of the core structure.

    PubMed

    Jagusch, Carsten; Negri, Matthias; Hille, Ulrike E; Hu, Qingzhong; Bartels, Marc; Jahn-Hoffmann, Kerstin; Pinto-Bazurco Mendieta, Mariano A E; Rodenwaldt, Barbara; Müller-Vieira, Ursula; Schmidt, Dirk; Lauterbach, Thomas; Recanatini, Maurizio; Cavalli, Andrea; Hartmann, Rolf W

    2008-02-15

    Novel chemical entities were prepared via Suzuki and S(N) reaction as AC-ring substrate mimetics of CYP17. The synthesised compounds 1-31 were tested for activity using human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against hepatic CYP enzymes (3A4, 2D6, 1A2, 2C9, 2C19, 2B6). Two potent inhibitors (27, IC50 = 373 nM/28, IC50 = 953 nM) were further examined in rats regarding their effects on plasma testosterone levels and their pharmacokinetic properties. Compound 28 was similarly active as abiraterone and showed better pharmacokinetic properties (higher bioavailability, t(1/2) 9.5 h vs 1.6 h). Docking studies revealed two new binding modes different from the one of the substrates and steroidal inhibitors.

  19. Effects of 6-paradol, an unsaturated ketone from gingers, on cytochrome P450-mediated drug metabolism.

    PubMed

    Kim, Hyeong Jun; Kim, In Sook; Rehman, Shaheed Ur; Ha, Sang Keun; Nakamura, Katsunori; Yoo, Hye Hyun

    2017-02-20

    Paradols are unsaturated ketones produced by biotransformation of shogaols in gingers. Among them, 6-paradol has been investigated as a new drug candidate due to its anti-inflammatory, apoptotic, and neuroprotective activities. In this study, the inhibitory effects of 6-paradol on the activities of cytochrome P450 (CYP) enzymes were investigated with human liver microsomes and recombinant CYP isozymes. 6-Paradol showed concentration-dependent inhibitory effects on CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 isozymes, with IC50 values ranging from 3.8 to 21.4µM in recombinant CYP isozymes. However, the inhibition was not potentiated following pre-incubation, indicating that 6-paradol is not a mechanism-based inhibitor. These results suggest that pharmacokinetic drug-drug interactions might occur with 6-paradol, which must be considered in the process of new drug development.

  20. Assessment of effect of Zhu-tan Tong-luo decoction on CYP450 isoforms activity of rats.

    PubMed

    Jin, Yongxi; Shao, Lingjiu; Li, Gaowen; Shao, Mengmeng; Zhi, Yinghao; Zhu, Wenzong

    2015-01-01

    In order to investigate the effects of Zhu-tan Tong-luo decoction on the metabolic capacity of cytochrome P450 (CYP) enzymes, a cocktail method was employed to evaluate the activities of CYP2B6, CYP2C19, CYP1A2, CYP3A4, CYP2C9, CYP2D6. The rats were randomly divided into acute Zhu-Tan Tong-Luo decoction group (Low, High), chronic Zhu-Tan Tong-Luo decoction group (Low, High) and control group. The acute group rats were given 0.6, 1.2 g/kg (Low, High) Zhu-tan Tong-luo decoction by intragastric administration for 1 day, and the chronic group for 14 days. Six probe drugs bupropion, omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol were given to rats through intragastric administration, and the plasma concentrations were determined by UPLC-MS/MS. There statistical pharmacokinetics differences for omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol in rats were observed by comparing acute Zhu-tan Tong-luo decoction group with control group; and statistical pharmacokinetics differences for bupropion, omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol were observed by comparing chronic Zhu-Tan Tong-Luo decoction group with control group. After intragastric administration of Zhu-Tan Tong-Luo decoction may slightly induce the activities of CYP2B6, CYP2C19, CYP1A2, CYP3A4, CYP2C9, CYP2D6 of rats. Induction of drug metabolizing enzyme by Zhu-Tan Tong-Luo decoction would reduce the efficacy of other drug. Additional, there no statistical difference for biochemical results after 1 or 14 intragastric administration of Zhu-Tan Tong-Luo decoction.

  1. Assessment of effect of Zhu-tan Tong-luo decoction on CYP450 isoforms activity of rats

    PubMed Central

    Jin, Yongxi; Shao, Lingjiu; Li, Gaowen; Shao, Mengmeng; Zhi, Yinghao; Zhu, Wenzong

    2015-01-01

    In order to investigate the effects of Zhu-tan Tong-luo decoction on the metabolic capacity of cytochrome P450 (CYP) enzymes, a cocktail method was employed to evaluate the activities of CYP2B6, CYP2C19, CYP1A2, CYP3A4, CYP2C9, CYP2D6. The rats were randomly divided into acute Zhu-Tan Tong-Luo decoction group (Low, High), chronic Zhu-Tan Tong-Luo decoction group (Low, High) and control group. The acute group rats were given 0.6, 1.2 g/kg (Low, High) Zhu-tan Tong-luo decoction by intragastric administration for 1 day, and the chronic group for 14 days. Six probe drugs bupropion, omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol were given to rats through intragastric administration, and the plasma concentrations were determined by UPLC-MS/MS. There statistical pharmacokinetics differences for omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol in rats were observed by comparing acute Zhu-tan Tong-luo decoction group with control group; and statistical pharmacokinetics differences for bupropion, omeprazole, phenacetin, testosterone, tolbutamide, and metroprolol were observed by comparing chronic Zhu-Tan Tong-Luo decoction group with control group. After intragastric administration of Zhu-Tan Tong-Luo decoction may slightly induce the activities of CYP2B6, CYP2C19, CYP1A2, CYP3A4, CYP2C9, CYP2D6 of rats. Induction of drug metabolizing enzyme by Zhu-Tan Tong-Luo decoction would reduce the efficacy of other drug. Additional, there no statistical difference for biochemical results after 1 or 14 intragastric administration of Zhu-Tan Tong-Luo decoction. PMID:26629097

  2. Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.

    PubMed

    Liu, Xidong; Hu, Lianghai; Ge, Guangbo; Yang, Bo; Ning, Jing; Sun, Shixin; Yang, Ling; Pors, Klaus; Gu, Jingkai

    2014-08-01

    Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

  3. Zearalenone activates pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor and corresponding phase I target genes mRNA in primary cultures of human hepatocytes.

    PubMed

    Ayed-Boussema, Imen; Pascussi, Jean Marc; Maurel, Patrick; Bacha, Hassen; Hassen, Wafa

    2011-01-01

    The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and β-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 μM.

  4. Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR.

    PubMed

    Kanno, Yuichiro; Tanuma, Nobuaki; Yazawa, Saki; Zhao, Shuai; Inaba, Miki; Nakamura, Satoshi; Nemoto, Kiyomitsu; Inouye, Yoshio

    2016-08-01

    The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

  5. Curcuminoids inhibit multiple human cytochromes P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes, while piperine is a relatively selective CYP3A4 inhibitor

    PubMed Central

    Volak, Laurie P.; Ghirmai, Senait; Cashman, John R.; Court, Michael H.

    2008-01-01

    Curcuminoid extract and piperine are being evaluated for beneficial effects in Alzheimer’s disease, among other intractable disorders. Consequently, we studied the potential for herb-drug interactions involving cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes. The curcuminoid extract inhibited SULT > CYP2C19 > CYP2B6 > UGT > CYP2C9 > CYP3A activities with IC50 values ranging from 0.99 ± 0.04 to 25.3 ± 1.3 μM, while CYP2D6, CYP1A2, and CYP2E1 activities were less affected (IC50 values >60 μM). Inhibition of CYP3A activity by curcuminoid extract was consistent with competitive inhibition (Ki = 11.0 ± 1.3 μM), while inhibition of both CYP2C9 and CYP2C19 activities were consistent with mixed competitive-noncompetitive inhibition (10.6 ± 1.1 μM and 7.8 ± 0.9 μM, respectively). Piperine was a relatively selective noncompetitive inhibitor of CYP3A (IC50 5.5 ± 0.7 μM, Ki = 5.4 ± 0.3 μM) with less effect on other enzymes evaluated (IC50 >29 μM). Curcuminoid extract and piperine inhibited recombinant CYP3A4 much more potently (by >5-fold) than CYP3A5. Pure synthetic curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) were also evaluated for their effects on CYP3A, CYP2C9, UGT, and SULT activities. All three curcuminoids had similar effects on CYP3A, UGT, and SULT activity, but demethoxycurcumin (IC50 = 8.8 ± 1.2 μM) was more active against CYP2C9 than either curcumin or bisdemethoxycurcumin (IC50 >50 μM). Based on these data and expected tissue concentrations of inhibitors, we predict that an orally administered curcuminoid/piperine combination is most likely to inhibit CYP3A, CYP2C9, UGT, and SULT metabolism within the intestinal mucosa. PMID:18480186

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

    PubMed

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

    2015-09-02

    In this study, we investigated the enzymes catalyzing the phase I metabolism 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.

  7. Effect of diphenoxylate on CYP450 isoforms activity in rats

    PubMed Central

    Geng, Peiwu; Cai, Jinzhang; Wang, Shuanghu; Yang, Suping; Liu, Zezheng; Lin, Yingying; Wen, Congcong; Wang, Xianqin; Zhou, Yunfang; Zhang, Meiling

    2015-01-01

    In order to investigate the effects of diphenoxylate on the metabolic capacity of cytochrome P450 (CYP) enzymes, a cocktail method was employed to evaluate the activities of CYP2B6, CYP2D6, CYP2C19, CYP1A2, CYP3A4, CYP2C9. The rats were randomly divided into diphenoxylate group (Low, Medium, High) and control group. The diphenoxylate group rats were given 12, 24, 48 mg/kg (Low, Medium, High) diphenoxylate by continuous intragastric administration for 7 days. Six probe drugs bupropion, metroprolol omeprazole, phenacetin, testosterone and tolbutamide were given to rats through intragastric administration, and the plasma concentrations were determined by UPLC-MS/MS. Statistical pharmacokinetics difference for omeprazole, phenacetin and tolbutamide in rats were observed by comparing diphenoxylate group with control group. Continuous 7 days-intragastric administration of diphenoxylate induces the activities of CYP2C19, CYP1A2 and CYP2C9 of rats. Induction of drug metabolizing enzyme by diphenoxylate would reduce the efficacy of other drug. Additionally, high dosage diphenoxylate may cause hepatotoxicity. PMID:26770498

  8. Activity levels of tamoxifen metabolites at the estrogen receptor and the impact of genetic polymorphisms of phase I and II enzymes on their concentration levels in plasma.

    PubMed

    Mürdter, T E; Schroth, W; Bacchus-Gerybadze, L; Winter, S; Heinkele, G; Simon, W; Fasching, P A; Fehm, T; Eichelbaum, M; Schwab, M; Brauch, H

    2011-05-01

    The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.

  9. Metabolic Drug-Drug Interaction Potential of Macrolactin A and 7-O-Succinyl Macrolactin A Assessed by Evaluating Cytochrome P450 Inhibition and Induction and UDP-Glucuronosyltransferase Inhibition In Vitro

    PubMed Central

    Bae, Soo Hyeon; Kwon, Min Jo; Park, Jung Bae; Kim, Doyun; Kim, Dong-Hee; Kang, Jae-Seon; Kim, Chun-Gyu; Oh, Euichaul

    2014-01-01

    Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, with Ki values of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, with Ki values of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of an in vitro-in vivo extrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolism in vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymes in vivo. Although further investigations will be required to clarify the in vivo interactions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice. PMID:24890600

  10. Metabolic drug-drug interaction potential of macrolactin A and 7-O-succinyl macrolactin A assessed by evaluating cytochrome P450 inhibition and induction and UDP-glucuronosyltransferase inhibition in vitro.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Park, Jung Bae; Kim, Doyun; Kim, Dong-Hee; Kang, Jae-Seon; Kim, Chun-Gyu; Oh, Euichaul; Bae, Soo Kyung

    2014-09-01

    Macrolactin A (MA) and 7-O-succinyl macrolactin A (SMA), polyene macrolides containing a 24-membered lactone ring, show antibiotic effects superior to those of teicoplanin against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. MA and SMA are currently being evaluated as antitumor agents in preclinical studies in Korea. We evaluated the potential of MA and SMA for the inhibition or induction of human liver cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in vitro to assess their safety as new molecular entities. We demonstrated that MA and SMA are potent competitive inhibitors of CYP2C9, with Ki values of 4.06 μM and 10.6 μM, respectively. MA and SMA also weakly inhibited UGT1A1 activity, with Ki values of 40.1 μM and 65.3 μM, respectively. However, these macrolactins showed no time-dependent inactivation of the nine CYPs studied. In addition, MA and SMA did not induce CYP1A2, CYP2B6, or CYP3A4/5. On the basis of an in vitro-in vivo extrapolation, our data strongly suggested that MA and SMA are unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most of the CYPs involved in drug metabolism in vivo, except for the inhibition of CYP2C9 by MA. Similarly, MA and SMA are unlikely to inhibit the activity of UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 enzymes in vivo. Although further investigations will be required to clarify the in vivo interactions of MA with CYP2C9-targeted drugs, our findings offer a clearer understanding and prediction of drug-drug interactions for the safe use of MA and SMA in clinical practice.

  11. Pharmacogenetics of plasma efavirenz exposure in HIV-infected adults and children in South Africa

    PubMed Central

    Sinxadi, Phumla Z; Leger, Paul D; McIlleron, Helen M; Smith, Peter J; Dave, Joel A; Levitt, Naomi S; Maartens, Gary; Haas, David W

    2015-01-01

    Aims Genetic factors, notably CYP2B6 516G→T [rs3745274] and 983T→C [rs28399499], explain much of the interindividual variability in efavirenz pharmacokinetics, but data from Africa are limited. We characterized relationships between genetic polymorphisms and plasma efavirenz concentrations in HIV-infected Black South African adults and children. Methods Steady-state mid-dosing interval efavirenz concentrations were measured. We genotyped 241 polymorphisms in genes potentially relevant to efavirenz metabolism and transport, including ABCB1, CYP2A6, CYP2B6, CYP3A4, CYP3A5, NR1I2 and NR1I3. Results Among 113 participants (59 adults and 54 children), minor allele frequencies for CYP2B6 516G→T, 983T→C, and 15582C→T [rs4803419] were 0.36, 0.07, and 0.09, respectively. Based on composite CYP2B6 15582/516/983 genotype, there were 33 extensive metabolizer, 62 intermediate metabolizer and 18 slow metabolizer genotypes. Median (IQR) mid-dose efavirenz concentrations were 1.44 (1.21–1.93) µg ml–1, 2.08 (1.68–2.94) µg ml–1 and 7.26 (4.82–8.34) µg ml–1 for extensive, intermediate and slow metabolizers, respectively. In univariate analyses, a model that included composite genotype best predicted efavirenz concentrations (β = 0.28, 95% CI 0.21, 0.35, P = 2.4 × 10–11). Among individual CYP2B6 polymorphisms, 516G→T best predicted efavirenz concentrations (β = 0.22, 95% CI 0.13, 0.30, P = 1.27 × 10−6). There was also associations with 983T→C (β = 0.27, 95% CI 0.10, 0.44, P = 0.002) and 15582C→T (β = 0.11, 95% CI 0.01, 0.22, P = 0.04). Associations were consistent in adults and children. No other polymorphisms were independently associated with efavirenz concentrations. Conclusions Composite CYP2B6 genotype based on CYP2B6 516G→T, 983T→C, and 15582C→T best described efavirenz exposure in HIV-infected Black South African adults and children. PMID:25611810

  12. Comparison of Liver Cell Models Using the Basel Phenotyping Cocktail

    PubMed Central

    Berger, Benjamin; Donzelli, Massimiliano; Maseneni, Swarna; Boess, Franziska; Roth, Adrian; Krähenbühl, Stephan; Haschke, Manuel

    2016-01-01

    Currently used hepatocyte cell systems for in vitro assessment of drug metabolism include hepatoma cell lines and primary human hepatocyte (PHH) cultures. We investigated the suitability of the validated in vivo Basel phenotyping cocktail (caffeine [CYP1A2], efavirenz [CYP2B6], losartan [CYP2C9], omeprazole [CYP2C19], metoprolol [CYP2D6], midazolam [CYP3A4]) in vitro and characterized four hepatocyte cell systems (HepG2 cells, HepaRG cells, and primary cryopreserved human hepatocytes in 2-dimensional [2D] culture or in 3D-spheroid co-culture) regarding basal metabolism and CYP inducibility. Under non-induced conditions, all CYP activities could be determined in 3D-PHH, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 in 2D-PHH and HepaRG, and CYP2C19 and CYP3A4 in HepG2 cells. The highest non-induced CYP activities were observed in 3D-PHH and HepaRG cells. mRNA expression was at least four-fold higher for all CYPs in 3D-PHH compared to the other cell systems. After treatment with 20 μM rifampicin, mRNA increased 3- to 50-fold for all CYPs except CYP1A2 and 2D6 for HepaRG and 3D-PHH, 4-fold (CYP2B6) and 17-fold (CYP3A4) for 2D-PHH and four-fold (CYP3A4) for HepG2. In 3D-PHH at least a two-fold increase in CYP activity was observed for all inducible CYP isoforms while CYP1A2 and CYP2C9 activity did not increase in 2D-PHH and HepaRG. CYP inducibility assessed in vivo using the same phenotyping probes was also best reflected by the 3D-PHH model. Our studies show that 3D-PHH and (with some limitations) HepaRG are suitable cell systems for assessing drug metabolism and CYP induction in vitro. HepG2 cells are less suited to assess CYP induction of the 2C and 3A family. The Basel phenotyping cocktail is suitable for the assessment of CYP activity and induction also in vitro. PMID:27917125

  13. 76 FR 15820 - Airworthiness Directives; B-N Group Ltd. Model BN-2, BN-2A, BN-2A-2, BN-2A-3, BN-2A-6, BN-2A-8...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-22

    ... reported where Glass Fibre Reinforced Plastic (GFRP) elevator tips have been found deformed on in-service... Fibre Reinforced Plastic (GFRP) elevator tips have been found deformed on in-service aircraft. The... stated above, the initial issue of this AD (AD 2009-0105) mandated inspection of the GFRP elevator...

  14. 75 FR 79990 - Airworthiness Directives; B-N Group Ltd. Model BN-2, BN-2A, BN-2A-2, BN-2A-3, BN-2A-6, BN-2A-8...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-21

    ... describes the unsafe condition as: An event has been reported where Glass Fibre Reinforced Plastic (GFRP... condition for the specified products. The MCAI states: An event has been reported where Glass Fibre... where Glass Fibre Reinforced Plastic (GFRP) elevator tips have been found deformed on...

  15. 'Smoking genes': a genetic association study.

    PubMed

    Verde, Zoraida; Santiago, Catalina; Rodríguez González-Moro, José Miguel; de Lucas Ramos, Pilar; López Martín, Soledad; Bandrés, Fernando; Lucia, Alejandro; Gómez-Gallego, Félix

    2011-01-01

    Some controversy exists on the specific genetic variants that are associated with nicotine dependence and smoking-related phenotypes. The purpose of this study was to analyse the association of smoking status and smoking-related phenotypes (included nicotine dependence) with 17 candidate genetic variants: CYP2A6*1×2, CYP2A6*2 (1799T>A) [rs1801272], CYP2A6*9 (-48T>G) [rs28399433], CYP2A6*12, CYP2A13*2 (3375C>T) [rs8192789], CYP2A13*3 (7520C>G), CYP2A13*4 (579G>A), CYP2A13*7 (578C>T) [rs72552266], CYP2B6*4 (785A>G), CYP2B6*9 (516G>T), CHRNA3 546C>T [rs578776], CHRNA5 1192G>A [rs16969968], CNR1 3764C>G [rs6928499], DRD2-ANKK1 2137G>A (Taq1A) [rs1800497], 5HTT LPR, HTR2A -1438A>G [rs6311] and OPRM1 118A>G [rs1799971]. We studied the genotypes of the aforementioned polymorphisms in a cohort of Spanish smokers (cases, N = 126) and ethnically matched never smokers (controls, N = 80). The results showed significant between-group differences for CYP2A6*2 and CYP2A6*12 (both P<0.001). Compared with carriers of variant alleles, the odds ratio (OR) for being a non-smoker in individuals with the wild-type genotype of CYP2A6*12 and DRD2-ANKK1 2137G>A (Taq1A) polymorphisms was 3.60 (95%CI: 1.75, 7.44) and 2.63 (95%CI: 1.41, 4.89) respectively. Compared with the wild-type genotype, the OR for being a non-smoker in carriers of the minor CYP2A6*2 allele was 1.80 (95%CI: 1.24, 2.65). We found a significant genotype effect (all P≤0.017) for the following smoking-related phenotypes: (i) cigarettes smoked per day and CYP2A13*3; (ii) pack years smoked and CYP2A6*2, CYP2A6*1×2, CYP2A13*7, CYP2B6*4 and DRD2-ANKK1 2137G>A (Taq1A); (iii) nicotine dependence (assessed with the Fagestrom test) and CYP2A6*9. Overall, our results suggest that genetic variants potentially involved in nicotine metabolization (mainly, CYP2A6 polymorphisms) are those showing the strongest association with smoking-related phenotypes, as opposed to genetic variants influencing the brain effects of nicotine, e

  16. Oxidation of Acenaphthene and Acenaphthylene by Human Cytochrome P450 Enzymes

    PubMed Central

    Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie; Yamazaki, Hiroshi; Kim, Joo-Hwan; Kim, Donghak; Yoshimoto, Francis K.; Guengerich, F. Peter; Komori, Masayuki

    2016-01-01

    Acenaphthene and acenaphthylene, two known environmental polycyclic aromatic hydrocarbon (PAH) pollutants, were incubated at 50 µM concentrations in a standard reaction mixture with human P450s 2A6, 2A13, 1B1, 1A2, 2C9, and 3A4 and the oxidation products were determined using HPLC and LC-MS. HPLC analysis showed that P450 2A6 converted acenaphthene and acenaphthylene to several mono- and di-oxygenated products. LC-MS analysis of acenaphthene oxidation by P450s indicated the formation of 1-acenaphthenol as a major product, with turnover rates of 6.7, 4.5, and 3.6 nmol product formed/min/nmol P450 for P450 2A6, 2A13, and 1B1, respectively. Acenaphthylene oxidation by P450 2A6 showed the formation of 1,2-epoxyacenaphthene as a major product (4.4 nmol epoxide formed/min/nmol P450) and also several mono- and di-oxygenated products. P450 2A13, 1B1, 1A2, 2C9, and 3A4 formed 1,2-epoxyacenaphthene at rates of 0.18, 5.3 2.4, 0.16, and 3.8 nmol/min nmol P450, respectively. 1-Acenaphthenol, which induced Type I binding spectra with P450 2A13, was further oxidized by P450 2A13 but not P450 2A6. 1,2-Epoxyacenaphthene induced Type I binding spectra with P450 2A6 and 2A13 (Ks 1.8 and 0.16 µM, respectively) and was also oxidized to several oxidation products by these P450s. Molecular docking analysis suggested different orientations of acenaphthene, acenaphthylene, 1-acenaphthenol, and 1,2-epoxyacenaphthene in their interactions with P450 2A6 and 2A13. Neither these four PAHs induced umu gene expression in a Salmonella typhimurium NM tester strain. These results suggest, for the first time, that acenaphthene and acenaphthylene are oxidized by human P450s 2A6 and 2A13 and other P450s to form several mono- and di-oxygenated products. The results are of use in considering the biological and toxicological significance of these environmental PAHs in humans. PMID:25642975

  17. Effects of zedoary turmeric oil on P450 activities in rats with liver cirrhosis induced by thioacetamide.

    PubMed

    Cheng, Jing-Jing; Yang, Nai-Bin; Wu, Liang; Lin, Jia-Le; Dai, Ge-Xin; Zhu, Jia-Yin

    2014-01-01

    The aim of this study was to elucidate the effects of zedoary turmeric oil (ZTO) on P450 activities (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) in rats with liver cirrhosis induced by thioacetamide (TAA). For the induction of liver cirrhosis, rats were given TAA in their drinking water at a concentration of 0.03% for consecutive 5 weeks and then 0.04% for the next consecutive 5 weeks throughout the establishment of cirrhosis. Then the cirrhotic rats were ip given saline, ZTO 100, 200 and 400 mg/kg, respectively, once daily for 2 weeks. When cirrhosis model was established at week 10, all rats of five groups were administered intragastrically with 15 mg/kg phenacetin, 0.6 mg/kg tolbutamide, 15 mg/kg omeprazole, 15 mg/kg bupropion, 15 mg/kg metoprolol, and 10 mg/kg midazolam. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The degree of liver cirrhosis was assessed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the model group increased by approximately 4-fold, and a decreased level of albumin (Alb) was also observed, as compared to the control group (P < 0.05). However, ZTO was found to reverse those changes of serum levels observed in the model group, and the 200 mg/kg ZTO treatment group showed the most obvious reverse tendency with significantly decreased ALT, AST and increased Alb levels (P < 0.05). The results indicated that ZTO with the dose of 100 mg/kg could inhibit the activities of CYP450 isoforms CYP2C9 and CYP2D6 in vivo in cirrhotic rats induced by TAA, while ZTO with the dose of 400 mg/kg could induce the activity of CYP2C19 in vivo in cirrhotic rats induced by TAA. However, ZTO showed no influence on cirrhotic rat hepatic CYP1A2, CYP2B6 and CYP3A4 activity in vivo. This has certain guiding significance to clinical treatment.

  18. Cytochrome P450 Drug Metabolizing Enzymes in Roma Population Samples: Systematic Review of the Literature.

    PubMed

    Szalai, Renata; Hadzsiev, Kinga; Melegh, Bela

    2016-01-01

    The cytochrome P450 drug metabolizing enzymes are highly polymorphic and show inter-individual differences in variability in drug response, which varies widely also with ethnicity. This study aims to summarize the available data on genetic polymorphisms associated with cytochrome enzymes conducted on Roma populations. Our goal was to compare the frequency of the variant alleles, genotypes and predicted phenotypes with corresponding rates from other populations. We carried out a systematic review including the papers published on the pharmacogenetically relevant variants of cytochrome P450 genes related to Roma population. The study was performed using several articles, websites and databases, including PubMed, Ensembl, dbSNP, HapMap and 1000 Genomes Project. This review attempts to summarize and discuss our current knowledge about the frequency distribution of the ever investigated 20 allelic variants of 9 cytochrome genes (CYP1A2, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2C8, CYP2D6, CYP3A5, CYP4F2) in Roma DNA samples and compare them with other populations. Differences between Roma and Hungarian samples are reported for 7 variant genotypes. CYP2C9 *2/*3 and CYP2C19 *2/*2 genotypes showed more than 3-fold differences. Additional differences are displayed for allele frequency of 7 variants (rs762551, rs3745274, rs1058930, rs1065852, rs3892097, rs1057910 and rs4244285) in Roma population samples. The interethnic variability in clinically relevant genetic polymorphisms of drug metabolizing enzymes, which may explain distinct drug response, highlights the need to allow for the ancestry of participants in pharmacogenetic studies.

  19. Enantioselectivity in the Metabolism of Cyclophosphamide in Patients With Multiple or Systemic Sclerosis.

    PubMed

    de Castro, Francine Attié; Simões, Belinda Pinto; Coelho, Eduardo Barbosa; Lanchote, Vera Lucia

    2017-01-13

    The aim of this study was to evaluate the enantioselective pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard in patients with systemic or multiple sclerosis. Patients with systemic sclerosis (n = 10) or multiple sclerosis (n = 10), genotyped for the allelic variants of CYP2C9*2 and CYP2C9*3 and of the CYP2B6 G516T polymorphism, were treated with 50 mg cyclophosphamide/kg daily for 4 days. Serial blood samples were collected up to 24 hours after administration of the last cyclophosphamide dose. Cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide enantiomers were analyzed in plasma samples using liquid chromatography-tandem mass spectrometry coupled to chiral column Chiralcel OD-R or Chiralpak AD-RH. Cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-17, TNF-α, and INT-δ in the plasma samples collected before cyclophosphamide infusion were analyzed by Milliplex MAP human cytokine/chemokine. Pharmacokinetic parameters showed higher plasma concentrations of (S)-(-)-cyclophosphamide (AUC 215.0 vs 186.2 μg·h/mL for multiple sclerosis patients and 219.1 vs 179.2 μg·h/mL for systemic sclerosis patients) and (R)-4-hydroxycyclophosphamide (AUC 5.6 vs 3.7 μg·h/mL for multiple sclerosis patients and 6.3 vs 5.6 μg·h/mL for systemic sclerosis patients) when compared to their enantiomers in both groups of patients, whereas the pharmacokinetics of the carboxyethylphosphoramide metabolite was not enantioselective. Cytokines' plasma concentrations were similar between multiple and systemic sclerosis groups. The pharmacokinetics of cyclophosphamide is enantioselective in patients with systemic sclerosis and multiple sclerosis, with higher plasma concentrations of the (S)-(-)-cyclophosphamide enantiomer due to the preferential formation of the (R)-4-hydroxycyclophosphamide metabolite.

  20. Evaluation of the inhibition potential of plumbagin against cytochrome P450 using LC-MS/MS and cocktail approach

    PubMed Central

    Chen, Ang; Zhou, Xiaojing; Tang, Shuowen; Liu, Mingyao; Wang, Xin

    2016-01-01

    Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural naphthoquinone compound isolated from roots of Plumbago zeylanica L., has drawn a lot of attention for its plenty of pharmacological properties including antidiabetes and anti-cancer. The aim of this study was to investigate the effects of plumbagin on CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver and evaluate the potential herb-drug interactions using the cocktail approach. All CYP substrates and their metabolites were analyzed using high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). Plumbagin presented non-time-dependent inhibition of CYP activities in both human and rat liver. In humans, plumbagin was not only a mixed inhibitor of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but also a non-competitive inhibitor of CYP1A2, with Ki values no more than 2.16 μM. In rats, the mixed inhibition of CYP1A2 and CYP2D1, and competitive inhibition for CYP2B1, CYP2C11 and CYP2E1 with Ki values less than 9.93 μM were observed. In general, the relatively low Ki values of plumbagin in humans would have a high potential to cause the toxicity and drug interactions involving CYP enzymes. PMID:27329697

  1. An Expanded Analysis of Pharmacogenetics Determinants of Efavirenz Response that Includes 3′-UTR Single Nucleotide Polymorphisms among Black South African HIV/AIDS Patients

    PubMed Central

    Swart, Marelize; Evans, Jonathan; Skelton, Michelle; Castel, Sandra; Wiesner, Lubbe; Smith, Peter J.; Dandara, Collet

    2016-01-01

    Introduction: Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor prescribed as part of first-line highly active antiretroviral therapy (HAART) in South Africa. Despite administration of fixed doses of EFV, inter-individual variability in plasma concentrations has been reported. Poor treatment outcomes such as development of adverse drug reactions or treatment failure have been linked to EFV plasma concentrations outside the therapeutic range (1–4 μg/mL) in some studies. The drug metabolizing enzyme (DME), CYP2B6, is primarily responsible for EFV metabolism with minor contributions by CYP1A2, CYP2A6, CYP3A4, CYP3A5, and UGT2B7. DME coding genes are also regulated by microRNAs through targeting the 3′-untranslated region. Expanded analysis of 30 single nucleotide polymorphisms (SNPs), including those in the 3′-UTR, was performed to identify pharmacogenetics determinants of EFV plasma concentrations in addition to CYP2B6 c.516G>T and c.983T>C SNPs. Methods: SNPs in CYP1A2, CYP2B6, UGT2B7, and NR1I2 (PXR) were selected for genotyping among 222 Bantu-speaking South African HIV-infected patients receiving EFV-containing HAART. This study is a continuation of earlier pharmacogenetics studies emphasizing the role of genetic variation in the 3′-UTR of genes which products are either pharmacokinetic or pharmacodynamic targets of EFV. Results: Despite evaluating thirty SNPs, CYP2B6 c.516G>T and c.983T>C SNPs remain the most prominent predictors of EFV plasma concentration. Conclusion: We have shown that CYP2B6 c.516G>T and c.983T>C SNPs are the most important predictors of EFV plasma concentration after taking into account all other SNPs, including genetic variation in the 3′-UTR, and variables affecting EFV metabolism. PMID:26779253

  2. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    PubMed

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each.

  3. Contribution of Cytochrome P450 and ABCB1 Genetic Variability on Methadone Pharmacokinetics, Dose Requirements, and Response

    PubMed Central

    Fonseca, Francina; de la Torre, Rafael; Díaz, Laura; Pastor, Antonio; Cuyàs, Elisabet; Pizarro, Nieves; Khymenets, Olha; Farré, Magí; Torrens, Marta

    2011-01-01

    Although the efficacy of methadone maintenance treatment (MMT) in opioid dependence disorder has been well established, the influence of methadone pharmacokinetics in dose requirement and clinical outcome remains controversial. The aim of this study is to analyze methadone dosage in responder and nonresponder patients considering pharmacogenetic and pharmacokinetic factors that may contribute to dosage adequacy. Opioid dependence patients (meeting Diagnostic and Statistical Manual of Mental Disorders, [4th Edition] criteria) from a MMT community program were recruited. Patients were clinically assessed and blood samples were obtained to determine plasma concentrations of (R,S)-, (R) and (S)- methadone and to study allelic variants of genes encoding CYP3A5, CYP2D6, CYP2B6, CYP2C9, CYP2C19, and P-glycoprotein. Responders and nonresponders were defined by illicit opioid consumption detected in random urinalysis. The final sample consisted in 105 opioid dependent patients of Caucasian origin. Responder patients received higher doses of methadone and have been included into treatment for a longer period. No differences were found in terms of genotype frequencies between groups. Only CYP2D6 metabolizing phenotype differences were found in outcome status, methadone dose requirements, and plasma concentrations, being higher in the ultrarapid metabolizers. No other differences were found between phenotype and responder status, methadone dose requirements, neither in methadone plasma concentrations. Pharmacokinetic factors could explain some but not all differences in MMT outcome and methadone dose requirements. PMID:21589866

  4. In vitro toxicological evaluation of NCS-382, a high-affinity antagonist of γ-hydroxybutyrate (GHB) binding.

    PubMed

    Vogel, K R; Ainslie, G R; Roullet, J-B; McConnell, A; Gibson, K M

    2017-01-22

    γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.

  5. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells

    PubMed Central

    Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P.; Walles, Heike

    2015-01-01

    In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. PMID:26488607

  6. Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo.

    PubMed

    Chen, Xiao-min; Wei, Min; Zhang, Hai-mou; Luo, Cheng-hao; Chen, Yi-kun; Chen, Yong

    2012-06-01

    Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 μM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction.

  7. Application of a higher throughput approach to derive apparent Michaelis-Menten constants of isoform-selective p450-mediated biotransformation reactions in human hepatocytes.

    PubMed

    Li, Albert P; Schlicht, Kari E

    2014-01-01

    A higher throughput platform was developed for the determination of K(M) values for isoformselective P450 substrates in human hepatocytes via incubation of the hepatocytes with substrates in 384- well plates and metabolite quantification by RapidFire™ mass spectrometry. Isoform-selective P450 substrates were incubated at 8 concentrations in triplicate with cryopreserved human hepatocytes from 16 donors. The metabolic pathways examined were the CYP1A2-catalyzed tacrine 1-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9- catalyzed diclofenac 4'-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, and CYP3A4-catalyzed midazolam 1'-hydroxylation. Typical saturation enzyme kinetics was observed for all the pathways evaluated. Individual differences in the apparent V(max) and K(M) values were observed among the human hepatocytes from each of the 16 individual donors, with no statistically significant gender- or age-associated differences. A "composite" K(M) value was calculated for each of the pathways via normalizing the individual activities to their respective V(max) values to develop "relative activities" followed by Michaelis-Menten analysis of the mean relative activities of the 16 donors at each of the 8 substrate concentrations. The resulting "composite" K(M) values for the P450 substrates may be used to guide in vitro P450 inhibition and induction studies and kinetic modeling of in vivo drug-drug interaction.

  8. Cobicistat versus ritonavir boosting and differences in the drug-drug interaction profiles with co-medications.

    PubMed

    Marzolini, Catia; Gibbons, Sara; Khoo, Saye; Back, David

    2016-07-01

    Nearly all HIV PIs and the integrase inhibitor elvitegravir require a pharmacokinetic enhancer in order to achieve therapeutic plasma concentrations at the desired dose and frequency. Whereas ritonavir has been the only available pharmacokinetic enhancer for more than a decade, cobicistat has recently emerged as an alternative boosting agent. Cobicistat and ritonavir are equally strong inhibitors of cytochrome P450 (CYP) 3A4 and consequently were shown to be equivalent pharmacokinetic enhancers for elvitegravir and for the PIs atazanavir and darunavir. Since cobicistat is a more selective CYP inhibitor than ritonavir and is devoid of enzyme-inducing properties, differences are expected in their interaction profiles with some co-medications. Drugs whose exposure might be altered by ritonavir but unaltered by cobicistat are drugs primarily metabolized by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 or drugs undergoing mainly glucuronidation. Thus, co-medications should be systematically reviewed when switching the pharmacokinetic enhancer to anticipate potential dosage adjustments.

  9. Chrysin, baicalein and galangin are indirect activators of the human constitutive androstane receptor (CAR).

    PubMed

    Carazo Fernández, Alejandro; Smutny, Tomas; Hyrsová, Lucie; Berka, Karel; Pavek, Petr

    2015-03-04

    The constitutive androstane receptor (CAR) is a crucial transcriptional regulator of key xenobiotic-metabolizing enzymes such as cytochrome P450 CYP3A4, CYP2C9 and CYP2B6. The flavonoids chrysin, baicalein and galangin have been reported to activate CAR and interfere with EGFR signaling. Nevertheless, it is not known if these flavonoids are direct CAR ligands or indirect phenobarbital-like CAR activators via the inhibition of epidermal growth factor receptor (EGFR) signaling. We analyze the interactions of chrysin, galangin and baicalein and its glycoside baicalin with human CAR. We have employed and validated methods that can study direct interaction with the CAR ligand binding pocket. Secondly, we determined if the compounds affect human EGFR signaling and interact with EGFR. Employing a TR-FRET coactivator assay with recombinant CAR or CAR assembly assay, a consistent activation of CAR with flavonoids and phenobarbital was not observed. It was determined, however, that galangin, chrysin, and baicalein may slightly repress EGFR-Tyr1068 autophosphorylation after EGF treatment, phosphorylation of downstream transcription factor ELK1 and stimulate EGFP-CAR nuclear translocation in primary human hepatocytes. These data suggest that flavonoids chrysin, galangin and baicalein are indirect human CAR activators. This study also demonstrates new approach how to test the direct CAR interaction with its ligands.

  10. The effects of antiepileptic inducers in neuropsychopharmacology, a neglected issue. Part II: Pharmacological issues and further understanding.

    PubMed

    de Leon, Jose

    2015-01-01

    The literature on inducers in epilepsy and bipolar disorder is seriously contaminated by false negative findings. Part II of this comprehensive review on antiepileptic drug (AED) inducers provides clinicians with further educational material about the complexity of interpreting AED drug-drug interactions. The basic pharmacology of induction is reviewed including the cytochrome P450 (CYP) isoenzymes, the Uridine Diphosphate Glucuronosyltransferases (UGTs), and P-glycoprotein (P-gp). CYP2B6 and CYP3A4 are very sensitive to induction. CYP1A2 is moderately sensitive while CYP2C9 and CYP2C19 are only mildly sensitive. CYP2D6 cannot be induced by medications. Induction of UGT and P-gp are poorly understood. The induction of metabolic enzymes such as CYPs and UGTs, and transporters such as P-gp, implies that the amount of these proteins increases when they are induced; this is almost always explained by increasing synthesis mediated by the so-called nuclear receptors (constitutive androstane, estrogen, glucocorticoid receptors and pregnaneX receptors). Although parti provides correction factors for AEDs, extrapolation from an average to an individual patient may be influenced by administration route, absence of metabolic enzyme for genetic reasons, and presence of inhibitors or other inducers. AED pharmacodynamic DDIs may also be important. Six patients with extreme sensitivity to AED inductive effects are described.

  11. Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

    PubMed

    Zielinski, Jana; Mevissen, Meike

    2015-04-01

    Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60 min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species.

  12. Metabolism of loratadine and further characterization of its in vitro metabolites.

    PubMed

    Ghosal, Anima; Gupta, Samir; Ramanathan, Ragu; Yuan, Yuan; Lu, Xiaowen; Su, Ai Duen Iris; Alvarez, Narciso; Zbaida, Shmuel; Chowdhury, Swapan K; Alton, Kevin B

    2009-08-01

    The present study demonstrated that in addition to CYP3A4 and CYP2D6, the metabolism of loratadine is also catalyzed by CYP1A1, CYP2C19, and to a lesser extent by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A5. The biotransformation of loratadine was associated with the formation of desloratadine (DL) and further hydroxylation of both DL and the parent drug (loratadine). Based on the inhibition and correlation studies contribution of CYP2C19 in the formation of the major circulating metabolite DL seems to be minor. Reported clinical results suggest that the steady state mean (%CV) plasma Cmax and AUC(24hr) of loratadine were 4.73 ng/ml (119%) and 24.1 ng.hr/ml (157%), respectively, after dosing with 10 mg loratadine tablets for 10 days. High inter-subject variability in loratadine steady-state data is probably due to the phenotypical characteristics of CYP2D6, CYP2C19, and CYP3A4. The relative abundance of CYP3A4 in the human liver exceeds that of CYP2C19 and CYP2D6 and therefore the contribution of CYP3A4 in the metabolism of loratadine should be major (approximately 70%).

  13. Pharmacokinetic and Pharmacodynamic Comparison of Once‐Daily Efavirenz (400 mg vs. 600 mg) in Treatment‐Naïve HIV‐Infected Patients: Results of the ENCORE1 Study

    PubMed Central

    Amin, J; Else, L; Boffito, M; Egan, D; Owen, A; Khoo, S; Back, D; Orrell, C; Clarke, A; Losso, M; Phanuphak, P; Carey, D; Cooper, DA; Emery, S

    2015-01-01

    Daily efavirenz 400 mg (EFV400) was virologically noninferior to 600 mg (EFV600) at 48 weeks in treatment‐naïve patients. We evaluated EFV400 and EFV600 pharmacokinetics (NONMEM v. 7.2), assessing patient demographics and genetic polymorphisms (CYP2B6, CYP2A6, CYP3A4, NR1I3) as covariates and explored relationships with efficacy (plasma HIV‐RNA (pVL) <200 copies/mL) and safety outcomes at 48 weeks in 606 randomized ENCORE1 patients (female = 32%, African = 37%, Asian = 33%; EFV400 = 311, EFV600 = 295). CYP2B6 516G>T/983T>C/CYP2A6*9B/*17 and weight were associated with efavirenz CL/F. Exposure was significantly lower for EFV400 (geometric mean ratio, GMR; 90% confidence interval, CI: 0.73 (0.68–0.78)) but 97% (EFV400) and 98% (EFV600) of evaluable pVL was <200 copies/mL at 48 weeks (P = 0.802). Four of 20 patients with mid‐dose concentrations <1.0 mg/L had pVL ≥200 copies/mL (EFV400 = 1; EFV600 = 3). Efavirenz exposure was similar between those with and without efavirenz‐related side effects (GMR; 90% CI: 0.95 (0.88–1.02)). HIV suppression was comparable between doses despite significantly lower EFV400 exposure. Comprehensive evaluation of efavirenz pharmacokinetics/pharmacodynamics revealed important limitations in the accepted threshold concentration. PMID:26044067

  14. Pharmacokinetic and Pharmacodynamic Comparison of Once-Daily Efavirenz (400 mg vs. 600 mg) in Treatment-Naïve HIV-Infected Patients: Results of the ENCORE1 Study.

    PubMed

    Dickinson, L; Amin, J; Else, L; Boffito, M; Egan, D; Owen, A; Khoo, S; Back, D; Orrell, C; Clarke, A; Losso, M; Phanuphak, P; Carey, D; Cooper, D A; Emery, S; Puls, R

    2015-10-01

    Daily efavirenz 400 mg (EFV400) was virologically noninferior to 600 mg (EFV600) at 48 weeks in treatment-naïve patients. We evaluated EFV400 and EFV600 pharmacokinetics (NONMEM v. 7.2), assessing patient demographics and genetic polymorphisms (CYP2B6, CYP2A6, CYP3A4, NR1I3) as covariates and explored relationships with efficacy (plasma HIV-RNA (pVL) <200 copies/mL) and safety outcomes at 48 weeks in 606 randomized ENCORE1 patients (female = 32%, African = 37%, Asian = 33%; EFV400 = 311, EFV600 = 295). CYP2B6 516G>T/983T>C/CYP2A6*9B/*17 and weight were associated with efavirenz CL/F. Exposure was significantly lower for EFV400 (geometric mean ratio, GMR; 90% confidence interval, CI: 0.73 (0.68-0.78)) but 97% (EFV400) and 98% (EFV600) of evaluable pVL was <200 copies/mL at 48 weeks (P = 0.802). Four of 20 patients with mid-dose concentrations <1.0 mg/L had pVL ≥200 copies/mL (EFV400 = 1; EFV600 = 3). Efavirenz exposure was similar between those with and without efavirenz-related side effects (GMR; 90% CI: 0.95 (0.88-1.02)). HIV suppression was comparable between doses despite significantly lower EFV400 exposure. Comprehensive evaluation of efavirenz pharmacokinetics/pharmacodynamics revealed important limitations in the accepted threshold concentration.

  15. Frequencies of 23 Functionally Significant Variant Alleles Related with Metabolism of Antineoplastic Drugs in the Chilean Population: Comparison with Caucasian and Asian Populations

    PubMed Central

    Roco, Ángela; Quiñones, Luis; Agúndez, José A. G.; García-Martín, Elena; Squicciarini, Valentina; Miranda, Carla; Garay, Joselyn; Farfán, Nancy; Saavedra, Iván; Cáceres, Dante; Ibarra, Carol; Varela, Nelson

    2012-01-01

    Cancer is a leading cause of death worldwide. The cancer incidence rate in Chile is 133.7/100,000 inhabitants and it is the second cause of death, after cardiovascular diseases. Most of the antineoplastic drugs are metabolized to be detoxified, and some of them to be activated. Genetic polymorphisms of drug-metabolizing enzymes can induce deep changes in enzyme activity, leading to individual variability in drug efficacy and/or toxicity. The present research describes the presence of genetic polymorphisms in the Chilean population, which might be useful in public health programs for personalized treatment of cancer, and compares these frequencies with those reported for Asian and Caucasian populations, as a contribution to the evaluation of ethnic differences in the response to chemotherapy. We analyzed 23 polymorphisms in a group of 253 unrelated Chilean volunteers from the general population. The results showed that CYP2A6*2, CYP2A6*3, CYP2D6*3, CYP2C19*3, and CYP3A4*17 variant alleles are virtually absent in Chileans. CYP1A1*2A allele frequency (0.37) is similar to that of Caucasians and higher than that reported for Japanese people. Allele frequencies for CYP3A5*3(0.76) and CYP2C9*3(0.04) are similar to those observed in Japanese people. CYP1A1*2C(0.32), CYP1A2*1F(0.77), CYP3A4*1B(0.06), CYP2D6*2(0.41), and MTHFR T(0.52) allele frequencies are higher than the observed either in Caucasian or in Japanese populations. Conversely, CYP2C19*2 allelic frequency (0.12), and genotype frequencies for GSTT1 null (0.11) and GSTM1 null (0.36) are lower than those observed in both populations. Finally, allele frequencies for CYP2A6*4(0.04), CYP2C8*3(0.06), CYP2C9*2(0.06), CYP2D6*4(0.12), CYP2E1*5B(0.14), CYP2E1*6(0.19), and UGT2B7*2(0.40) are intermediate in relation to those described in Caucasian and in Japanese populations, as expected according to the ethnic origin of the Chilean population. In conclusion, our findings support the idea that ethnic variability must be

  16. Further Characterization of the Metabolism of Desloratadine and Its Cytochrome P450 and UDP-glucuronosyltransferase Inhibition Potential: Identification of Desloratadine as a Relatively Selective UGT2B10 Inhibitor.

    PubMed

    Kazmi, Faraz; Yerino, Phyllis; Barbara, Joanna E; Parkinson, Andrew

    2015-09-01

    Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating antihistamine used for the treatment of seasonal allergies and hives. Previously we reported that the formation of 3-hydroxydesloratadine, the major human metabolite of desloratadine, involves three sequential reactions, namely N-glucuronidation by UGT2B10 followed by 3-hydroxylation by CYP2C8 followed by deconjugation (rapid, nonenzymatic hydrolysis of the N-glucuronide). In this study we assessed the perpetrator potential of desloratadine based on in vitro studies of its inhibitory effects on cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes (HLM). Desloratadine (10 µM) caused no inhibition (<15%) of CYP1A2, CYP2C8, CYP2C9, or CYP2C19 and weak inhibition (32-48%) of CYP2B6, CYP2D6, and CYP3A4/5. In cryopreserved human hepatocytes (CHH), which can form the CYP2C8 substrate desloratadine N-glucuronide, desloratadine did not inhibit the CYP2C8-dependent metabolism of paclitaxel or amodiaquine. Assessment of UGT inhibition identified desloratadine as a potent and relatively selective competitive inhibitor of UGT2B10 (Ki value of 1.3 μM). Chemical inhibition of UGT enzymes in HLM demonstrated that nicotine (UGT2B10 inhibitor) but not hecogenin (UGT1A4 inhibitor) completely inhibited the conversion of desloratadine (1 µM) to 3-hydroxydesloratadine in HLM fortified with both NADPH and UDP-glucuronic acid. 3-Hydroxydesloratadine formation correlated well with levomedetomidine glucuronidation (UGT2B10 marker activity) with a panel of individual CHH (r(2) = 0.72). Overall, the results of this study confirm the role of UGT2B10 in 3-hydroxydesloratadine formation and identify desloratadine as a relatively selective in vitro inhibitor of UGT2B10.

  17. Evaluation of Mutual Drug-Drug Interaction within Geneva Cocktail for Cytochrome P450 Phenotyping using Innovative Dried Blood Sampling Method.

    PubMed

    Bosilkovska, Marija; Samer, Caroline; Déglon, Julien; Thomas, Aurélien; Walder, Bernhard; Desmeules, Jules; Daali, Youssef

    2016-09-01

    Cytochrome P450 (CYP) activity can be assessed using a 'cocktail' phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low-dose probes with a low-invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre-treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug-drug interactions. In a randomized, four-way, Latin-square crossover study, 30 healthy volunteers received low-dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug-drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self-sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8-1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post-cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.

  18. Diphenidine, a new psychoactive substance: metabolic fate elucidated with rat urine and human liver preparations and detectability in urine using GC-MS, LC-MS(n) , and LC-HR-MS(n).

    PubMed

    Wink, Carina S D; Michely, Julian A; Jacobsen-Bauer, Andrea; Zapp, Josef; Maurer, Hans H

    2016-10-01

    Diphenidine is a new psychoactive substance (NPS) sold as a 'legal high' since 2013. Case reports from Sweden and Japan demonstrate its current use and the necessity of applying analytical procedures in clinical and forensic toxicology. Therefore, the phase I and II metabolites of diphenidine should be identified and based on these results, the detectability using standard urine screening approaches (SUSAs) be elucidated. Urine samples were collected after administration of diphenidine to rats and analyzed using different sample workup procedures with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-(high resolution)-mass spectrometry (LC-(HR)-MS). With the same approaches incubates of diphenidine with pooled human liver microsomes (pHLM) and cytosol (pHLC) were analyzed. According to the identified metabolites, the following biotransformation steps were proposed in rats: mono- and bis-hydroxylation at different positions, partly followed by dehydrogenation, N,N-bis-dealkylation, and combinations of them followed by glucuronidation and/or methylation of one of the bis-hydroxy-aryl groups. Mono- and bis-hydroxylation followed by dehydrogenation could also be detected in pHLM or pHLC. Cytochrome-P450 (CYP) isozymes CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were all capable of forming the three initial metabolites, namely hydroxy-aryl, hydroxy-piperidine, and bis-hydroxy-piperidine. In incubations with CYP2D6 hydroxy-aryl and hydroxy-piperidine metabolites were detected. After application of a common users' dose, diphenidine metabolites could be detected in rat urine by the authors' GC-MS as well as LC-MS(n) SUSA. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Metabolite Identification, Reaction Phenotyping and Retrospective Drug-Drug Interaction Predictions of 17-deacetylnorgestimate, the Active Component of the Oral Contraceptive Norgestimate.

    PubMed

    Ahire, Deepak; Sinha, Sarmistha; Brock, Barry; Iyer, Ramaswamy; Mandlekar, Sandhya; Subramanian, Murali

    2017-03-10

    Ortho-Tri-Cyclen® (OTC), a two drug cocktail comprising of ethinylestradiol (EE) and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450s (rCYPs), recombinant uridine 5'-diphospho-glucuronosyltransferases (rUGTs) and human liver microsomes (HLM) in the presence and absence of selective cytochrome P450 (CYP) inhibitors were conducted. It was found that cytochrome P450 3A4 (CYP3A4) plays a key role in NGNM metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm, the predicted area under the plasma concentration vs. time curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm of 1. The data presented in this manuscript will lead to a better understanding and management of NGMN based drug-drug interactions (DDIs) when norgestimate is co-administered with CYP3A4 modulators.

  20. Expression of cytochromes p450 in fetal, infant, and juvenile liver of cynomolgus macaques.

    PubMed

    Ise, Ryota; Kondo, Satoshi; Kato, Hiroto; Imai, Noritaka; Akiyama, Hideo; Iwasaki, Kazuhide; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-01-01

    Preclinical data of fetal, infant, and juvenile animals are important for the prediction of drug toxicity in fetuses and children. However, expression of drug-metabolizing enzymes, including cytochromes P450 (CYPs), have not been fully investigated in fetal, infant, or juvenile liver of the cynomolgus macaque, an animal species important for preclinical studies. In this study, hepatic expression of 20 cynomolgus macaque CYPs (mfCYPs) in the CYP1-4 subfamilies that are relevant to drug metabolism was measured in fetuses, infants, and juveniles using DNA microarrays. Expression of most mfCYPs, including those moderately or abundantly expressed in postnatal livers such as mfCYP2A23, mfCYP2A24, mfCYP2B6, mfCYP2C9, mfCYP2C19, mfCYP2C76, mfCYP2D17, mfCYP2E1 mfCYP3A4, and mfCYP3A5, was much less abundant in fetal livers, but increased substantially after birth. In contrast, expression of mfCYP2C8 in fetal livers was not substantially different from postnatal livers. Since human CYP3A7 is expressed more abundantly in fetal livers than in adult livers, mfCYP3A7, an ortholog of human CYP3A7, was analyzed by quantitative polymerase chain reaction. Expression of mfCYP3A7 in fetal livers was much lower than that in postnatal livers, and greatly increased after birth, unlike the expression of human CYP3A7. These results indicate that expression of most mfCYPs examined was low in fetal livers, but increased greatly in postnatal livers, with a few exceptions such as mfCYP2C8.

  1. Association study of genetic polymorphism in ABCC4 with cyclophosphamide-induced adverse drug reactions in breast cancer patients.

    PubMed

    Low, Siew-Kee; Kiyotani, Kazuma; Mushiroda, Taisei; Daigo, Yataro; Nakamura, Yusuke; Zembutsu, Hitoshi

    2009-10-01

    Cyclophosphamide (CPA)-based combination treatment has known to be effective for breast cancer, but often causes adverse drug reactions (ADRs). Hence, the identification of patients at risk for toxicity by CPA is clinically significant. In this study, a stepwise case-control association study was conducted using 403 patients with breast cancer who received the CPA combination therapy. A total of 143 genetic polymorphisms in 13 candidate genes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, ALDH1A1, ALDH3A1, GSTA1, GSTM1, GSTP1, GSTT1, ABCC2 and ABCC4), possibly involved in the activation, metabolism and transport of CPA, were genotyped using 184 cases who developed either > or =grade 3 leukopenia/neutropenia or > or =grade 2 gastrointestinal toxicity and 219 controls who did not show any ADRs throughout the treatment. The association study revealed that one SNP, rs9561778 in ABCC4, showed a significant association with CPA-induced ADRs (Cochran-Armitage trend's P-value=0.00031; odds ratio (OR)=2.06). Subgroup analysis also indicated that the SNP rs9561778 was significantly associated with two major ADR subgroups; gastrointestinal toxicity and leukopenia/neutropenia (Cochran-Armitage trend's P-value=0.00019 and 0.014; OR=2.31 and 1.83). Furthermore, the SNP rs9561778 showed an association with breast cancer patients who were treated with CA(F) drug regimen-induced ADR (Cochran-Armitage trend's P-value=0.00028; OR=3.13). The SNPs in ABCC4 might be applicable in predicting the risk of ADRs in patients receiving CPA combination chemotherapy.

  2. Genotype and allele frequencies of drug-metabolizing enzymes and drug transporter genes affecting immunosuppressants in the Spanish white population.

    PubMed

    Bosó, Virginia; Herrero, María J; Buso, Enrique; Galán, Juan; Almenar, Luis; Sánchez-Lázaro, Ignacio; Sánchez-Plumed, Jaime; Bea, Sergio; Prieto, Martín; García, María; Pastor, Amparo; Sole, Amparo; Poveda, José Luis; Aliño, Salvador F

    2014-04-01

    Interpatient variability in drug response can be widely explained by genetically determined differences in metabolizing enzymes, drug transporters, and drug targets, leading to different pharmacokinetic and/or pharmacodynamic behaviors of drugs. Genetic variations affect or do not affect drug responses depending on their influence on protein activity and the relevance of such proteins in the pathway of the drug. Also, the frequency of such genetic variations differs among populations, so the clinical relevance of a specific variation is not the same in all of them. In this study, a panel of 33 single nucleotide polymorphisms in 14 different genes (ABCB1, ABCC2, ABCG2, CYP2B6, CYP2C19, CYP2C9, CYP3A4, CYP3A5, MTHFR, NOD2/CARD15, SLCO1A2, SLCO1B1, TPMT, and UGT1A9), encoding for the most relevant metabolizing enzymes and drug transporters relating to immunosuppressant agents, was analyzed to determine the genotype profile and allele frequencies in comparison with HapMap data. A total of 570 Spanish white recipients and donors of solid organ transplants were included. In 24 single nucleotide polymorphisms, statistically significant differences in allele frequency were observed. The largest differences (>100%) occurred in ABCB1 rs2229109, ABCG2 rs2231137, CYP3A5 rs776746, NOD2/CARD15 rs2066844, TPMT rs1800462, and UGT1A9 rs72551330. In conclusion, differences were recorded between the Spanish and other white populations in terms of allele frequency and genotypic distribution. Such differences may have implications in relation to dose requirements and drug-induced toxicity. These data are important for further research to help explain interindividual pharmacokinetic and pharmacodynamic variability in response to drug therapy.

  3. Comparison of hepatic-like cell production from human embryonic stem cells and adult liver progenitor cells: CAR transduction activates a battery of detoxification genes.

    PubMed

    Funakoshi, Natalie; Duret, Cédric; Pascussi, Jean-Marc; Blanc, Pierre; Maurel, Patrick; Daujat-Chavanieu, Martine; Gerbal-Chaloin, Sabine

    2011-09-01

    In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate.

  4. Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

    SciTech Connect

    Kanno, Yuichiro Inajima, Jun; Kato, Sayaka; Matsumoto, Maika; Tokumoto, Chikako; Kure, Yuki; Inouye, Yoshio

    2015-03-27

    The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that protein arginine methyltransferase 5 (PRMT5) is a novel CAR-interacting protein. Furthermore, the PRMT-dependent induction of a CAR reporter gene, which was independent of methyltransferase activity, was enhanced in the presence of steroid receptor coactivator 1 (SRC1), peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) or DEAD box DNA/RNA helicase DP97. Using tetracycline inducible-hCAR system in HepG2 cells, we showed that knockdown of PRMT5 with small interfering RNA suppressed tetracycline -induced mRNA expression of CYP2B6 but not of CYP2C9 or CYP3A4. PRMT5 enhanced phenobarbital-mediated transactivation of a phenobarbital-responsive enhancer module (PBREM)-driven reporter gene in co-operation with PGC-1α in rat primary hepatocytes. Based on these findings, we suggest PRMT5 to be a gene (or promoter)-selective coactivator of CAR by mediating the formation of complexes between hCAR and appropriate coactivators. - Highlights: • Nuclear receptor CAR interact with PRMT5. • PRMT5 enhances transcriptional activity of CAR. • PRMT5 synergistically enhances transactivity of CAR by the co-expression of SRC-1, DP97 or PGC1α. • PRMT5 is a gene-selective co-activator for hCAR.

  5. Pharmacogenetics of nicotine and associated smoking behaviors.

    PubMed

    Tanner, Julie-Anne; Chenoweth, Meghan J; Tyndale, Rachel F

    2015-01-01

    This chapter summarizes genetic factors that contribute to variation in nicotine pharmacokinetics and nicotine's pharmacological action in the central nervous system (CNS), and how this in turn influences smoking behaviors. Nicotine, the major psychoactive compound in cigarette smoke, is metabolized by a number of enzymes, including CYP2A6, CYP2B6, FMOs, and UGTs, among others. Variation in the genes encoding these enzymes, in particular CYP2A6, can alter the rate of nicotine metabolism and smoking behaviors. Faster nicotine metabolism is associated with higher cigarette consumption and nicotine dependence, as well as lower quit rates. Variation in nicotine's CNS targets and downstream signaling pathways can also contribute to interindividual differences in smoking patterns. Binding of nicotine to neuronal nicotinic acetylcholine receptors (nAChRs) mediates the release of several neurotransmitters including dopamine and serotonin. Genetic variation in nAChRs, and in transporter and enzyme systems that leads to altered CNS levels of dopamine and serotonin, is associated with a number of smoking behaviors. To date, the precise mechanism underpinning many of these findings remains unknown. Considering the complex etiology of nicotine addiction, a more comprehensive approach that assesses the contribution of multiple gene variants, and their interaction with environmental factors, will likely improve personalized therapeutic approaches and increase smoking cessation rates.

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

    PubMed

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

    2015-12-25

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

  7. Cytochrome P4502C9: an enzyme of major importance in human drug metabolism

    PubMed Central

    Miners, John O; Birkett, Donald J

    1998-01-01

    Accumulating evidence indicates that CYP2C9 ranks amongst the most important drug metabolizing enzymes in humans. Substrates for CYP2C9 include fluoxetine, losartan, phenytoin, tolbutamide, torsemide, S-warfarin, and numerous NSAIDs. CYP2C9 activity in vivo is inducible by rifampicin. Evidence suggests that CYP2C9 substrates may also be induced variably by carbamazepine, ethanol and phenobarbitone. Apart from the mutual competitive inhibition which may occur between alternate substrates, numerous other drugs have been shown to inhibit CYP2C9 activity in vivo and/or in vitro. Clinically significant inhibition may occur with coadministration of amiodarone, fluconazole, phenylbutazone, sulphinpyrazone, sulphaphenazole and certain other sulphonamides. Polymorphisms in the coding region of the CYP2C9 gene produce variants at amino acid residues 144 (Arg144Cys) and 359 (Ile359Leu) of the CYP2C9 protein. Individuals homozygous for Leu359 have markedly diminished metabolic capacities for most CYP2C9 substrates, although the frequency of this allele is relatively low. Consistent with the modulation of enzyme activity by genetic and other factors, wide interindividual variability occurs in the elimination and/or dosage requirements of prototypic CYP2C9 substrates. Individualisation of dose is essential for those CYP2C9 substrates with a narrow therapeutic index. PMID:9663807

  8. Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction.

    PubMed

    Oh, Hyun-A; Lee, Hyunbeom; Kim, Donghak; Jung, Byung Hwa

    2017-02-15

    As drug interactions with cytochrome P450 enzymes become increasingly important in the field of drug discovery, a high-throughput screening method for analysing the effects of a drug is needed. We have developed a simple and rapid simultaneous analytical method using a cocktail approach for measuring the activities of seven cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). Human liver microsomes were used as a source for the seven cytochrome P450 enzymes, and a gas chromatography-mass spectrometry (GC-MS) was used for analysing their activities. Kinetic studies and inhibition assays of CYP enzymes were performed using known substrates and inhibitors for validating and comparing the reaction rates and time-dependent activities between methods using each substrate versus a method using a cocktail solution. The optimized cocktail method was successfully applied to evaluate the effects of the decoction of Socheongryong-tang (SCRT) on cytochrome P450 enzymes. Our cocktail method provides a simultaneous high-throughput activity assay using GC-MS for the first time. This method is applicable for analysing the drug interactions of various plant-derived mixtures.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  11. Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells

    SciTech Connect

    Du Liping; Neis, Mark M.; Ladd, Patricia A.; Yost, Garold S.; Keeney, Diane S. . E-mail: diane.keeney@vanderbilt.edu

    2006-06-01

    Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

  12. Relationship between clopidogrel-related polymorphisms and variable platelet reactivity at 1 year: A cohort study from Han Chinese

    PubMed Central

    Wang, Xiaodong; Lai, Yan; Luo, Yu; Zhang, Xumin; Zhou, Hua; Ye, Zi; Tang, Jiani; Liu, Xuebo

    2016-01-01

    Background: This study was designed to investigate the effect of clopidogrel-related gene polymorphisms on platelet reactivity and clinical outcome in Chinese Han patients. Materials and Methods: Three hundred and thirty-six percutaneous coronary intervention - treated patients were recruited and followed for 1 year. Blood samples were collected from all patients for DNA genotyping. The platelet reactivity unit was measured by the VerifyNow technique. The CYP2C19*2, CYP2C19*3, CYP2C19*17, ATP-binding cassette subfamily B member 1, ITGB3, CYP2C9*3, CYP2B6*9, and P2Y12 alleles were assessed. Results: The clinical endpoints were related to previous heart disease history (11.90% vs. 28.57%, P = 0.017), stroke (12.24% vs. 16.67%, P = 0.039), and diabetes (27.55% vs. 52.38%, P = 0.047). High on-treatment platelet reactivity (HTPR) was frequent in advanced age (P = 0.019), male gender (P = 0.016), hypertension (P = 0.033), and chronic renal failure (P = 0.040). There were more endpoints in the CYP2C19*2 and P2Y12 mutant carriers (76.19% vs. 43.20%, P < 0.001; 50.00% vs. 35.71%, P = 0.001, respectively), whereas fewer in the CYP2C19*17 mutant carriers (11.90% vs. 56.46%, P = 0.001). CYP2C19*2 and P2Y12 polymorphism manifested HTPR (194.25 ± 45.91 vs. 151.38 ± 58.14, P < 0.001; 180.33 ± 67.25 vs. 161.89 ± 56.49, P = 0.008, respectively), whereas CYP2C19*17 mutant improved platelet reactivity (97.17 ± 45.38 vs. 169.08 ± 57.15, P = 0.003). However, there were no further cardiovascular deaths in endpoint patients. Conclusion: In Han Chinese people of mainland China, clopidogrel-related gene polymorphisms are related to variable platelet reactivity after clopidogrel maintenance dosing, which influences major adverse cardiovascular events, without an effect on cardiac death. PMID:28255319

  13. Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds.

    PubMed

    Hultman, Ia; Vedin, Charlotta; Abrahamsson, Anna; Winiwarter, Susanne; Darnell, Malin

    2016-08-01

    Design of slowly metabolized compounds is an important goal in many drug discovery projects. Standard hepatocyte suspension intrinsic clearance (CLint) methods can only provide reliable CLint values above 2.5 μL/min/million cells. A method that permits extended incubation time with maintained performance and metabolic activity of the in vitro system is warranted to allow in vivo clearance predictions and metabolite identification of slowly metabolized drugs. The aim of this study was to evaluate the static HμREL coculture of human hepatocytes with stromal cells to be set up in-house as a standard method for in vivo clearance prediction and metabolite identification of slowly metabolized drugs. Fourteen low CLint compounds were incubated for 3 days, and seven intermediate to high CLint compounds and a cocktail of cytochrome P450 (P450) marker substrates were incubated for 3 h. In vivo clearance was predicted for 20 compounds applying the regression line approach, and HμREL coculture predicted the human intrinsic clearance for 45% of the drugs within 2-fold and 70% of the drugs within 3-fold of the clinical values. CLint values as low as 0.3 μL/min/million hepatocytes were robustly produced, giving 8-fold improved sensitivity of robust low CLint determination, over the cutoff in hepatocyte suspension CLint methods. The CLint values of intermediate to high CLint compounds were at similar levels both in HμREL coculture and in freshly thawed hepatocytes. In the HμREL coculture formation rates for five P450-isoform marker reactions, paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), and 1-OH-midazolam (3A4) were within the range of literature values for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation rate was lower. Further, both phase I and phase II metabolites were detected and an increased number of metabolites were observed in the HμREL coculture compared to hepatocyte suspension. In conclusion, HμREL coculture can

  14. An Appraisal of Drug-Drug Interactions with Green Tea (Camellia sinensis).

    PubMed

    Albassam, Ahmed A; Markowitz, John S

    2017-01-24

    This review summarizes published in vitro, animal, and clinical studies investigating the effects of green tea (Camellia sinensis) extract and associated catechins on drug-metabolizing enzymes and drug transporters. In vitro studies suggest that green tea extract and its main catechin, (-)-epigallocatechin-3-gallate, to varying degrees, inhibit the activity of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4. UGT1A1 and UGT1A4 isoforms were also inhibited by (-)-epigallocatechin-3-gallate. Animal studies suggest green tea extract and/or (-)-epigallocatechin-3-gallate significantly increase the bioavailability of diltazem, verapamil, tamoxifen simvastatin, 5-fluorouracil, and nicardipine. Conversely, green tea extract and/or (-)-epigallocatechin-3-gallate reduce the bioavailability of quetiapine, sunitinib, clozapine, and nadolol. Of the few clinical studies available for review, it appears neither green tea extract nor (-)-epigallocatechin-3-gallate inhibit any major cytochrome P450 enzyme. Regarding drug transporters, in vitro studies indicate P-glycoprotein, organic anion transporting polypeptide 1A1, organic anion transporting polypeptide 1B1, organic anion transporting polypeptide 1B3, organic anion transporting polypeptide 2B1, organic cation transporter 1, organic cation transporter 2, multidrug and toxin extrusion 1, and multidrug and toxin extrusion 2-K are potentially inhibited by green tea extract. A clinical study indicates the organic anion transporting polypeptide 1A1 transporter is inhibited by (-)-epigallocatechin-3-gallate while P-glycoprotein is unaffected. In conclusion, the ingestion of green tea extract or its associated catechins is not expected to result in clinically significant influences on major cytochrome P450 or uridine 5'-diphospho-glucuronosyltransferase enzyme substrates or drugs serving as substrates of P-glycoprotein. However, some caution is advised in the consumption of significant amounts of green tea beverages or green

  15. Highly sensitive LC-MS/MS methods for the determination of seven human CYP450 activities using small oral doses of probe-drugs in human.

    PubMed

    Grangeon, Alexia; Gravel, Sophie; Gaudette, Fleur; Turgeon, Jacques; Michaud, Veronique

    2017-01-01

    Cocktails composed of several Cytochrome P450 (CYP450)-selective probe drugs have been shown of value to characterize in vivo drug-metabolism activities. Our objective was to develop and validate highly sensitive and selective LC-MS/MS assays allowing the determination of seven major human CYP450 isoenzyme activities following administration of low oral doses of a modified CYP450 probe-drug cocktail in patients. The seven-drug cocktail was composed of caffeine, bupropion, tolbutamide, omeprazole, dextromethorphan, midazolam (all administered concomitantly) and chlorzoxazone (administered separately) to phenotype for CYP1A2, 2B6, 2C9, 2C19, 2D6, 3A4/5 and 2E1, respectively. Serial plasma and urine samples were collected over an 8h period. The probe-drugs and their respective metabolites were measured in both human plasma and urine, except for omeprazole (plasma only) and chlorzoxazone (urine only). Samples were analyzed by high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using a Phenomenex Luna PFP (2) analytical column (3μm PFP(2) 150×3mm) for chromatographic separation. Optimal detection was achieved based on 3 different analytical methods; (1) isocratic elution with a mobile phase consisting of acetonitrile and water both fortified with 0.01% formic acid for the analysis of bupropion, tolbutamide, chlorzoxazone and their respective metabolites; (2) isocratic elution with a mobile phase composed of acetonitrile and ammonium formate (pH 3; 10mM) for omeprazole, dextromethorphan, midazolam and their metabolites; (3) for caffeine and paraxanthine, gradient elution using acetonitrile and 0.01% formic acid in water was used. All calibration functions were linear for all probe drugs and metabolites in both matrices over wide analytical ranges. The main advantages of our methods are the use of specific probe drugs available in most countries, the administration of small doses of probe drugs, small

  16. Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2015-02-01

    1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

  17. Biotransformation and detectability of the new psychoactive substances N,N-diallyltryptamine (DALT) derivatives 5-fluoro-DALT, 7-methyl-DALT, and 5,6-methylenedioxy-DALT in urine using GC-MS, LC-MS(n), and LC-HR-MS/MS.

    PubMed

    Michely, Julian A; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

    2017-02-01

    Derivatives of N,N-diallyltryptamine (DALT) can be classified as new psychoactive substances. Biotransformation and detectability of 5-fluoro-DALT (5-F-DALT), 7-methyl-DALT (7-Me-DALT), and 5,6-methylenedioxy-DALT (5,6-MD-DALT) are described here. Their metabolites detected in rat urine and pooled human liver microsomes were identified by liquid chromatography (LC)-high resolution (HR)-tandem mass spectrometry (MS/MS). In addition, the human cytochrome-P450 (CYP) isoenzymes involved in the main metabolic steps were identified and detectability tested in urine by the authors' urine screening approaches using GC-MS, LC-MS(n), or LC-HR-MS/MS. Aromatic and aliphatic hydroxylations, N-dealkylation, N-oxidation, and combinations could be proposed for all compounds as main pathways. Carboxylation after initial hydroxylation of the methyl group could also be detected for 7-Me-DALT and O-demethylenation was observed for 5,6-MD-DALT. All phase I metabolites were extensively glucuronidated or sulfated. Initial phase I reactions were catalyzed by CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5. Rat urine samples were analyzed following two different low-dose administrations. GC-MS was not able to monitor consumption reliably, but all three compounds are predicted to be detectable in cases of overdose. The LC-MS(n) and LC-HR-MS/MS approaches were suitable for detecting an intake of all three compounds mainly via their metabolites. However, after the lowest dose, a reliable monitoring could only be achieved for 5-F-DALT via LC-MS(n) and LC-HR-MS/MS and for 7-Me-DALT via LC-HR-MS/MS. The most abundant targets in both LC-MS screenings were one of two hydroxy-aryl metabolites and both corresponding glucuronides for 5-F-DALT, one N-deallyl hydroxy-aryl, the carboxy, and one dihydroxy-aryl metabolite for 7-Me-DALT, and the demethylenyl metabolite, its oxo metabolite, and glucuronide for 5,6-MD-DALT.

  18. Inhibition selectivity of grapefruit juice components on human cytochromes P450.

    PubMed

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

    2000-06-15

    Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19

  19. Cytochrome P450 expression and activities in human tongue cells and their modulation by green tea extract

    SciTech Connect

    Yang, S.-P.; Raner, Gregory M. . E-mail: gmraner@uncg.edu

    2005-01-15

    The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3. The results were negative for CYP2A6, 2B6/7, 3A3/4, and 3A7. Both cell lines displayed identical expression and induction profiles for all of the isoforms examined in this study except 3A7 and 2B6/7, which were produced constitutively in HepG2 but not Cal-27 cells. CYP1A1 and 1A2 were both induced by treatment with {beta}-napthoflavone as indicated by RT-PCR and Western blotting, while CYP2C mRNA was upregulated by all-trans retinoic acid and farnesol. RT-PCR and Western blot analysis showed that the expressions of CYP1A1 and 1A2 were induced by green tea extract (GTE), which also caused an increase in mRNA for CYP2E1, CYP2D6, and CYP2C isoforms. The four tea catechins, EGC, EC, EGCG and ECG, applied to either HepG2 or Cal-27 cells at the concentration found in GTE failed to induce CYP1A1 or CYP1A2, as determined by RT-PCR. Of the isoforms that were apparently induced by GTE, only 7-ethoxycoumarin deethylase (ECOD) activity could be detected in CAL 27 or HepG2 cells. Interestingly, mRNA and protein for CYP1A1 and CYP1A2 were detected in both cell lines, and although protein and mRNA levels of CYP1A1 and CYP1A2 were increased by GTE, the observed ECOD activity in both cell lines was decreased.

  20. A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism

    PubMed Central

    Loukola, Anu; Buchwald, Jadwiga; Gupta, Richa; Palviainen, Teemu; Hällfors, Jenni; Tikkanen, Emmi; Korhonen, Tellervo; Ollikainen, Miina; Sarin, Antti-Pekka; Ripatti, Samuli; Lehtimäki, Terho; Raitakari, Olli; Salomaa, Veikko; Rose, Richard J.; Tyndale, Rachel F.; Kaprio, Jaakko

    2015-01-01

    Individuals with fast nicotine metabolism typically smoke more and thus have a greater risk for smoking-induced diseases. Further, the efficacy of smoking cessation pharmacotherapy is dependent on the rate of nicotine metabolism. Our objective was to use nicotine metabolite ratio (NMR), an established biomarker of nicotine metabolism rate, in a genome-wide association study (GWAS) to identify novel genetic variants influencing nicotine metabolism. A heritability estimate of 0.81 (95% CI 0.70–0.88) was obtained for NMR using monozygotic and dizygotic twins of the FinnTwin cohort. We performed a GWAS in cotinine-verified current smokers of three Finnish cohorts (FinnTwin, Young Finns Study, FINRISK2007), followed by a meta-analysis of 1518 subjects, and annotated the genome-wide significant SNPs with methylation quantitative loci (meQTL) analyses. We detected association on 19q13 with 719 SNPs exceeding genome-wide significance within a 4.2 Mb region. The strongest evidence for association emerged for CYP2A6 (min p = 5.77E-86, in intron 4), the main metabolic enzyme for nicotine. Other interesting genes with genome-wide significant signals included CYP2B6, CYP2A7, EGLN2, and NUMBL. Conditional analyses revealed three independent signals on 19q13, all located within or in the immediate vicinity of CYP2A6. A genetic risk score constructed using the independent signals showed association with smoking quantity (p = 0.0019) in two independent Finnish samples. Our meQTL results showed that methylation values of 16 CpG sites within the region are affected by genotypes of the genome-wide significant SNPs, and according to causal inference test, for some of the SNPs the effect on NMR is mediated through methylation. To our knowledge, this is the first GWAS on NMR. Our results enclose three independent novel signals on 19q13.2. The detected CYP2A6 variants explain a strikingly large fraction of variance (up to 31%) in NMR in these study samples. Further, we provide evidence

  1. A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism.

    PubMed

    Loukola, Anu; Buchwald, Jadwiga; Gupta, Richa; Palviainen, Teemu; Hällfors, Jenni; Tikkanen, Emmi; Korhonen, Tellervo; Ollikainen, Miina; Sarin, Antti-Pekka; Ripatti, Samuli; Lehtimäki, Terho; Raitakari, Olli; Salomaa, Veikko; Rose, Richard J; Tyndale, Rachel F; Kaprio, Jaakko

    2015-01-01

    Individuals with fast nicotine metabolism typically smoke more and thus have a greater risk for smoking-induced diseases. Further, the efficacy of smoking cessation pharmacotherapy is dependent on the rate of nicotine metabolism. Our objective was to use nicotine metabolite ratio (NMR), an established biomarker of nicotine metabolism rate, in a genome-wide association study (GWAS) to identify novel genetic variants influencing nicotine metabolism. A heritability estimate of 0.81 (95% CI 0.70-0.88) was obtained for NMR using monozygotic and dizygotic twins of the FinnTwin cohort. We performed a GWAS in cotinine-verified current smokers of three Finnish cohorts (FinnTwin, Young Finns Study, FINRISK2007), followed by a meta-analysis of 1518 subjects, and annotated the genome-wide significant SNPs with methylation quantitative loci (meQTL) analyses. We detected association on 19q13 with 719 SNPs exceeding genome-wide significance within a 4.2 Mb region. The strongest evidence for association emerged for CYP2A6 (min p = 5.77E-86, in intron 4), the main metabolic enzyme for nicotine. Other interesting genes with genome-wide significant signals included CYP2B6, CYP2A7, EGLN2, and NUMBL. Conditional analyses revealed three independent signals on 19q13, all located within or in the immediate vicinity of CYP2A6. A genetic risk score constructed using the independent signals showed association with smoking quantity (p = 0.0019) in two independent Finnish samples. Our meQTL results showed that methylation values of 16 CpG sites within the region are affected by genotypes of the genome-wide significant SNPs, and according to causal inference test, for some of the SNPs the effect on NMR is mediated through methylation. To our knowledge, this is the first GWAS on NMR. Our results enclose three independent novel signals on 19q13.2. The detected CYP2A6 variants explain a strikingly large fraction of variance (up to 31%) in NMR in these study samples. Further, we provide evidence

  2. Production and immunohistochemical characterization of monoclonal antibodies against ovarian antigen of the common tree shrew (Tupaia glis).

    PubMed

    Kimura, J; Mukawa, H; Endo, H; Rerkamnuaychoke, W; Kurohmaru, M; Yamada, J; Tsukise, A; Nishida, T

    1998-02-01

    To study the ovarian function of the common tree shrew (Tupaia glis), monoclonal antibodies (MAb) against its ovarian antigen were produced. Several positive hybridomas were cloned and MAb-T2C9 (IgG) was examined for reactivity. Strong immunoreactivity of MAb-T2C9 was localized within the cytoplasm of luteal cells and in the theca interna cells of Graafian follicles. MAb-T2C9 reacted also with testicular interstitial cells and adrenocortical cells, except those of the zona glomerulosa. Similar reactions were seen in the goat and sheep. Western blotting analysis of the ovine corpus luteum after reaction with MAb-T2C9 revealed a single positive band of approximately 60 kDa. These findings suggest that MAb-T2C9 recognized protein molecules related to steroid synthesis.

  3. Drug-Induced Gingival Overgrowth: The Genetic Dimension

    PubMed Central

    Charles, Noronha Shyam Curtis; Chavan, Rahul; Moon, Ninad Joshirao; Nalla, Srinivas; Mali, Jaydeepchandra; Prajapati, Anchal

    2014-01-01

    Background: Currently, the etiology of drug-induced gingival overgrowth is not entirely understood but is clearly multifactorial. Phenytoin, one of the common drugs implicated in gingival enlargement, is metabolized mainly by cytochrome P450 (CYP)2C9 and partly by CYP2C19. The CYP2C9 and CYP2C19 genes are polymorphically expressed and most of the variants result in decreased metabolism of the respective substrates. Aims: The present study was undertaken to investigate the influence of the CYP2C9*2 and *3 variant genotypes on phenytoin hydroxylation in subjects diagnosed with epilepsy from South India, thus establishing the genetic polymorphisms leading to its defective hydroxylation process. Materials and Methods: Fifteen epileptic subjects, age 9 to 60 years were included in the study. Among the study subjects, 8 were males and 7 were females. Genomic DNA was extracted from patients’ blood using Phenol-chloroform method and genotyping was done for CYP2C9 using customized TaqMan genotyping assays on a real time thermocycler, by allelic discrimination method. The genetic polymorphisms *1, *2 and *3 on CYP2C9 were selected based on their function and respective allele frequencies in Asian subcontinent among the Asian populations. Results: CYP2C9*1*2 and CYP2C9*3/*3 were identified with equal frequency in the study population. There were seven subjects with CYP2C9*1/*2 genotype (heterozygous mutant), one subject with CYP2C9*1/*1 (wild type) and seven study subjects with CYP2C9*3/*3 (homozygous mutant). Conclusion: The results obtained in the present study will be helpful in the medical prescription purposes of phenytoin, and a more personalized patient approach with its administration can be advocated. PMID:25317394

  4. CYP2E1 hydroxylation of aniline involves negative cooperativity.

    PubMed

    Hartman, Jessica H; Knott, Katie; Miller, Grover P

    2014-02-01

    CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n=0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K(s)=30 μM) followed by a second weaker binding event (K(ss)=1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis-Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.

  5. A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

    PubMed

    Qin, Chong-Zhen; Ren, Xian; Tan, Zhi-Rong; Chen, Yao; Yin, Ji-Ye; Yu, Jing; Qu, Jian; Zhou, Hong-Hao; Liu, Zhao-Qian

    2014-02-01

    A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD < 6.20%) and intra-day (RSD < 6.13%) precision. Also, the incubation extracts of the sample were stable at room temperature (20 °C) for 48 h and for 96 h in the autosampler (4 °C). The presented method is the first HPLC-MS/MS method of this combination for simultaneous detection of the five metabolites 7-hydroxycoumarin, 4-hydroxytolbutamide, 4'-hydroxymephenytoin, α-hydroxymetoprolol and 1-hydroxymidazolam in a single-run process. It is possible that the high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities.

  6. Xenobiotic metabolizing genes, meat-related exposures, and risk of advanced colorectal adenoma

    PubMed Central

    Ferrucci, Leah M.; Cross, Amanda J.; Gunter, Marc J.; Ahn, Jiyoung; Mayne, Susan T.; Ma, Xiaomei; Chanock, Stephen J.; Yeager, Meredith; Graubard, Barry I.; Berndt, Sonja I.; Huang, Wen-Yi; Hayes, Richard B.; Sinha, Rashmi

    2011-01-01

    Objectives Carcinogenic action of meat-related exposures, such as heterocyclic amines (HCAs), polycyclic aromatic hydrocarbons (PAHs), and N-nitroso compounds (NOCs), might explain positive associations between red and processed meat and colorectal neoplasia. Single nucleotide polymorphisms in xenobiotic metabolizing enzyme (XME) genes could alter activation/detoxfication of these compounds. Methods We evaluated interactions between several XME genes (CYP1A1, CYP1B1, CYP2A6, CYP2C9, CYP2E1, CYP3A4, EPHX1, GSTM1, GSTM2, GSTT1, NAT1, NAT2, NQO1, SULT1A1, and SULT1A2) and meat-related exposures using a pathway-based approach in 720 advanced colorectal adenoma cases of the distal colon or rectum and 746 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Using meat-related databases, we estimated intake of the HCAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), the PAH, benzo[a]pyrene (B[a]P), and nitrate/nitrite, as NOC precursors. Results There were possible interactions between PhIP and CYP1B1 (Pinteraction=0.019) and NQO1 (Pinteraction=0.007), B[a]P and CYP1B1 (Pinteraction=0.005) and CYP3A4 (Pinteraction=0.021), and nitrate/nitrite and CYP1A1 (Pinteraction=0.022) in relation to colorectal adenoma. However, none of these interactions were statistically significant using a false discovery rate threshold of 0.20. Conclusions Common variants in XME genes may modify the association of HCAs, PAHs, and nitrate/nitrite with advanced colorectal adenoma, but investigation in other populations is required, especially within consortia. PMID:20436251

  7. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites.

    PubMed

    Bu, H Z; Magis, L; Knuth, K; Teitelbaum, P

    2000-01-01

    A highly efficient direct injection/on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. This method employed an extremely short C(18) cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2. 0-2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on-line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode).

  8. Characterization of in vitro biotransformation of new, orally active, direct thrombin inhibitor ximelagatran, an amidoxime and ester prodrug.

    PubMed

    Clement, Bernd; Lopian, Katrin

    2003-05-01

    N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b(5), NADH-cytochrome b(5) reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b(5) and b(5) reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.