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  1. 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

  2. CYP2C subfamily, primarily CYP2C9, catalyses the enantioselective demethylation of the endocrine disruptor pesticide methoxychlor in human liver microsomes: use of inhibitory monoclonal antibodies in P450 identification.

    PubMed

    Hu, Y; Krausz, K; Gelboin, H V; Kupfer, D

    2004-02-01

    1. The endocrine disruptor pesticide methoxychlor undergoes O-demethylation by mammalian liver microsomes forming chiral mono-phenolic (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane, i.e. mono-OH-M) and achiral bis-phenolic oestrogenic metabolites. Human liver microsomes (HLM) generated primarily the S-mono-OH-M. 2. Inhibitory monoclonal antibodies (MAb) identified those P450s catalysing the enantioselective O-demethylation of methoxychlor. In HLM, O-demethylation was inhibited by MAb anti-2C9 (30-40%), diminishing the per cent of S-mono-OH-M from about 80 to 55-60%. MAb anti-CYP1A2, 2A6, 2B6, 2C8, 2C19, 2D6 and 3A4 did not affect the demethylation rate in HLM. Nevertheless, MAb anti-CYP1A2 decreased the formation of R-mono-OH-M from 21-23 to 10-17%, indicating that CYP1A2 exhibits a role in generating the R-enantiomer. 3. Among cDNA-expressed human P450s (supersomes), CYP2C19 was the most active in demethylation, but in HLM, CYP2C19 appeared inactive (no inhibition by MAb anti-CYP2C19). There was a substantial difference in the per cent inhibition of demethylation by MAb anti-CYP2C9 and anti-rat CYP2C (MAb inhibiting all human CYP2C forms) and in altering the enantioselectivity, suggesting that demethylation by combined CYP2C8, 2C18 and 2C19 was significant (20-30%). 4. Polymorphism of methoxychlor demethylation was examined with supersomes and HLM-expressing CYP2C9 allelic variants. CYP2C9*1 and 2C9*2 were highly active; however, CYP2C9*3 appeared inactive.

  3. Biotransformation of BDE-47 to Potentially Toxic Metabolites Is Predominantly Mediated by Human CYP2B6

    PubMed Central

    Feo, Maria Luisa; Gross, Michael S.; McGarrigle, Barbara P.; Eljarrat, Ethel; Barceló, Damià; Olson, James R.

    2012-01-01

    Background: Previous studies have indicated that cytochrome P450s (CYPs) are involved in the metabolism of polybrominated diphenyl ether (PBDE) flame retardants in humans, resulting in the formation of hydroxylated PBDEs (OH-PBDEs) that are potentially more toxic than the parent PBDEs. However, the specific enzymes responsible for the formation of OH-PBDEs are unknown. Objectives: The purposes of this study were to characterize the in vitro metabolism of 2,2´,4,4´-tetrabromodiphenyl ether (BDE-47) by human liver microsomes (HLM) and recombinant human CYPs, and to identify the CYP(s) that are active in the oxidative metabolism of BDE-47. Methods: Recombinant human CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) were incubated with BDE-47 (20 µM), and the metabolites were measured and characterized using gas chromatography with tandem mass spectrometry (GC-MS/MS). For kinetic studies, CYP2B6 and pooled human liver microsomes (HLMs) were incubated with BDE-47 (0–60 µM). Results: CYP2B6 was the predominant CYP capable of forming six OH-BDEs, including 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4´-OH-BDE-49, and a metabolite tentatively identified as 2´-OH-BDE-66. On the basis of full-scan GC-MS analysis, we hypothesized the formation of two other metabolites: di-OH-tetra-BDE and di-OH-tetrabrominated dioxin. In kinetic studies of BDE-47 metabolism by CYP2B6 and pooled HLMs, we found Km values ranging from 3.8 to 6.4 µM and 7.0 to 11.4 µM, respectively, indicating the high affinity toward the formation of OH-BDEs. Conclusion: Our findings support a predominant role of CYP2B6 in the metabolism of BDE-47 to potentially toxic metabolites, including a hypothesized di-OH-tetrabrominated dioxin metabolite. These results will assist future epidemiological studies investigating the potential of PBDEs and their metabolites to produce neurobehavioral/neurodevelopmental disorders. PMID:23249762

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

    PubMed

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

    2015-07-01

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

  5. 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.

  6. The effect of the cytochrome P450 CYP2C8 polymorphism on the disposition of (R)-ibuprofen enantiomer in healthy subjects

    PubMed Central

    Martínez, Carmen; García-Martín, Elena; Blanco, Gerardo; Gamito, Francisco J G; Ladero, José M; Agúndez, José A G

    2005-01-01

    Aims To study the effect of CYP2C8*3, the most common CYP2C8 variant allele on the dis-position of (R)-ibuprofen and the association of CYP2C8*3 with variant CYP2C9 alleles. Methods Three hundred and fifty-five randomly selected Spanish Caucasians were screened for the common CYP2C8 and CYP2C9 mutations. The pharmacokinetics of (R)-ibuprofen were studied in 25 individuals grouped into different CYP2C8 genotypes. Results The allele frequency of CYP2C8*3 (0.17) was found to be higher than that reported for other Caucasian populations (P = 0.0001). The frequencies of CYP2C9*2 and CYP2C9*3 were 0.19 (0.16–0.21) and 0.10 (0.08–0.12), respectively. An association between CYP2C8*3 and CYP2C9*2 alleles was observed, occurring together at a frequency 2.4-fold higher than expected for a random association of alleles (P = 0.0001). The presence of the CYP2C8*3 allele was found to influence the pharmacokinetics of (R)-ibuprofen in a gene–dose effect manner. Thus, after administration of 400 mg ibuprofen, the plasma half-life (95% confidence intervals) for individuals with genotypes CYP2C8*1/*1, CYP2C8*1/*3 and CYP2C8*3/*3, was 2.0 h (1.8–2.2), 4.2 h (1.9–6.5; P < 0.05) and 9.0 h (7.8–10.2; P < 0.002), respectively. A statistically significant trend with respect to the number of variant CYP2C8*3 alleles was also observed for the area under the concentration-time curve (P < 0.025), and drug clearance (P < 0.03). Conclusion Polymorphism of the CYP2C8 gene was found to be common, with nearly 30% of the population studied carrying the variant CYP2C8*3 allele. The presence of the latter caused a significant effect on the disposition of (R)-ibuprofen. This suggests that a substantial proportion of Caucasian subjects may show alterations in the disposition of drugs that are CYP2C8 substrates. PMID:15606441

  7. Allele frequency distribution of CYP2C9 2 and CYP2C9 3 polymorphisms in six Mexican populations.

    PubMed

    Castelán-Martínez, Osvaldo D; Hoyo-Vadillo, Carlos; Sandoval-García, Emmanuel; Sandoval-Ramírez, Lucila; González-Ibarra, Miriam; Solano-Solano, Gloria; Gómez-Díaz, Rita A; Parra, Esteban J; Cruz, Miguel; Valladares-Salgado, Adán

    2013-07-10

    Allele frequency differences of functional CYP2C9 polymorphisms are responsible for some of the variation in drug response observed in human populations. The most relevant CYP2C9 functional variants are CYP2C9*2 (rs1799853) and CYP2C9 3 (rs1057910). These polymorphisms show variation in allele frequencies among different population groups. The present study aimed to analyze these polymorphisms in 947 Mexican-Mestizo from Mexico City and 483 individuals from five indigenous Mexican populations: Nahua, Teenek, Tarahumara, Purepecha and Huichol. The CYP2C9*2 allele frequencies in the Mestizo, Nahua and Teenek populations were 0.051, 0.007 and 0.005, respectively. As for CYP2C9 3, the allelic frequencies in the Mestizo, Nahua and Teenek populations were 0.04, 0.005 and 0.005, respectively. The CYP2C9 2 and CYP2C9 3 alleles were not observed in the Tarahumara, Purepecha and Huichol populations. These findings are in agreement with previous studies reporting very low allele frequencies for these polymorphisms in American Indigenous populations.

  8. Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6.

    PubMed

    Sridar, Chitra; Kent, Ute M; Notley, Lisa M; Gillam, Elizabeth M J; Hollenberg, Paul F

    2002-06-01

    Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K(I) of 0.9 microM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity. PMID:12023523

  9. Analysis of the Functional Polymorphism in the Cytochrome P450 CYP2C8 Gene rs11572080 with Regard to Colorectal Cancer Risk

    PubMed Central

    Ladero, José M.; Agúndez, José A. G.; Martínez, Carmen; Amo, Gemma; Ayuso, Pedro; García-Martín, Elena

    2012-01-01

    In addition to the known effects on drug metabolism and response, functional polymorphisms of genes coding for xenobiotic-metabolizing enzymes (XME) play a role in cancer. Genes coding for XME act as low-penetrance genes and confer modest but consistent and significant risks for a variety of cancers related to the interaction of environmental and genetic factors. Consistent evidence supports a role for polymorphisms of the cytochrome P450 CYP2C9 gene as a protecting factor for colorectal cancer susceptibility. It has been shown that CYP2C8 and CYP2C9 overlap in substrate specificity. Because CYP2C8 has the common functional polymorphisms rs11572080 and rs10509681 (CYP2C8*3), it could be speculated that part of the findings attributed to CYP2C9 polymorphisms may actually be related to the presence of polymorphisms in the CYP2C8 gene. Nevertheless, little attention has been paid to the role of the CYP2C8 polymorphism in colorectal cancer. We analyzed the influence of the CYP2C8*3 allele in the risk of developing colorectal cancer in genomic DNA from 153 individuals suffering colorectal cancer and from 298 age- and gender-matched control subjects. Our findings do not support any effect of the CYP2C8*3 allele (OR for carriers of functional CYP2C8 alleles = 0.50 (95% CI = 0.16–1.59; p = 0.233). The absence of a relative risk related to CYP2C8*3 did not vary depending on the tumor site. We conclude that the risk of developing colorectal cancer does not seem to be related to the commonest functional genetic variation in the CYP2C8 gene. PMID:23420707

  10. Effects of Angiotensin II Receptor Blockers on Metabolism of Arachidonic Acid via CYP2C8.

    PubMed

    Senda, Asuna; Mukai, Yuji; Toda, Takaki; Hayakawa, Toru; Yamashita, Miki; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2015-01-01

    Arachidonic acid (AA) is metabolized to epoxyeicosatrienoic acids (EETs) via cytochrome enzymes such as CYP 2C9, 2C8 and 2J2. EETs play a role in cardioprotection and regulation of blood pressure. Recently, adverse reactions such as sudden heart attack and fatal myocardial infarction were reported among patients taking angiotensin II receptor blockers (ARBs). As some ARBs have affinity for these CYP enzymes, metabolic inhibition of AA by ARBs is a possible cause for the increase in cardiovascular events. In this study, we quantitatively investigated the inhibitory effects of ARBs on the formation of EETs and further metabolites, dihydroxyeicosatrienoic acids (DHETs), from AA via CYP2C8. In incubations with recombinant CYP2C8 in vitro, the inhibitory effects were compared by measuring EETs and DHETs by HPLC-MS/MS. Inhibition of AA metabolism by ARBs was detected in a concentration-dependent manner with IC50 values of losartan (42.7 µM), telmisartan (49.5 µM), irbesartan (55.6 µM), olmesartan (66.2 µM), candesartan (108 µM), and valsartan (279 µM). Losartan, telmisartan and irbesartan, which reportedly accumulate in the liver and kidneys, have stronger inhibitory effects than other ARBs. The lower concentration of EETs leads to less protective action on the cardiovascular system and a higher incidence of adverse effects such as sudden heart attack and myocardial infarction in patients taking ARBs. PMID:26632190

  11. High-throughput screening assays for the assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 metabolism using fluorogenic Vivid substrates.

    PubMed

    Marks, Bryan D; Thompson, David V; Goossens, Tony A; Trubetskoy, Olga V

    2004-08-01

    CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

  12. Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance

    PubMed Central

    Zanger, Ulrich M.; Klein, Kathrin

    2013-01-01

    Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. CYP2B6 is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is CYP2B6*6 [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, CYP2B6*18 [I328T], occurs predominantly in Africans (4–12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The CYP2B6 polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems. PMID

  13. Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

    PubMed

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

    2016-07-01

    Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26581561

  14. Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

    PubMed

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

    2016-07-01

    Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd.

  15. 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. PMID:27417918

  16. Effect of CYP2C9 genetic polymorphism on the metabolism of flurbiprofen in vitro.

    PubMed

    Wang, Li; Bao, Shi-Hui; Pan, Pei-Pei; Xia, Meng-Ming; Chen, Meng-Chun; Liang, Bing-Qing; Dai, Da-Peng; Cai, Jian-Ping; Hu, Guo-Xin

    2015-01-01

    CYP2C9 is an important member of the cytochrome P450 enzyme superfamily, and 57 cytochrome P450 2C9 alleles have been previously reported. To examine the enzymatic activity of the CYP2C9 alleles, kinetic parameters for 4'-hydroxyflurbiprofen were determined using recombinant human P450s CYP2C9 microsomes from insect cells Sf21 carrying wild-type CYP2C9*1 and other variants. The results showed that the enzyme activity of most of the variants decreased comparing with the wild type as the previous studies reported, while the enzyme activity of some of them increased, which were not in accordance with the previous researches. Of the 36 tested CYP2C9 allelic isoforms, two variants (CYP2C9*53 and CYP2C9*56) showed a higher intrinsic clearance value than the wild-type protein, especially for CYP2C9*56, exhibited much higher intrinsic clearance (197.3%) relative to wild-type CYP2C9*1, while the remaining 33 CYP2C9 allelic isoforms exhibited significantly decreased clearance values (from 0.6 to 83.8%) compared to CYP2C9*1. This study provided the most comprehensive data on the enzymatic activities of all reported CYP2C9 variants in the Chinese population with regard to the commonly used non-steroidal anti-inflammatory drug, flurbiprofen (FP). The results indicated that most of the tested rare alleles decreased the catalytic activity of CYP2C9 variants toward FP hydroxylation in vitro. This is the first report of all these rare alleles for FP metabolism providing fundamental data for further clinical studies on CYP2C9 alleles for FP metabolism in vivo.

  17. Cytochrome P450 2C8 pharmacogenetics: a review of clinical studies

    PubMed Central

    Daily, Elizabeth B; Aquilante, Christina L

    2009-01-01

    Cytochrome P450 (CYP) 2C8 is responsible for the oxidative metabolism of many clinically available drugs from a diverse number of drug classes (e.g., thiazolidinediones, meglitinides, NSAIDs, antimalarials and chemotherapeutic taxanes). The CYP2C8 enzyme is encoded by the CYP2C8 gene, and several common nonsynonymous polymorphisms (e.g., CYP2C8*2 and CYP2C8*3) exist in this gene. The CYP2C8*2 and *3 alleles have been associated in vitro with decreased metabolism of paclitaxel and arachidonic acid. Recently, the influence of CYP2C8 polymorphisms on substrate disposition in humans has been investigated in a number of clinical pharmacogenetic studies. Contrary to in vitro data, clinical data suggest that the CYP2C8*3 allele is associated with increased metabolism of the CYP2C8 substrates, rosiglitazone, pioglitazone and repaglinide. However, the CYP2C8*3 allele has not been associated with paclitaxel pharmacokinetics in most clinical studies. Furthermore, clinical data regarding the impact of the CYP2C8*3 allele on the disposition of NSAIDs are conflicting and no definitive conclusions can be made at this time. The purpose of this review is to highlight these clinical studies that have investigated the association between CYP2C8 polymorphisms and CYP2C8 substrate pharmacokinetics and/or pharmacodynamics in humans. In this review, CYP2C8 clinical pharmacogenetic data are provided by drug class, followed by a discussion of the future of CYP2C8 clinical pharmacogenetic research. PMID:19761371

  18. Frequency of CYP2C9 alleles in Koreans and their effects on losartan pharmacokinetics

    PubMed Central

    Bae, Jung-woo; Choi, Chang-ik; Kim, Mi-jeong; Oh, Da-hee; Keum, Seul-ki; Park, Jung-in; Kim, Bo-hye; Bang, Hye-kyoung; Oh, Sung-gon; Kang, Byung-sung; Park, Hyun-joo; Kim, Hae-deun; Ha, Ji-hey; Shin, Hee-jung; Kim, Young-hoon; Na, Han-sung; Chung, Myeon-woo; Jang, Choon-gon; Lee, Seok-yong

    2011-01-01

    Aim: CYP2C9 enzyme metabolizes numerous clinically important drugs. The aim of this study is to investigate the frequencies of CYP2C9 genotypes and the effects of selected alleles on losartan pharmacokinetics in a large sample of the Korean population. Methods: The CYP2C9 gene was genotyped in 1796 healthy Korean subjects. CYP2C9 alleles (CYP2C9*1, *2, *3 and *13 alleles) were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and direct sequencing assay. The enzymatic activity of each CYP2C9 genotype was evaluated using losartan as the substrate. Results: The frequencies of CYP2C9*1, *3 and *13 allele were 0.952 (95% confidence interval 0.945–0.959), 0.044 (95% CI 0.037–0.051) and 0.005 (95% CI 0.003–0.007), respectively. The frequencies of the CYP2C9*1/*1, *1/*3, *1/*13 and *3/*3 genotypes were 0.904 (95% CI 0.890–0.918), 0.085 (95% CI 0.072–0.098), 0.009 (95% CI 0.005–0.013) and 0.001 (95% CI 0.000–0.002), respectively. In the pharmacokinetics studies, the AUC0–∞ of losartan in CYP2C9*3/*3 subjects was 1.42-fold larger than that in CYP2C9*1/*1 subjects, and the AUC0–∞ of E-3174, a more active metabolite of losartan, in CYP2C9*3/*3 subjects was only 12% of that in CYP2C9*1/*1 subjects. Conclusion: The results confirmed the frequencies of CYP2C9 genotypes in a large cohort of Koreans, and detected the CYP2C9*3/*3 genotype. CYP2C9*3/*3 subjects metabolized much less losartan into E-3174 than CYP2C9*1/*1 subjects. PMID:21841812

  19. Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions.

    PubMed

    Backman, Janne T; Filppula, Anne M; Niemi, Mikko; Neuvonen, Pertti J

    2016-01-01

    During the last 10-15 years, cytochrome P450 (CYP) 2C8 has emerged as an important drug-metabolizing enzyme. CYP2C8 is highly expressed in human liver and is known to metabolize more than 100 drugs. CYP2C8 substrate drugs include amodiaquine, cerivastatin, dasabuvir, enzalutamide, imatinib, loperamide, montelukast, paclitaxel, pioglitazone, repaglinide, and rosiglitazone, and the number is increasing. Similarly, many drugs have been identified as CYP2C8 inhibitors or inducers. In vivo, already a small dose of gemfibrozil, i.e., 10% of its therapeutic dose, is a strong, irreversible inhibitor of CYP2C8. Interestingly, recent findings indicate that the acyl-β-glucuronides of gemfibrozil and clopidogrel cause metabolism-dependent inactivation of CYP2C8, leading to a strong potential for drug interactions. Also several other glucuronide metabolites interact with CYP2C8 as substrates or inhibitors, suggesting that an interplay between CYP2C8 and glucuronides is common. Lack of fully selective and safe probe substrates, inhibitors, and inducers challenges execution and interpretation of drug-drug interaction studies in humans. Apart from drug-drug interactions, some CYP2C8 genetic variants are associated with altered CYP2C8 activity and exhibit significant interethnic frequency differences. Herein, we review the current knowledge on substrates, inhibitors, inducers, and pharmacogenetics of CYP2C8, as well as its role in clinically relevant drug interactions. In addition, implications for selection of CYP2C8 marker and perpetrator drugs to investigate CYP2C8-mediated drug metabolism and interactions in preclinical and clinical studies are discussed. PMID:26721703

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

    PubMed

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

    2016-02-01

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

  1. Mechanism of CYP2C9 inhibition by flavones and flavonols.

    PubMed

    Si, Dayong; Wang, Ying; Zhou, Yi-Han; Guo, Yingjie; Wang, Juan; Zhou, Hui; Li, Ze-Sheng; Fawcett, J Paul

    2009-03-01

    This article describes an in vitro investigation of the inhibition of cytochrome P450 (P450) 2C9 by a series of flavonoids made up of flavones (flavone, 6-hydroxyflavone, 7-hydroxyflavone, chrysin, baicalein, apigenin, luteolin, scutellarein, and wogonin) and flavonols (galangin, fisetin, kaempferol, morin, and quercetin). With the exception of flavone, all flavonoids were shown to inhibit CYP2C9-mediated diclofenac 4'-hydroxylation in the CYP2C9 RECO system, with K(i) value 2C9, whereas the other flavonoids were competitive inhibitors. Computer docking simulation and constructed mutants substituted at residue 100 of CYP2C9.1 indicate that the noncompetitive binding site of 6-hydroxyflavone lies beside Phe100, similar to the reported allosteric binding site of warfarin. The other flavonoids exert competitive inhibition through interaction with the substrate binding site of CYP2C9 accessed by flurbiprofen. These results suggest flavonoids can participate in interactions with drugs that act as substrates for CYP2C9 and provide a possible molecular basis for understanding cooperativity in human P450-mediated drug-drug interactions. PMID:19074529

  2. A Case of Intolerance to Warfarin Dosing in an Intermediate Metabolizer of CYP2C9

    PubMed Central

    Lee, Soo-Youn; Kim, June Soo

    2005-01-01

    We report a case of intolerance to warfarin dosing due to impaired drug metabolism in a patient heterozygous for the CYP2C9*3 allele. A 30-year-old woman with an artificial cardiac pacemaker was taking warfarin to prevent thromboembolism. This patient had an extremely elevated international normalized ratio (INR) of prothrombin time (PT) following standard doses of warfarin and experienced difficulties during the induction of anticoagulation. Genotyping for CYP2C9 revealed that this patient was an intermediate metabolizer with genotype CYP2C9*1/*3. This case suggests the clinical usefulness of pharmacogenetic testing for individualized dosage adjustments of warfarin. PMID:16385662

  3. 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.

  4. Impact of CYP2C9 genetic polymorphism on warfarin dose requirements in Pakistani population.

    PubMed

    Siddiqi, Aisha; Khan, Dilshad Ahmad; Khan, Farooq Ahmed; Naveed, Abdul Khaliq

    2010-10-01

    Variations of cytochrome-P450 enzyme system (CYP2CP) are associated with impaired metabolism of warfarin. The objective of our study was to estimate the frequency of genetic and allelic variants of CYP2C9 in Punjabi population of Pakistan and their effects on warfarin dose requirement. One hundred and twenty unrelated Pakistani subjects belong to Punjab province, were randomly included from the registry of National Institute of Heart Disease Rawalpindi, Pakistan. The patients had stable international normalized ratio (INR) of 2 to 3 for last 3 months with warfarin therapy after heart valves replacement. The detection of CYP2C9 variant was done on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Total 120 patients (73 males; 47 females) of mean age of 37 years participated in the study. Nine patients had mutant allele CYP2C9*3 (7.5%), one CYP2C9*2 (0.8%) and 110 patients exhibited wild type CYP2C9*1 (91.7%). The frequency of CYP2C9 genotype was *1/*1 (0.858) ; *1/*3 (0.117) ; 2/*20 (0.08 ) and *3/*3 (0.017) in our study population. A high dose of warfarin (42.2+9.56) mg/week is required for patients with *1/*1 genotype as compared to patients with *2/*2 (17.5+1.9) and *1/*3 (16.6+2.3) allele (p<0.001). Individuals with CYP2C9*3/3* need lowest (8.75±1.76 mg/week) daily warfarin dose. In conclusion, the genetic variations in the CYP2C9 occur in 14% of Punjabi ethnic group in Pakistan. Presence of CYP2C9*2 or *3 variants is an independent predictor of low warfarin dose requirement in our patients. CYP2C9 variants assay may be used in high risk groups for appropriate dose adjustment to avoid complications on long term basis.

  5. Inhibitory effects of fruit juices on cytochrome P450 2C9 activity in vitro.

    PubMed

    Hidaka, Muneaki; Nagata, Masashi; Kawano, Yohei; Sekiya, Hiroshi; Kai, Hirofumi; Yamasaki, Keishi; Okumura, Manabu; Arimori, Kazuhiko

    2008-02-01

    There is limited information on the effect of fruits on human cytochrome P450 (CYP) 2C9 activity. The objective of this study was to determine the effect of fruit juice on CYP2C9-mediated drug metabolism. Nine citrus fruits and eight tropical fruits were chosen. We investigated effects of the fruits on diclofenac 4'-hydroxylation and tolbutamide hydroxylation by human liver microsomes. Among the fruits, pineapple juice showed potent inhibition of CYP2C9 activity. The addition of 25 microl (5.0% v/v) of pineapple juice resulted in almost complete inhibition. Next we examined the inhibitory effect of bromelain, a cysteine protease in pineapple. Bromelain also strongly inhibited CYP2C9 activity. In addition, E-64, a cysteine protease inhibitor, almost entirely blocked inhibition by pineapple juice and bromelain. Thus we found that pineapple juice was a potent inhibitor of CYP2C9, and that the inhibitory effect might be due to the bromelain contained in pineapple.

  6. 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.

  7. Influence of CYP2C8 polymorphisms on the hydroxylation metabolism of paclitaxel, repaglinide and ibuprofen enantiomers in vitro.

    PubMed

    Yu, Lushan; Shi, Da; Ma, Liping; Zhou, Quan; Zeng, Su

    2013-07-01

    CYP2C8 plays an important role in the metabolism of various drugs, such as paclitaxel, repaglinide and ibuprofen. Polymorphisms in the CYP2C8 gene were shown to influence interindividual differences in the pharmacokinetics of paclitaxel, repaglinide and ibuprofen enantiomers. In this study, three CYP2C8 allelic variants (CYP2C8.2, CYP2C8.3 and CYP2C8.4) and wild-type CYP2C8 (CYP2C8.1) were co-expressed for the first time with human cytochrome P450 oxidoreductase (POR) and cytochrome b5 by using a baculovirus-assisted insect cell expression system. Further, the effects of genotype-phenotype correlations of CYP2C8 alleles on the metabolism of paclitaxel, repaglinide and ibuprofen enantiomers were evaluated. The CLint values of CYP2C8.2, CYP2C8.3 and CYP2C8.4 for paclitaxel were 47.7%, 64.3% and 30.2% of that of CYP2C8.1 (p<0.01). The CLint values of CYP2C8.2 and CYP2C8.4 for repaglinide were 77.9% and 80.2% of that of CYP2C8.1 (p<0.05), respectively, while the CLint value of CYP2C8.3 was 1.31-fold higher than that of CYP2C8.1 (p<0.05). The relative CLint values of CYP2C8.2, CYP2C8.3 and CYP2C8.4 were 110.5%, 72.3% and 49.7% of that of CYP2C8.1 and were 124.6%, 83.4% and 47.4% of that of CYP2C8.1 for R-ibuprofen and S-ibuprofen, respectively. Comparing hydroxylation by CYP2C8.1 and CYP2C8.3 resulted in higher and lower intrinsic clearance of repaglinide and ibuprofen enantiomers, respectively. These in vitro findings were consistent with the pharmacokinetics in volunteers who were heterozygous or homozygous carriers of CYP2C8*3. The results of this study provide useful information for predicting CYP2C8 phenotypes and may contribute to individualized drug therapy in the future.

  8. Enzyme source effects on CYP2C9 kinetics and inhibition.

    PubMed

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

    2006-11-01

    When choosing a recombinant cytochrome P450 (P450) enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. Although literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes, Baculosomes, RECO system, and in-house purified, reconstituted enzyme) to determine whether the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher K(m) values, reduced substrate affinity, and lower calculated intrinsic clearance values compared with baculovirus microsomal preparations. Six- to 25-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. In addition, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of 12 inhibitors, decreased inhibition potency was observed across 11 of those inhibitors in the RECO purified, reconstituted enzyme compared with the Supersomes baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of an enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9. PMID:16928789

  9. Cytochrome P450 2C9 gene polymorphism in phenytoin induced gingival enlargement: A case report.

    PubMed

    Babu, S P K Kennedy; Ramesh, V; Samidorai, Agila; Charles, N S C

    2013-07-01

    Gingival enlargement comprises any clinical condition in which an increase in the size of the gingiva is observed. Among the drugs that induce gingival enlargement, the antiepileptic agent phenytoin has been widely related to this condition. The Cytochrome P450(CYP) superfamily is the most commonly involved enzymes in metabolism of drugs. Common coding region CYP variants that affects drug elimination and response has been studied in great detail. Pharmacogenetic influences on drug metabolism have been widely reviewed and gene polymorphism of cytochrome P450 2C9 appeared to be responsible for much of the interindividual variability on drug elimination. Genetic variation in the CYP2C9 gene can affect metabolism, leading to altered phenotypes. Individuals with poor metaboliser alleles of CYP2C9 gene were shown to have a reduced metabolism of phenytoin compared with wild-type alleles. Thus identification of patients genotype prior to anti-epileptic drug administration could potentially prevent higher serum drug concentrations leading to adverse side effects such as gingival enlargement. This case report addresses the influence of CYP2C9 genetic polymorphism on Phenytoin drug metabolism thereby causing gingival enlargement. PMID:24082701

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

    PubMed Central

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

    2011-01-01

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix. PMID:21852944

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

    SciTech Connect

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

    2011-08-11

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

  12. The tyrosine kinase inhibitor nilotinib selectively inhibits CYP2C8 activities in human liver microsomes.

    PubMed

    Kim, Min-Jung; Lee, Jae-Won; Oh, Kyung-Suk; Choi, Chang-Soo; Kim, Kwang Hee; Han, Won Seok; Yoon, Chang-No; Chung, Eun Sook; Kim, Dong-Hyun; Shin, Jae-Gook

    2013-01-01

    The tyrosine kinase inhibitor nilotinib was examined for its inhibition of cytochrome P450s (CYPs) in human liver microsomes and in human CYPs expressed in a baculovirus-insect cell system. Nilotinib demonstrated preferential inhibition of CYP2C8-mediated paclitaxel 6α-hydroxylation, rosiglitazone hydroxylation and amodiaquine N-deethylation in human liver microsomes, with IC₅₀ values of 0.4, 7.5 and 0.7 µM, respectively. The IC₅₀ value of nilotinib for paclitaxel 6α-hydroxylation was 20-fold lower than that of the other five tyrosine-kinase inhibitors tested. Nilotinib appears to display competitive inhibition against paclitaxel 6α-hydroxylation and amodiaquine N-deethylation, with estimated mean Ki values of 0.90 and 0.15 µM in human liver microsomes and 0.10 and 0.61 µM in recombinant human CYP2C8, respectively. These results are consistent with those of molecular docking simulations, where paclitaxel could not access the CYP2C8 catalytic site in the presence of nilotinib, but the binding of midazolam, a substrate of CYP3A4, to the catalytic site of CYP3A4 was not affected by nilotinib. The demonstrated inhibitory activity of nilotinib against CYP2C8 at concentrations less than those observed in patients who received nilotinib therapy is of potential clinical relevance and further in vivo exploration is warranted.

  13. The impact of CYP2C8 polymorphism and grapefruit juice on the pharmacokinetics of repaglinide

    PubMed Central

    Bidstrup, Tanja Busk; Damkier, Per; Olsen, Anette Kristensen; Ekblom, Marianne; Karlsson, Anders; Brøsen, Kim

    2006-01-01

    Aims The primary aim of the study was to investigate the possible effect of the CYP2C8 3 allele and of grapefruit juice on the pharmacokinetics of repaglinide. Furthermore, the impact of a single dose of grapefruit juice on the pharmacokinetics of repaglinide in relation to dose. Methods Thirty-six healthy male subjects, genotyped for CYP2C8 3 (11 genotyped as CYP2C8 1/ 3, one as CYP2C8 3/ 3 and 24 as CYP2C8 1/ 1), participated in a randomized, cross-over trial. In the two phases, the subjects drank 300 mL water or 300 mL grapefruit juice, in randomized order, 2 h before administration of a single dose of either 0.25 mg or 2 mg repaglinide. Results Neither the mean AUC0−∞ (geometric mean ratio: 1.01; 95% CI: 0.93–1.1, P = 0.88) nor the mean Cmax (geometric mean ratio: 1.05; 95% CI: 0.94–1.2, P = 0.35) of repaglinide were statistically significantly different in the group carrying the CYP2C8 3 mutant allele compared with wild-types. Grapefruit juice caused a 19% decrease in the geometric mean ratio of the 3-hydroxyquinidine to quinidine ratio (difference: 0.81; 95% CI: 0.75–0.87, P < 0.0001), which was used as an index of CYP3A4 activity, and an increase in the mean AUC0−∞ of repaglinide (geometric mean ratio: 1.13; 95% CI: 1.04–1.2, P = 0.0048), but had no statistically significant effect on the t1/2. There was no statistically significant difference in blood glucose concentration in subjects who had or had not ingested grapefruit juice. The effect was more pronounced at the low dose of repaglinide (0.25 mg) than at the therapeutic dose of 2 mg. Conclusions The pharmacokinetics of repaglinide in subjects carrying the CYP2C8*3 mutant allele did not differ significantly from those in the wild-types. Grapefruit juice increased the bioavailability of repaglinide, suggesting significant intestinal elimination of the drug which was assumed to be primarily mediated by CYP3A4 in the gut. PMID:16390351

  14. A potential therapeutic effect of CYP2C8 overexpression on anti-TNF-α activity

    PubMed Central

    LIU, WANJUN; WANG, BEI; DING, HU; WANG, DAO WEN; ZENG, HESONG

    2014-01-01

    Epoxyeicosatrienoic acids (EETs) are generated from arachidonic acid catalysed by cytochrome P450 (CYP) epoxygenases. In addition to regulating vascular tone EETs may alleviate inflammation and ROS. The present study was conducted to determine whether CYP2C8 gene overexpression was able to increase the level of EETs, and subsequently prevent TNF-α induced inflammation and reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) and macrophages. Peroxisome proliferator-activated receptor γ (PPARγ) activation, nuclear factor-κB (NF-κB) activation, endothelial nitric oxide synthase (eNOS) activation, gp-91 activation, and inflammatory cytokine expression were detected by western blot analysis or enzyme-linked immunosorbent assay. Intracellular reactive oxygen species (ROS) was measured by flow cytometry, while the migration of vascular smooth muscle cells (VSMCs) was detected by Transwell assay. pCMV-mediated CYP2C8 overexpression and its metabolites, EETs, markedly suppressed TNF-α induced inflammatory cytokines IL-6 and MCP-1 expression via the activation of NF-κB and degradation of IκBα. Moreover, pretreatment with 11,12-EET significantly blocked TNF-α-induced ROS production. CYP2C8-derived EETs also effectively alleviated the migration of VSMCs and improved the function of endothelial cells through the upregulation of eNOS, which was significantly decreased under the stimulation of TNF-α. Furthermore, these protective effects observed were mediated by PPARγ activation. To the best of our knowledge, the results of the present study demonstrated for the first time that CYP2C8-derived EETs exerted antivascular inflammatory and anti-oxidative effects, at least in part, through the activation of PPARγ. Thus, the CYP2C8 gene may be useful in the prevention and treatment of vascular inflammatory diseases. PMID:25017038

  15. CYP2C9 Mutation Affecting the Individual Variability of Warfarin Dose Requirement.

    PubMed

    Kim, Young Bum; Ko, Moon Ju; Lee, Dae Gu; Do, Jong Gul; Hwang, Ji Hye

    2012-12-01

    Warfarin is a frequently prescribed anticoagulant in rehabilitation patients. Adverse drug reactions of warfarin were reported as bleeding and cutaneous microvascular thrombosis. Major bleeding, such as intracranial hemorrhage and psoas hematoma, in patients receiving anticoagulation therapy is a rare condition, but sometimes very serious complication that can even be fatal. Patient-specific factors (eg, age, body size, race, concurrent diseases, and medications) explain some of the individual variability in warfarin dose, but genetic factors, which influence warfarin response, explain a significantly higher proportion of the variability in the dose. There are two identified genes that are responsible for the main proportion of the genetic effect: CYP2C9, which codes for the enzyme cytochrome P450 2C9 that metabolizes S-warfarin, and VKORC1, which codes for warfarin's target, vitamin K epoxide reductase. We report a case of intolerance to warfarin dosing, due to impaired drug metabolism in a patient with CYP2C9(*)1/(*)3 and VKORC 1173TT. Fortunately, there are no severe complications.

  16. Efavirenz intoxication due to a new CYP2B6 constellation.

    PubMed

    Anagnostopoulos, Alexia; Rotger, Margalida; Aouri, Manel; Kuster, Stefan P; Telenti, Amalio; Décosterd, Laurent A; Günthard, Huldrych F

    2013-01-01

    Here, we describe severe neuropsychiatric symptoms in an HIV-positive Asian man with extremely high efavirenz plasma levels while receiving standard treatment with efavirenz/tenofovir/emtricitabine fixed-dose regimen. Genetic examination revealed compound homozygosity for loss-of-function alleles of CYP2B6, including coding for a rare truncated protein. Neuropsychiatric symptoms resolved completely after efavirenz discontinuation.

  17. 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.

  18. 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

  19. Effects of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen in Korean subjects.

    PubMed

    Lee, Yun-Jeong; Byeon, Ji-Yeong; Kim, Young-Hoon; Kim, Se-Hyung; Choi, Chang-Ik; Bae, Jung-Woo; Sohn, Uy-Dong; Jang, Choon-Gon; Lee, Jeongmi; Lee, Seok-Yong

    2015-06-01

    The aim of this study was to investigate the impact of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen and its metabolite. The CYP2C9 genotypes were determined with the use of polymerase chain reaction and restriction fragment and DNA sequencing analysis in 358 healthy Koreans. Among them, twenty individuals with CYP2C9*1/*1 (n = 12) or CYP2C9*1/*3 (n = 8) genotypes received a single 40 mg oral dose of flurbiprofen. The plasma concentrations of flurbiprofen and its metabolite, 4'-hydroxyflurbiprofen were measured by HPLC. AUCinf of flurbiprofen was significantly higher and its clearance was significantly lower in the CYP2C9*1/*3 individuals than in those with CYP2C9*1/*1. The AUC ratio of 4'-hydroxyflurbiprofen to flurbiprofen was significantly lower in the CYP2C9*1/*3 individuals than in those with CYP2C9*1/*1. These results indicate that the individuals carrying of CYP2C9*3 have significant reduction in flurbiprofen metabolism. The clinical use of this information may allow for more efficient personalized pharmacotherapy.

  20. Influence of Surface Preparation for Different Groups of A2B6 Mixed Crystals

    NASA Astrophysics Data System (ADS)

    Zakrzewski, J.; Maliński, M.; Strzałkowski, K.; Firszt, F.; Łęgowski, S.; Męczyńska, H.

    2010-01-01

    Piezoelectric photothermal spectroscopy has been used for measurements of the optical and thermal parameters of semiconductors. The investigated crystals were grown by the high-pressure Bridgman method under argon overpressure. The obtained photoacoustic (PA) spectra show the complexity of the effects observed for the different groups of selected A2B6 crystals. These effects comprise ideal samples and samples with damaged surfaces. The spectra show the influence of the surface treatment on the PA amplitude and phase spectra.

  1. 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

  2. Influence of admixture components on CYP2C9*2 allele frequency in eight indigenous populations from Northwest Mexico.

    PubMed

    Sosa-Macías, M; Lazalde-Ramos, B P; Galaviz-Hernández, C; Rangel-Villalobos, H; Salazar-Flores, J; Martínez-Sevilla, V M; Martínez-Fierro, M L; Dorado, P; Wong, M L; Licinio, J; LLerena, A

    2013-12-01

    We previously documented the lowest frequency of CYP2C9*2 in Mexican indigenous Tepehuanos followed by Mestizos and Mexican-Americans populations, suggesting a negative correlation between the CYP2C9*2 frequency and the degree of Asian ancestry in indigenous Americans. We determined the influence of ethnic admixture components on the CYP2C9 allele distribution in 505 Amerindian from eight indigenous populations through genotyping CYP2C9*2, *3 and *6 alleles by real-time PCR and molecular evaluation of ancestry. The frequencies for CYP2C9*2 were 0.026 in Seris and 0.057 in Mayos, being higher than in Asians (P<0.001). CYP2C9*3 was found in Tarahumaras (0.104), Mayos (0.091), Tepehuanos (0.075), Guarijíos (0.067), Huicholes (0.033) and Coras (0.037), with East Asians having lower frequencies than the former three groups (P<0.001). CYP2C9*6 was not found. The frequency of CYP2C9*2 was lower in Amerindians than in European populations, and higher than their Asian ancestors. The presence of this allele in ethnic groups in Mexico can be explained by European admixture.

  3. 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

  4. High-throughput screening assays for CYP2B6 metabolism and inhibition using fluorogenic vivid substrates.

    PubMed

    Marks, Bryan D; Goossens, Tony A; Braun, Heidi A; Ozers, Mary S; Smith, Ronald W; Lebakken, Connie; Trubetskoy, Olga V

    2003-01-01

    CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo- and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme's involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (K(i), K(d), or K(m) values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.

  5. [Cytochrome P4502C9(CYP2C9) gene polymorphism and safety of therapy with warfarin].

    PubMed

    Mikheeva, Iu A; Kropacheva, E S; Ignat'ev, I V; Bulytova, Iu M; Ramenskaia, G V; Sychev, D A; Dobrovol'skiĭ, A B; Panchenko, E P

    2008-01-01

    Aim of the study was to investigate frequency and influence of alleles CYP2C9*2 and CYP2C9*3 on pharmacokinetics, pharmacodynamics and dosing regimen of warfarin and on development of hemorrhagic complications. We included 84 patients (mean age 62,8 +/- 10,5 years). Duration of follow-up varied between 1 month and 1 year. Carriage of allele variants was determined by polymerase chain reaction, measurement of plasma wafarin concentration was carried out with the help of high performance liquid chromatography. Wild type (CYP2C9*1/*1) was found in 68% of patients; overall frequency of 2C9*1/*1, *l/*3, *2/*2, *3/*3, *2/*3 genotypes was 32%. Average maintenance doses of warfarin for patients with allele variants CYP 2C9 *2 and 2C9 *3 were 3.6 and 3.1 mg/day, respectively, what was significantly lower than in wild type homozygotes (6.1 mg/day). Wild type homozygotes (1) had the highest warfarin clearance (3,51 ml/min). In carriers of 2C9 *2(2) and 2C9 *3(3) warfarin clearance was significantly lower (2.42 and 1.82 ml/min; p1 - 2 = 0,05; p1 - 3 = 0,0008). In carriers of allele variants CYP2C9*2, CYP2C9*3 values of international normalized ratio > 3,0 were met more often, especially in carriers of CYP2C9*3 (in 100% of cases) vs. 28% in wild type homozygotes (p=0,02). Carriers of CYP2C9*3 compared with wild type homozygotes had more hemorrhagic complications (67% and 16%, respectively, p=0,0008). Thus cytochrome P450 2C9 gene polymorphism influences frequency of development of hemorrhagic complications, metabolic clearance, and magnitude of warfarin maintenance dose. PMID:18429757

  6. Comparative QSAR analyses of competitive CYP2C9 inhibitors using three-dimensional molecular descriptors.

    PubMed

    Lather, Viney; Fernandes, Miguel X

    2011-07-01

    One of the biggest challenges in QSAR studies using three-dimensional descriptors is to generate the bioactive conformation of the molecules. Comparative QSAR analyses have been performed on a dataset of 34 structurally diverse and competitive CYP2C9 inhibitors by generating their lowest energy conformers as well as additional multiple conformers for the calculation of molecular descriptors. Three-dimensional descriptors accounting for the spatial characteristics of the molecules calculated using E-Dragon were used as the independent variables. The robustness and the predictive performance of the developed models were verified using both the internal [leave-one-out (LOO)] and external statistical validation (test set of 12 inhibitors). The best models (MLR using GETAWAY descriptors and partial least squares using 3D-MoRSE) were obtained by using the multiple conformers for the calculation of descriptors and were selected based upon the higher external prediction ( values of 0.65 and 0.63, respectively) and lower root mean square error of prediction (0.48 and 0.48, respectively). The predictive ability of the best model, i.e., MLR using GETAWAY descriptors was additionally verified on an external test set of quinoline-4-carboxamide analogs and resulted in an value of 0.6. These simple and alignment-independent QSAR models offer the possibility to predict CYP2C9 inhibitory activity of chemically diverse ligands in the absence of X-ray crystallographic information of target protein structure and can provide useful insights about the ADMET properties of candidate molecules in the early phases of drug discovery.

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed Central

    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. PMID:26930610

  9. Influence of CYP2C9 and VKORC1 polymorphisms on warfarin and acenocoumarol in a sample of Lebanese people.

    PubMed

    Esmerian, Maria O; Mitri, Zahi; Habbal, Mohammad-Zuheir; Geryess, Eddy; Zaatari, Ghazi; Alam, Samir; Skouri, Hadi N; Mahfouz, Rami A; Taher, Ali; Zgheib, Nathalie K

    2011-10-01

    The authors assessed the impact of CYP2C9*2, CYP2C9*3, and/or VKORC1-1639G>A/1173C>T single-nucleotide polymorphisms on oral anticoagulants in a Lebanese population. This study recruited 231 Lebanese participants on long-term warfarin or acenocoumarol maintenance therapy with an international normalized ratio (INR) monitored at the American University of Beirut Medical Center. CYP2C9 and VKORC1 variant alleles were screened by real-time PCR. Plasma R- and S-warfarin and R- and S-acenocoumarol levels were assayed using high-performance liquid chromatography. The variant allele frequencies of CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A/1173C>T were 15.4%, 7.8%, and 52.4%, respectively. Fifty-five participants were excluded from analysis because of nontherapeutic INR values at recruitment, leaving 43 participants taking warfarin and 133 taking acenocoumarol. There was a significant decrease in the weekly maintenance dose of both drugs with CYP2C9 and VKORC1 variants when compared with wild-type patients. CYP2C9*2 had the least impact on the response to both drugs. The concentrations of R- and S-warfarin in plasma were significantly correlated with CYP2C9 genotypes. For acenocoumarol, time to reach target INR was more prolonged in patients carrying any CYP2C9 variant allele but failed to reach statistical significance because of low numbers of patients. There was no association between allelic variants and bleeding events. This is the first pharmacogenetic study of oral anticoagulants in Arabs. The authors showed that both CYP2C9 and VKORC1 polymorphisms are common in Lebanon and influence warfarin and acenocoumarol dose requirements, with the CYP2C9*2 polymorphism having less effect on acenocoumarol, the most commonly used oral anticoagulant in Lebanon. PMID:21148049

  10. 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

  11. Effects of pomegranate juice on human cytochrome P450 2C9 and tolbutamide pharmacokinetics in rats.

    PubMed

    Nagata, Masashi; Hidaka, Muneaki; Sekiya, Hiroshi; Kawano, Yohei; Yamasaki, Keishi; Okumura, Manabu; Arimori, Kazuhiko

    2007-02-01

    In this study, we investigated whether pomegranate juice could inhibit CYP2C9 activity. The ability of pomegranate juice to inhibit the diclofenac 4'-hydroxylase activity of human CYP2C9 was examined using human liver microsomes. Pomegranate juice was shown to be a potent inhibitor of human CYP2C9. The addition of 25 microl (5% v/v) of pomegranate juice resulted in almost complete inhibition of human CYP2C9 activity. In addition, we investigated the effect of pomegranate juice on the pharmacokinetics of tolbutamide (substrate for CYP2C9) in rats. Relative to the control group, the area under the concentration-time curve was approximately 1.2-fold greater when pomegranate juice (3 ml) was injected p.o. 1 h before the p.o. administration of the tolbutamide (20 mg/kg). The elimination half-life of tolbutamide was not altered by pomegranate juice administration. These results suggest pomegranate juice ingestion inhibits the intestinal metabolism of tolbutamide without inhibiting the hepatic metabolism in rats. Thus, we discovered that pomegranate juice inhibited human CYP2C9 activity and furthermore increased tolbutamide bioavailability in rats.

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

    PubMed Central

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

    2014-01-01

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

  13. QM/MM modeling of benzene hydroxylation in human cytochrome P450 2C9.

    PubMed

    Bathelt, Christine M; Mulholland, Adrian J; Harvey, Jeremy N

    2008-12-18

    The mechanism of benzene hydroxylation was investigated in the realistic enzyme environment of the human CYP 2C9 by using quantum mechanical/molecular mechanical (QM/MM) calculations of the whole reaction profile using the B3LYP method to describe the QM region. The calculated QM/MM barriers for addition of the active species Compound I to benzene are consistent with experimental rate constants for benzene metabolism in CYP 2E1. In contrast to gas-phase model calculations, our results suggest that competing side-on and face-on geometries of arene addition may both occur in the case of aromatic ring oxidation in cytochrome P450s. QM/MM profiles for three different rearrangement pathways of the initially formed sigma-adduct, leading to formation of epoxide, ketone, and an N-protonated porphyrin species, were calculated. Our results suggest that epoxide and ketone products form with comparable ease in the face-on pathway, whereas epoxide formation is preferred in the side-on pathway. Additionally, rearrangement to the N-protonated porphyrin species was found to be competitive with side-on epoxide formation. This suggests that overall, the competition between formation of epoxide and phenol final products in P450 oxidation of aromatic substrates is quite finely balanced. PMID:18754597

  14. 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.

  15. Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

    PubMed Central

    Thomas, Maria; Winter, Stefan; Klumpp, Britta; Turpeinen, Miia; Klein, Kathrin; Schwab, Matthias; Zanger, Ulrich M.

    2015-01-01

    The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype. PMID:26582990

  16. High-temperature Raman spectroscopic study of vanadoborate Na3VO2B6O11

    NASA Astrophysics Data System (ADS)

    Ji, Zhang; De-Ming, Zhang; Qing-Li, Zhang; Shao-Tang, Yin

    2016-03-01

    Raman spectra of a vanadoborate (Na3VO2B6O11) crystal from room temperature up to the melting point have been recorded. The main internal vibrational modes of the crystal have been assigned. It was found that all the Raman bands exhibit decreases in frequency and the widths of the Raman bands increase with the increase of temperature. However, no phase transition was observed under 525 °C. The micro-structure of its melt was studied by quantum chemistry ab initio calculation. The continuous three-dimensional network of the crystal collapsed and transformed into VO4 and VBO6 clusters during the melting process with an isomerization reaction from four-coordinated boron to a three-coordinated species. Project supported by the National Natural Science Foundation of China (Grant Nos. 51302268 and 51102239) and the Natural Science Foundation of Anhui Province, China (Grant No. KJ2015A339).

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

    PubMed

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

    2010-03-01

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

  18. 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

  19. 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

  20. Prevalence of combinatorial CYP2C9 and VKORC1 genotypes in Puerto Ricans: implications for warfarin management in Hispanics.

    PubMed

    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

    2009-01-01

    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 C>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.

  1. CYP2C8 Is a Novel Target of Peroxisome Proliferator-Activated Receptor α in Human Liver

    PubMed Central

    Makia, Ngome L.

    2016-01-01

    Human cytochrome P450 (CYP) 2C enzymes metabolize ∼30% of clinically prescribed drugs and various environmental chemicals. CYP2C8, an important member of this subfamily, metabolizes the anticancer drug paclitaxel, certain antidiabetic drugs, and endogenous substrates, including arachidonic acid, to physiologically active epoxyeicosatrienoic acids. Previous studies from our laboratory showed that microRNA 107 (miR107) and microRNA 103 downregulate CYP2C8 post-transcriptionally. miR107 is located in intron 5 of the pantothenate kinase 1 (PANK1) gene. p53 has been reported to coregulate the induction of PANK1 and miR107. Here, we examine the possible downregulation of CYP2C8 by drugs capable of inducing miR107. Hypolipidemic drugs, such as bezafibrate, known activators of the peroxisome proliferator-activated receptor α (PPARα), induce both the PANK1 gene and miR107 (∼2.5-fold) in primary human hepatocytes. Surprisingly, CYP2C8 mRNA and protein levels were induced by bezafibrate. CYP2C8 promoter activity was increased by ectopic expression of PPARα in HepG2 cells, with a further increase after bezafibrate (∼18-fold), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (∼10-fold) treatment, or the antidiabetic drug rosiglitazone, all known PPAR activators. Promoter sequence analyses, deletion studies, mutagenesis studies, and electrophoretic mobility shift assays identified a PPARα response element located at position −2109 base pair relative to the translation start site of CYP2C8. Chromatin immunopreciptation assay analysis confirmed recruitment of PPARα to this PPARα response element after bezafibrate treatment of human hepatocytes. Thus, we show for the first time that CYP2C8 is transcriptionally regulated by PPARα, suggesting the potential for drug-drug interactions due to upregulation of CYP2C8 by PPAR activators. PMID:26467040

  2. CYP2C8 Is a Novel Target of Peroxisome Proliferator-Activated Receptor α in Human Liver.

    PubMed

    Makia, Ngome L; Goldstein, Joyce A

    2016-01-01

    Human cytochrome P450 (CYP) 2C enzymes metabolize ∼30% of clinically prescribed drugs and various environmental chemicals. CYP2C8, an important member of this subfamily, metabolizes the anticancer drug paclitaxel, certain antidiabetic drugs, and endogenous substrates, including arachidonic acid, to physiologically active epoxyeicosatrienoic acids. Previous studies from our laboratory showed that microRNA 107 (miR107) and microRNA 103 downregulate CYP2C8 post-transcriptionally. miR107 is located in intron 5 of the pantothenate kinase 1 (PANK1) gene. p53 has been reported to coregulate the induction of PANK1 and miR107. Here, we examine the possible downregulation of CYP2C8 by drugs capable of inducing miR107. Hypolipidemic drugs, such as bezafibrate, known activators of the peroxisome proliferator-activated receptor α (PPARα), induce both the PANK1 gene and miR107 (∼2.5-fold) in primary human hepatocytes. Surprisingly, CYP2C8 mRNA and protein levels were induced by bezafibrate. CYP2C8 promoter activity was increased by ectopic expression of PPARα in HepG2 cells, with a further increase after bezafibrate (∼18-fold), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (∼10-fold) treatment, or the antidiabetic drug rosiglitazone, all known PPAR activators. Promoter sequence analyses, deletion studies, mutagenesis studies, and electrophoretic mobility shift assays identified a PPARα response element located at position -2109 base pair relative to the translation start site of CYP2C8. Chromatin immunopreciptation assay analysis confirmed recruitment of PPARα to this PPARα response element after bezafibrate treatment of human hepatocytes. Thus, we show for the first time that CYP2C8 is transcriptionally regulated by PPARα, suggesting the potential for drug-drug interactions due to upregulation of CYP2C8 by PPAR activators. PMID:26467040

  3. Drug-drug Interaction between Losartan and Paclitaxel in Human Liver Microsomes with Different CYP2C8 Genotypes.

    PubMed

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

    2015-06-01

    The cytochrome P450 (CYP) 2C8*3 allele is associated with reduced metabolic activity of paclitaxel. This study was aimed to investigate the inhibitory effect of losartan on paclitaxel metabolism in human liver microsomes (HLMs) and to determine the impact of the CYP2C8*3 polymorphism. HLMs that contained the CYP2C8*1 homozygote (HL60) or CYP2C8*3 heterozygote (HL54) genotype were used for the inhibition study. Losartan, at a concentration of 50 μmol/L, significantly inhibited paclitaxel metabolism by 29% and 57% in the HL60 (p < 0.001) and HL54 (p < 0.01), respectively. When using HL60, losartan and the CYP3A4-selective inhibitors, erythromycin and ketoconazole, caused a greater inhibition of the paclitaxel metabolism than quercetin, a CYP2C8-selective inhibitor. This demonstrated that the paclitaxel metabolism was mainly catalysed by CYP3A4 in HL60. There were no significant differences found for the inhibitory effects caused by the four inhibitors of the paclitaxel metabolism in HL54, indicating that both CYP2C8 and CYP3A4 play important roles in paclitaxel metabolism in HL54. These findings suggest that 50 μmol/L of losartan inhibits both CYP2C8 and CYP3A4 in HLMs. In summary, losartan inhibited paclitaxel metabolism, with concentrations over 50 μmol/L in HLMs. The CYP2C8*3 allele carriers are likely susceptible to the interactions of losartan and CYP3A4 inhibitors to paclitaxel metabolism.

  4. Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

    PubMed Central

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

    2014-01-01

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

  5. Systematic characterization and comparison of the CYP2C9 variability of the Orang Asli in Malaysia with 12 populations.

    PubMed

    Teh, Lay Kek; Subramaniam, Vinothini; Tuan Abdu Aziz, Tuan Azlin; Lee, Lian Shien; Ismail, Mohamed Izwan; Yu, Choo Yee; Ang, Geik Yong; James Johari, Richard; Ismet, Rose Iszati; Sahak, Noor Saadah; Ahmad, Aminuddin; Rahman, Thuhairah Abdul; Nor Ghazali, Fadzilah Mohd; Shaari, SyahrulAzlin; Omar, Mustaffa; Ismail, Adzrool Idzwan; Md Isa, Kamarudzaman; Salleh, Hood; Salleh, Mohd Zaki

    2016-08-01

    We conducted a systematic characterization of CYP2C9 variants in 61 Orang Asli and 96 Singaporean Malays using the whole genome sequences data and compared the variants with the other 11 HapMap populations. The frequency of rs1057910 (CYP2C9*3) is the highest in the Orang Asli compared to other populations. Three alleles with clinical implication were detected in the Orang Asli while 2 were found in the Singaporean Malays. Large numbers of the Orang Asli are predicted to have reduced metabolic capacity and therefore they would require a lower dose of drugs which are metabolized by CYP2C9. They are also at increased risks of adverse effects and therapeutic failures. A large number of CYP2C9 variants in the Orang Asli were not in the Hardy Weinberg Equilibrium which could be due to small sample size or mutations that disrupt the equilibrium of allele frequencies. In conclusion, different polymorphism patterns, allele frequencies, genotype frequencies and LD blocks are observed between the Orang Asli, the Singaporean Malays and the other populations. The study provided new information on the genetic polymorphism of CYP2C9 which is important for the implementation of precision medicine for the Orang Asli. PMID:27325019

  6. CYP2B6rs2279343 Is Associated with Improved Survival of Pediatric Rhabdomyosarcoma Treated with Cyclophosphamide

    PubMed Central

    A. Abdelrahim, Mohamed E.; Elnadi, Enas; Hesham, Reem M.; Yassin, Dina

    2016-01-01

    Background Rhabdomyosarcoma (RMS) is a small round blue cell malignant tumor, representing 7% of childhood malignancies, and over 50% of all soft tissue sarcomas. Cyclophosphamide (CPA) is a prodrug and is the mainstay of RMS treatment. CYP2B6 is a highly polymorphic drug metabolizing enzyme involved in CPA bioactivation. The influence of CYP2B6 single nucleotide polymorphisms (SNPs) on the survival of RMS is still unknown. Methods We genotyped CYP2B6SNPs rs2279343, rs3745274, and rs3211371 by restriction fragment polymorphism (RFLP) after PCR amplification in a cohort of 73 pediatric RMS patients treated with CPA-based first line treatment. We then analyzed the association between those genotypes and survival outcome of RMS. Results The frequencies of CYP2B6 rs2279343, rs3745274, and rs3211371 were 63%, 45.2%, and 5.5%, respectively. There was no association between rs3745274, rs3211371 genotypes and survival outcomes of RMS. However, the carriers of at least one mutant allele CYP2B6rs2279343 had significantly longer event-free survival (p-value = 0.03). Conclusion Our results demonstrated that CYP2B6 rs2279343 may predict EFS in RMS patients and warrants future studies to clarify the pharmacogenetics of CPA in pediatrics. If validated, integration of genetic factors with clinical and molecular characteristics could be used for a composite algorithm to better stratify risk prior to treatment. PMID:27388155

  7. The influence of CYP2C8*3 on carbamazepine serum concentration in epileptic pediatric patients

    PubMed Central

    Milovanovic, DD; Milovanovic, JR; Radovanovic, M; Radosavljevic, I; Obradovic, S; Jankovic, S; Milovanovic, D

    2016-01-01

    Abstract The aim of the present study was to investigate the distribution of CYP2C8 variants *3 and *5, as well as their effect on carbamazepine pharmacokinetic properties, in 40 epileptic pediatric patients on carbamazepine treatment. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and allele-specific (AS)-PCR methods, and steady-state carbamazepine plasma concentrations were determined by high performance liquid chromatography (HPLC). The CYP2C8 *3 and *5 polymorphisms were found at frequencies of 17.5 and 0.0%, respectively. After dose adjustment, there was a difference in daily dose in CYP2C8*3 carriers compared to non carriers [mean ± standard deviation (SD): 14.19 ± 5.39 vs. 15.46 ± 4.35 mg/kg; p = 0.5]. Dose-normalized serum concentration of carbamazepine was higher in CYP2C8*3 (mean ± SD: 0.54 ± 0.18 vs. 0.43 ± 0.11 mg/mL, p = 0.04), and the observed correlation between weight-adjusted carbamazepine dose and carbamazepine concentration after dose adjustment was significant only in CYP2C8*3 non carriers (r = 0.52, p = 0.002). However, the population pharmacokinetic analysis failed to demonstrate any significant effect of CYP2C8 *3 polymorphism on carbamazepine clearance [CL L/h = 0.215 + 0.0696*SEX+ 0.000183*DD]. The results indicated that the CYP2C8*3 polymorphism might not be of clinical importance for epilepsy treatment in pediatric populations.

  8. Differential Oxidation of Two Thiophene-Containing Regioisomers to Reactive Metabolites by Cytochrome P450 2C9

    PubMed Central

    Rademacher, Peter M; Woods, Caleb M; Huang, Qingbiao; Szklarz, Grazyna D; Nelson, Sidney D

    2012-01-01

    The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this manuscript was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 18O from 18O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product, but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver

  9. Bonding in Complexes of Bis(pentalene)dititanium, Ti2(C8H6)2

    PubMed Central

    2015-01-01

    Bonding in the bis(pentalene)dititanium “double-sandwich” species Ti2Pn2 (Pn = C8H6) and its interaction with other fragments have been investigated by density functional calculations and fragment analysis. Ti2Pn2 with C2v symmetry has two metal–metal bonds and a low-lying metal-based empty orbital, all three frontier orbitals having a1 symmetry. The latter may be regarded as being derived by symmetric combinations of the classic three frontier orbitals of two bent bis(cyclopentadienyl) metal fragments. Electrochemical studies on Ti2Pn†2 (Pn† = 1,4-{SiiPr3}2C8H4) revealed a one-electron oxidation, and the formally mixed-valence Ti(II)–Ti(III) cationic complex [Ti2Pn†2][B(C6F5)4] has been structurally characterized. Theory indicates an S = 1/2 ground-state electronic configuration for the latter, which was confirmed by EPR spectroscopy and SQUID magnetometry. Carbon dioxide binds symmetrically to Ti2Pn2, preserving the C2v symmetry, as does carbon disulfide. The dominant interaction in Ti2Pn2CO2 is σ donation into the LUMO of bent CO2, and donation from the O atoms to Ti2Pn2 is minimal, whereas in Ti2Pn2CS2 there is significant interaction with the S atoms. The bridging O atom in the mono(oxo) species Ti2Pn2O, however, employs all three O 2p orbitals in binding and competes strongly with Pn, leading to weaker binding of the carbocyclic ligand, and the sulfur analogue Ti2Pn2S behaves similarly. Ti2Pn2 is also capable of binding one, two, or three molecules of carbon monoxide. The bonding demands of a single CO molecule are incompatible with symmetric binding, and an asymmetric structure is found. The dicarbonyl adduct Ti2Pn2(CO)2 has Cs symmetry with the Ti2Pn2 unit acting as two MCp2 fragments. Synthetic studies showed that in the presence of excess CO the tricarbonyl complex Ti2Pn†2(CO)3 is formed, which optimizes to an asymmetric structure with one semibridging and two terminal CO ligands. Low-temperature 13C NMR spectroscopy revealed a rapid

  10. Genetic Heterogeneity Beyond CYP2C8*3 Does Not Explain Differential Sensitivity to Paclitaxel-Induced Neuropathy

    PubMed Central

    Hertz, Daniel L.; Roy, Siddharth; Jack, John; Motsinger-Reif, Alison A.; Drobish, Amy; Clark, L. Scott; Carey, Lisa A.; Dees, E. Claire; McLeod, Howard L.

    2014-01-01

    Purpose The development of paclitaxel-induced peripheral neuropathy (PIPN) is influenced by drug exposure and patient genetics. The purpose of this analysis was to expand on a previous reported association of CYP2C8*3 and PIPN risk by investigating additional polymorphisms in CYP2C8 and in hundreds of other genes potentially relevant to paclitaxel pharmacokinetics. Methods Clinical data was collected prospectively in an observational registry of newly diagnosed breast cancer patients. Patients treated with paclitaxel-containing regimens were genotyped using the Affymetrix DMET™ Plus chip. Patients who carried the CYP2C8*2, *3 or *4 variant were collapsed into a low-metabolizer CYP2C8 phenotype for association with PIPN. Separately, all SNPs that surpassed quality control were assessed individually and as a composite of genetic ancestry for associations with PIPN. Results 412 paclitaxel-treated patients and 564 genetic markers were included in the analysis. The risk of PIPN was significantly greater in the CYP2C8 low-metabolizer group (HR=1.722, p=0.018), however, the influence of the *2 and *4 SNPs were not independently significant (*2: p=0.847, *4: p=0.408). One intronic SNP in ABCG1 (rs492338) surpassed the exploratory significance threshold for an association with PIPN in the Caucasian cohort (p=0.0008) but not in the non-Caucasian replication group (p=0.54). Substantial genetic variability was observed within self-reported racial groups but this genetic variability was not associated with risk of grade 2+ PIPN. Conclusions The pharmacogenetic heterogeneity within a cohort of breast cancer patients is dramatic, though we did not find evidence that this heterogeneity directly influences the risk of PIPN beyond the contribution of CYP2C8*3. PMID:24706167

  11. 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.

  12. 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

  13. Pharmacogenetic-Based Efavirenz Dose Modification: Suggestions for an African Population and the Different CYP2B6 Genotypes

    PubMed Central

    Mukonzo, Jackson K.; Owen, Joel S.; Ogwal-Okeng, Jasper; Kuteesa, Ronald B.; Nanzigu, Sarah; Sewankambo, Nelson; Thabane, Lehana; Gustafsson, Lars L.; Ross, Colin; Aklillu, Eleni

    2014-01-01

    Background Pharmacogenetics contributes to inter-individual variability in pharmacokinetics (PK) of efavirenz (EFV), leading to variations in both efficacy and toxicity. The purpose of this study was to assess the effect of genetic factors on EFV pharmacokinetics, treatment outcomes and genotype based EFV dose recommendations for adult HIV-1 infected Ugandans. Methods In total, 556 steady-state plasma EFV concentrations from 99 HIV infected patients (64 female) treated with EFV/lamivudine/zidovidine were analyzed. Patient genotypes for CYP2B6 (*6 & *11), CYP3A5 (*3,*6 & *7) and ABCB1 c.4046A>G, baseline biochemistries and CD4 and viral load change from baseline were determined. A one-compartment population PK model with first-order absorption (NONMEM) was used to estimate genotype effects on EFV pharmacokinetics. PK simulations were performed based upon population genotype frequencies. Predicted AUCs were compared between the product label and simulations for doses of 300 mg, 450 mg, and 600 mg. Results EFV apparent clearance (CL/F) was 2.2 and 1.74 fold higher in CYP2B6*6 (*1/*1) and CYP2B6*6 (*1/*6) compared CYP2B6*6 (*6/*6) carriers, while a 22% increase in F1 was observed for carriers of ABCB1 c.4046A>G variant allele. Higher mean AUC was attained in CYP2B6 *6/*6 genotypes compared to CYP2B6 *1/*1 (p<0.0001). Simulation based AUCs for 600 mg doses were 1.25 and 2.10 times the product label mean AUC for the Ugandan population in general and CYP2B6*6/*6 genotypes respectively. Simulated exposures for EFV daily doses of 300 mg and 450 mg are comparable to the product label. Viral load fell precipitously on treatment, with only six patients having HIV RNA >40 copies/mL after 84 days of treatment. No trend with exposure was noted for these six patients. Conclusion Results of this study suggest that daily doses of 450 mg and 300 mg might meet the EFV treatment needs of HIV-1 infected Ugandans in general and individuals homozygous for CYP2B6*6 mutation, respectively

  14. Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes.

    PubMed

    Albassam, Ahmed A; Mohamed, Mohamed-Eslam F; Frye, Reginald F

    2015-05-01

    1. Herbal supplements widely used in the US were screened for the potential to inhibit CYP2C8 activity in human liver microsomes. The herbal extracts screened were garlic, echinacea, saw palmetto, valerian, black cohosh and cranberry. N-desethylamodiaquine (DEAQ) and hydroxypioglitazone metabolite formation were used as indices of CYP2C8 activity. 2. All herbal extracts showed inhibition of CYP2C8 activity for at least one of three concentrations tested. A volume per dose index (VDI) was calculated to determine the volume in which a dose should be diluted to obtain IC50 equivalent concentration. Cranberry and saw palmetto had a VDI value > 5.0 l per dose unit, suggesting a potential for interaction. 3. Inhibition curves were constructed and the IC50 (mean ± SE) values were 24.7 ± 2.7 μg/ml for cranberry and 15.4 ± 1.7 μg/ml for saw palmetto. 4. The results suggest a potential for cranberry or saw palmetto extracts to inhibit CYP2C8 activity. Clinical studies are needed to evaluate the significance of this interaction. PMID:25430798

  15. A haplotype of CYP2C9 associated with warfarin sensitivity in mechanical heart valve replacement patients

    PubMed Central

    Lee, Su-Jun; Jang, Yin Jin; Cha, Eun-Young; Kim, Ho-Sook; Lee, Sang Seop; Shin, Jae-Gook

    2010-01-01

    AIMS The objectives of this study were to determine the distribution of CYP2C9 variants in Koreans and investigate their association with warfarin dose requirements in patients who received MHVRs. METHODS All nine exons, intron–exon junction, and promoter region of CYP2C9 were amplified and directly sequenced in 50 healthy normal Koreans. Additional direct DNA sequencing of the CYP2C9 gene was conducted in 36 of the 267 MHVR patients who required low maintenance warfarin doses without carrying CYP2C9*3 and VKORC1 1173T mutations. The effects of CYP2C9 genetics on warfarin maintenance dose were assessed in 267 MHVR patients. RESULTS Thirty-nine single nucleotide polymorphisms (SNPs) including seven previously unidentified SNPs were identified in 50 Koreans by direct DNA sequencing. One of the CYP2C9 haplotypes exhibited an association with warfarin low dose requirement. The adjusted odds ratio for the haplotype between the low dose group and the normal subjects was 2.5 (95% confidence interval 1.05, 6.16). This haplotype consisting of -1565C>T, -1188T>C, IVS3+197G>A, IVS3-334C>T, IVS3-65G>C, IVS4-115A>G, and IVS5-73A>G was found in 15% of 36 MHVR patients who required low warfarin doses, while 4% of 50 normal healthy subjects exhibited this haplotype. One of the SNPs comprising this haplotype, -1565C>T, apparently changed a protein binding pattern as observed in electrophoretic mobility shift assay. CONCLUSION The haplotype including -1565C>T, -1188T>C, IVS3+197G>A, IVS3-334C>T, IVS3-65G>C, IVS4-115A>G, and IVS5-73A>G seems to be associated with low warfarin dose requirement and this haplotype could be considered in the development of a warfarin dose prediction model for Asian populations. PMID:20653674

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2016-10-01

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

  18. 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.

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

    SciTech Connect

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

    2010-06-15

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

  20. Effect of Genetic Variants, Especially CYP2C9 and VKORC1, on the Pharmacology of Warfarin

    PubMed Central

    Fung, Erik; Patsopoulos, Nikolaos A.; Belknap, Steven M.; O’Rourke, Daniel J.; Robb, John F.; Anderson, Jeffrey L.; Shworak, Nicholas W.; Moore, Jason H.

    2014-01-01

    The genes encoding the cytochrome P450 2C9 enzyme (CYP2C9) and vitamin K-epoxide reductase complex unit 1 (VKORC1) are major determinants of anticoagulant response to warfarin. Together with patient demographics and clinical information, they account for approximately one-half of the warfarin dose variance in individuals of European descent. Recent prospective and randomized controlled trial data support pharmacogenetic guidance with their use in warfarin dose initiation and titration. Benefits from pharmacogenetics-guided warfarin dosing have been reported to extend beyond the period of initial dosing, with supportive data indicating benefits to at least 3 months. The genetic effects of VKORC1 and CYP2C9 in African and Asian populations are concordant with those in individuals of European ancestry; however, frequency distribution of allelic variants can vary considerably between major populations. Future randomized controlled trials in multiethnic settings using population-specific dosing algorithms will allow us to further ascertain the generalizability and cost-effectiveness of pharmacogenetics-guided warfarin therapy. Additional genome-wide association studies may help us to improve and refine dosing algorithms and potentially identify novel biological pathways. PMID:23041981

  1. CYP2B6 Variants and Plasma Efavirenz Concentrations during Antiretroviral Therapy in Port-au-Prince, Haiti

    PubMed Central

    Leger, Paul; Dillingham, Rebecca; Beauharnais, Carole Anne; Kashuba, Angela D. M.; Rezk, Naser L.; Fitzgerald, Daniel W.; Pape, Jean William; Haas, David W.

    2009-01-01

    Background Polymorphisms in CYP2B6 are known to predict increased steady-state plasma concentrations of efavirenz. We characterized relationships between genetic polymorphisms and plasma efavirenz concentrations among 45 Haitians who initiated antiretroviral therapy in Port-au-Prince. Methods An observational study characterized relationships between clinical factors, pharmacokinetics, and treatment response among antiretroviral-naïve patients initiating once-daily efavirenz plus twice-daily AZT/3TC. Plasma drug concentrations were determined at weeks 2 and 4. Drug doses were directly observed by field workers or designated family members. We retrospectively characterized relationships between efavirenz concentrations and 50 single nucleotide polymorphisms in CYP2B6, and several polymorphisms in CYP2A6, CYP3A4, CYP3A5 and ABCB1. Results Plasma for efavirenz assay was obtained 13.9 ±1.6 hours (mean ± SD) post-dose. As expected, CYP2B6 516G→T was associated with increased plasma efavirenz concentrations (Spearman’s rho=0.71, P<0.0001), as were 10 polymorphisms in linkage disequilibrium with 516G→T. Distinct CYP2B6 polymorphisms were associated with decreased plasma efavirenz concentrations (greatest absolute rho=0.48, P=0.0008). Associations were replicated by results from a recent pharmacokinetic study involving 34 healthy, HIV-negative African Americans. Conclusions Relatively frequent CYP2B6 polymorphisms may predict decreased plasma efavirenz exposure in patients of African descent. If replicated in other cohorts, the implications of these novel associations for treatment response warrant further study. PMID:19659438

  2. Mechanisms of interaction between persistent organic pollutants (POPs) and CYP2B6: An in silico approach.

    PubMed

    Maldonado-Rojas, Wilson; Rivera-Julio, Karen; Olivero-Verbel, Jesus; Aga, Diana S

    2016-09-01

    Human Cytochrome P450s (CYP450) are a group of heme-containing metalloenzymes responsible for recognition and metabolism of numerous xenobiotics, including drugs and environmental contaminants. CYP2B6, a member of CYP450, is well known for being a highly inducible and polymorphic enzyme and for its important role in the oxidative metabolism of environmental pollutants, such as the Polybrominated Diphenyl Ethers (PBDEs) and Polychlorinated Biphenyls (PCBs). However the mechanisms of interaction of PBDEs and PCBs with CYP2B6 is not entirely known. In this work, a computational approach was carried out to study the interactions of 41 POPs (17 PBDEs, 17 PCBs, and 7 Dioxins) with four CYP2B6 protein structures downloaded from PDB data base (PDB: 3UA5, 3QOA, 3QU8 and 4I91) using molecular docking protocols with AutoDock Vina. The best binding affinity values (kcal/mol) were obtained for PBDE-99 (-8.5), PCB-187 (-9.6), and octachloro-dibenzo-dioxin (-9.8) that can be attributed to the hydrophobic interactions with important residues, such as Phe-363, in the catalytic site of CYP2B6. Molecular docking validation revealed the best values for PDB: 3UA5 (R = 0.622, p = 0.001) demonstrating the reliability of molecular docking predictions. The information obtained in this work can be useful in evaluating the modes of interaction of xenobiotic compounds with the catalytic site of CYP2B6 and provide insights on the important role of these enzymes in the metabolism of potentially toxic compounds in humans.

  3. Mechanisms of interaction between persistent organic pollutants (POPs) and CYP2B6: An in silico approach.

    PubMed

    Maldonado-Rojas, Wilson; Rivera-Julio, Karen; Olivero-Verbel, Jesus; Aga, Diana S

    2016-09-01

    Human Cytochrome P450s (CYP450) are a group of heme-containing metalloenzymes responsible for recognition and metabolism of numerous xenobiotics, including drugs and environmental contaminants. CYP2B6, a member of CYP450, is well known for being a highly inducible and polymorphic enzyme and for its important role in the oxidative metabolism of environmental pollutants, such as the Polybrominated Diphenyl Ethers (PBDEs) and Polychlorinated Biphenyls (PCBs). However the mechanisms of interaction of PBDEs and PCBs with CYP2B6 is not entirely known. In this work, a computational approach was carried out to study the interactions of 41 POPs (17 PBDEs, 17 PCBs, and 7 Dioxins) with four CYP2B6 protein structures downloaded from PDB data base (PDB: 3UA5, 3QOA, 3QU8 and 4I91) using molecular docking protocols with AutoDock Vina. The best binding affinity values (kcal/mol) were obtained for PBDE-99 (-8.5), PCB-187 (-9.6), and octachloro-dibenzo-dioxin (-9.8) that can be attributed to the hydrophobic interactions with important residues, such as Phe-363, in the catalytic site of CYP2B6. Molecular docking validation revealed the best values for PDB: 3UA5 (R = 0.622, p = 0.001) demonstrating the reliability of molecular docking predictions. The information obtained in this work can be useful in evaluating the modes of interaction of xenobiotic compounds with the catalytic site of CYP2B6 and provide insights on the important role of these enzymes in the metabolism of potentially toxic compounds in humans. PMID:27281544

  4. VKORC1 and CYP2C9 Genotype Variations in Relation to Warfarin Dosing in Korean Stroke Patients

    PubMed Central

    Park, Sea Mi; Lee, Jong-Keuk; Chun, Sa Il; Lee, Hae In; Kwon, Sun U.; Kang, Dong-Wha

    2013-01-01

    Background and Purpose Variant alleles of CYP2C9 and VKORC1 account for differences in anticoagulation response. We sought to establish a warfarin dosing formula for individualized target International Normalization Ratio of Prothrombin Times (INRs) using data from single nucleotide polymorphisms (SNPs) in VKORC1 and CYP2C9 in Korean patients. Methods Ischemic stroke patients displaying stable target INR for at least 3 months before enrollment were analyzed. Warfarin and vitamin K levels were measured to adjust for confounders. Phenotypes were defined using the 'warfarin response index' (WRI) defined as INR divided by the daily maintenance warfarin dose. We tested SNPs in CYP2C9 (3 sites: 430C>T (rs1799853), 1075A>C (rs1057910), 1076T>C) and VKORC1 (14 sites: 381C>T, 861C>A (rs17880887), 2653G>C, 3673A>G, 5496G>T, 5808T>G (r17882154), 6009C>T, 6484T>C (rs9934438), 6853C>T (rs17886369), 7566T>C, 8767G>C, 8814T>C, 9041G>A (rs17880624), and 9071G>T) using a standard sequencing method. Multivariate linear regression analysis was applied to establish the formula for warfarin dosage. Results All 204 patients had excellent drug compliance. The mean INR was 2.22 (+0.56) and mean daily maintenance dose of warfarin was 3.92 mg (+1.54). Patients with low WRI were younger (P<0.001) with high body mass index (P=0.003), high prevalence of wild-type CYP2C9 polymorphism (1075A>C, P<0.001), and six heterozygote SNPs in VRORC1 (P<0.001), which were tightly interlinked (381T>C, 3673G>A, 6484T>C, 6853C>G. 7566C>T, 9041G>A) (r2=1). Based on these data, a warfarin dosing formula was established. Conclusions WRI is influenced by age, body mass index and SNPs in VKORC1 and CYP2C9 in Korean stroke patients. The obtained warfarin dosing formula may be clinically applicable. PMID:24324947

  5. Absence of the human CYP2C8*3 allele in Ugandan children exposed to Plasmodium falciparum malaria.

    PubMed

    Paganotti, Giacomo Maria; Cosentino, Valentina; Russo, Gianluca; Tabacchi, Francesca; Gramolelli, Silvia; Coluzzi, Mario; Romano, Rita

    2014-10-01

    Study of host pharmacogenetics can improve our knowledge of mechanisms of drug resistance selection and spread. This issue has recently been addressed with respect to chloroquine and amodiaquine in malaria endemic areas of West and East Africa. Here we report, from surveys performed in two different areas of Uganda, that the human CYP2C8*3 allele, which had been reported to be strongly associated with parasite drug resistance in Zanzibar, is absent, being a marker of genetic admixture of the Zanzibari population with a Caucasoid component. Moreover, a retrospective analysis of CYP2C8*2 and the Plasmodium falciparum drug resistant pfmdr1 86Y allele does not show any association, which may be related to the high level of drug resistance.

  6. Dose Optimization of Efavirenz Based on Individual CYP2B6 Polymorphisms in Chinese Patients Positive for HIV.

    PubMed

    Hui, K H; Lee, S S; Lam, T N

    2016-04-01

    The purpose of this study was to investigate the impact of CYP2B6-G516T polymorphisms on the pharmacokinetics (PKs) of efavirenz among the Chinese population and to propose doses for different genotypic populations that optimize therapeutic outcomes. Nonlinear mixed-effect modeling was applied to describe PKs of efavirenz in Chinese patients with human immunodeficiency virus (HIV). Probabilities of successful treatment at different doses were obtained by simulations using the developed model to identify the optimal doses. The model was based on data from 163 individuals. Efavirenz clearance was found to be significantly influenced by CYP2B6-G516T polymorphisms and body weight. The typical values of oral clearance were 10.2 L/h, 7.33 L/h, and 2.38 L/h and simulation results suggested that the optimal daily oral doses are 550 mg, 350 mg, and 100 mg for the GG, GT, and TT populations, respectively. The effect of CYP2B6-G516T polymorphisms on efavirenz clearance was successfully quantified. Pharmacogenetics-based dose individualization of efavirenz may optimize patient outcomes by promoting efficacy while minimizing central nervous system (CNS) side effects. PMID:27299708

  7. Effect of atorvastatin on CYP2C9 metabolic activity as measured by the formation rate of losartan metabolite in hypercholesterolaemic patients.

    PubMed

    Yasar, Umit; Sain-Guven, Gulay; Yardimci, Yildiz; Kilicarslan, Alpaslan; Babaoglu, Melih O; Bozkurt, Atilla

    2011-08-01

    HMG-CoA reductase inhibitors (statins) have a potential to interact with substrates of the drug-metabolizing enzyme cytochrome P450 2C9 (CYP2C9). This may lead to concentration-dependent toxicity such as skeletal muscle side effects. Atorvastatin, a widely used statin, is presently inadequately investigated in vivo with regard to effects on CYP2C9 activity in human beings. The aim of this study was to determine the effect of atorvastatin on the activity of CYP2C9 in a group of Turkish hypercholesterolaemic patients. We prospectively investigated the atorvastatin effect on CYP2C9 activity in a sample of Turkish hypercholesterolaemia patients (11 women, 7 men) who commenced atorvastatin (10 mg/day). Losartan was used as a probe drug to determine CYP2C9 metabolic activity. A single 25-mg oral dose of losartan was given to the patients before, on the first day and after the fourth week of the atorvastatin treatment. Urinary concentrations of losartan and its metabolite, E3174, were measured by high-pressure liquid chromatography (HPLC). Urinary losartan/E3174 ratios were used as an index of CYP2C9 activity. As the baseline enzyme activity may influence the extent of drug-drug interactions, the CYP2C9*2 and 2C9*3 alleles were identified by using PCR-RFLP. In the patients with the CYP2C9*1*1 genotype (n = 12), atorvastatin treatment did not cause a significant change in losartan/E3174 ratios (medians; 95% CI) neither after the first day (0.73; 0.34-1.61) nor at the fourth week (0.71; 0.36-1.77) of the treatment as compared with the baseline activity (0.92; 0.57-1.74, p = 0.38). Similarly, no significant change in the baseline CYP2C9 activity (0.91; 0.30-1.60) was observed in patients with the CYP2C9*1*2 genotype as compared with those of the first day (1.08; 0.08-2.72) and fourth week (0.64; 0.0-3.82) of the atorvastatin treatment (n = 4, p = 0.86). These observations in a hypercholesterolaemic patient sample suggest that atorvastatin does not have a significant effect

  8. 2D QSAR Study for Gemfibrozil Glucuronide as the Mechanism-based Inhibitor of CYP2C8

    PubMed Central

    Taxak, N.; Bharatam, P. V.

    2013-01-01

    Mechanism-based inhibition of cytochrome P450 involves the bioactivation of the drug to a reactive metabolite, which leads to cytochrome inhibition via various mechanisms. This is generally seen in the Phase I of drug metabolism. However, gemfibrozil (hypolipidemic drug) leads to mechanism-based inhibition after generating glucuronide conjugate (gemfibrozil acyl-β-glucuronide) in the Phase II metabolism reaction. The mechanism involves the covalent binding of the benzyl radical (generated from the oxidation of aromatic methyl group in conjugate) to the heme of CYP2C8. This article deals with the development of a 2D QSAR model based on the inhibitory potential of gemfibrozil, its analogues and corresponding glucuronide conjugates in inhibiting the CYP2C8-catalysed amodiaquine N-deethylation. The 2D QSAR model was developed using multiple linear regression analysis in Accelrys Discovery Studio 2.5 and helps in identifying the descriptors, which are actually contributing to the inhibitory potency of the molecules studied. The built model was further validated using leave one out method. The best quantitative structure activity relationship model was selected having a correlation coefficient (r) of 0.814 and cross-validated correlation coefficient (q2) of 0.799. 2D QSAR revealed the importance of volume descriptor (Mor15v), shape descriptor (SP09) and 3D matrix-based descriptor (SpMax_RG) in defining the activity for this series of molecules. It was observed that volume and 3D matrix-based descriptors were crucial in imparting higher potency to gemfibrozil glucuronide conjugate, as compared with other molecules. The results obtained from the present study may be useful in predicting the inhibitory potential (IC50 for CYP2C8 inhibition) of the glucuronide conjugates of new molecules and compare with the standard gemfibrozil acyl-β-glucuronide (in terms of pIC50 values) in early stages of drug discovery and development. PMID:24591743

  9. P450 (Cytochrome) Oxidoreductase Gene (POR) Common Variant (POR*28) Significantly Alters CYP2C9 Activity in Swedish, But Not in Korean Healthy Subjects.

    PubMed

    Hatta, Fazleen H M; Aklillu, Eleni

    2015-12-01

    CYP2C9 enzyme contributes to the metabolism of several pharmaceuticals and xenobiotics and yet displays large person-to-person and interethnic variation. Understanding the mechanisms of CYP2C9 variation is thus of immense importance for personalized medicine and rational therapeutics. A genetic variant of P450 (cytochrome) oxidoreductase (POR), a CYP450 redox partner, is reported to influence CYP2C9 metabolic activity in vitro. We investigated the impact of a common variant, POR*28, on CYP2C9 metabolic activity in humans. 148 healthy Swedish and 146 healthy Korean volunteers were genotyped for known CYP2C9 defective variant alleles (CYP2C9*2, *3). The CYP2C9 phenotype was determined using a single oral dose of 50 mg losartan. Excluding oral contraceptive (OC) users and carriers of 2C9*2 and *3 alleles, 117 Korean and 65 Swedish were genotyped for POR*5, *13 and *28 using Taqman assays. The urinary losartan to its metabolite E-3174 metabolic ratio (MR) was used as an index of CYP2C9 metabolic activity. The allele frequency of the POR*28 variant allele in Swedes and Koreans was 29% and 44%, respectively. POR*5 and *13 were absent in both study populations. Considering the CYP2C9*1/*1 genotypes only, the CYP2C9 metabolic activity was 1.40-fold higher in carriers of POR*28 allele than non-carriers among Swedes (p = 0.02). By contrast, no influence of the POR*28 on CYP2C9 activity was found in Koreans (p = 0.68). The multivariate analysis showed that ethnicity, POR genotype, and smoking were strong predictors of CYP2C9 MR (p < 0.05). This is the first report to implicate the importance of POR*28 genetic variation for CYP2C9 metabolic activity in humans. These findings contribute to current efforts for global personalized medicine and using medicines by taking into account pharmacogenetic and phenotypic variations. PMID:26669712

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

    PubMed Central

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

    2016-01-01

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

  11. Quantum mechanics/molecular mechanics modeling of regioselectivity of drug metabolism in cytochrome P450 2C9.

    PubMed

    Lonsdale, Richard; Houghton, Kerensa T; Żurek, Jolanta; Bathelt, Christine M; Foloppe, Nicolas; de Groot, Marcel J; Harvey, Jeremy N; Mulholland, Adrian J

    2013-05-29

    Cytochrome P450 enzymes (P450s) are important in drug metabolism and have been linked to adverse drug reactions. P450s display broad substrate reactivity, and prediction of metabolites is complex. QM/MM studies of P450 reactivity have provided insight into important details of the reaction mechanisms and have the potential to make predictions of metabolite formation. Here we present a comprehensive study of the oxidation of three widely used pharmaceutical compounds (S-ibuprofen, diclofenac, and S-warfarin) by one of the major drug-metabolizing P450 isoforms, CYP2C9. The reaction barriers to substrate oxidation by the iron-oxo species (Compound I) have been calculated at the B3LYP-D/CHARMM27 level for different possible metabolism sites for each drug, on multiple pathways. In the cases of ibuprofen and warfarin, the process with the lowest activation energy is consistent with the experimentally preferred metabolite. For diclofenac, the pathway leading to the experimentally observed metabolite is not the one with the lowest activation energy. This apparent inconsistency with experiment might be explained by the two very different binding modes involved in oxidation at the two competing positions. The carboxylate of diclofenac interacts strongly with the CYP2C9 Arg108 side chain in the transition state for formation of the observed metabolite-but not in that for the competing pathway. We compare reaction barriers calculated both in the presence and in the absence of the protein and observe a marked improvement in selectivity prediction ability upon inclusion of the protein for all of the substrates studied. The barriers calculated with the protein are generally higher than those calculated in the gas phase. This suggests that active-site residues surrounding the substrate play an important role in controlling selectivity in CYP2C9. The results show that inclusion of sampling (particularly) and dispersion effects is important in making accurate predictions of drug

  12. 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. PMID:21811894

  13. Generation and Characterization of a CYP2A13/2B6/2F1-Transgenic Mouse Model

    PubMed Central

    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

    2012-01-01

    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. PMID:22397853

  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. A Case Report of a Patient Carrying CYP2C9*3/4 Genotype with Extremely Low Warfarin Dose Requirement

    PubMed Central

    Lee, Soo-Youn; Nam, Myung-Hyun; Kim, June Soo

    2007-01-01

    We report a case of intolerance to warfarin dosing due to impaired drug metabolism in a patient with CYP2C9*3/*4. A 73-yr-old woman with atrial fibrilation was taking warfarin. She attained a high prothrombin time international normalized ratio (INR) at the standard doses during the induction of anticoagulation and extremely low dose of warfarin (6.5 mg/week) was finally chosen to reach the target INR. Genotyping for CYP2C9 revealed that this patient had a genotype CYP2C9*3/*4. This is the first Korean compound heterozygote for CYP2C9*3 and *4. This case suggests the clinical usefulness of pharmacogenetic testing for individualized dosage adjustments of warfarin. PMID:17596671

  16. Dimetallaborane analogues of the octaboranes of the type Cp2M2B6H10: structural variations with changes in the skeletal electron count.

    PubMed

    Brânzanic, Adrian M V; Lupan, Alexandru; King, R Bruce

    2016-05-31

    The structures and energetics of the complete series of hydrogen-rich dimetallaboranes Cp2M2B6H10 and Cp*2M2B6H10 (Cp = η(5)-C5H5; Cp* = η(5)-Me5C5; M = Pd, Pt; Rh, Ir; Ru, Os; Re; Mo, W; Ta), including the experimentally known Cp*2Rh2B6H10 and Cp*2W2B6H10 (Cp* = η(5)-Me5C5), have been investigated by density functional theory. The lowest energy structures of the hyperelectronic Cp2M2B6H10 (M = Pd, Pt; Rh, Ir) systems have central M2B6 frameworks with a hexagonal open face similar to the B8 networks in arachno-B8H14 and nido-B8H12. The two lowest energy structures for Cp2Rh2B6H10 and Cp*2Rh2B6H10, lying within 1 kcal mol(-1) of energy, differ only in the locations of the bridging hydrogen atoms around the hexagonal hole consistent with the experimentally observed fluxionality of the hydrogen atoms in Cp*2Rh2B6H10. Most of the lowest energy Cp2M2B6H10 (M = Ru, Os) structures also have a central M2B6 framework similar to B8H12, typically with such additional features as an additional metal-metal bond or a formal metal-metal double bond. A common motif for the low-energy structures of the hypoelectronic Cp2M2B6H10 (M = Re; Mo, W; Ta) systems, including the experimentally known Cp*2W2B6H10, is a central M2B4 octahedron with its two M2B faces capped by the remaining boron atoms and with four M-B edges bridged by hydrogen atoms. Such structures can also be considered as oblatonido structures derived from the experimentally known 9-vertex oblatocloso Cp*2Re2B7H7 structure by removal of the unique degree 4 vertex atom. PMID:27186632

  17. The effect of CYP2C9, VKORC1 genotypes and old age on warfarin pharmacologic sensitivity in korean patients with thromboembolic disease.

    PubMed

    Moon, Hee-Won; Noh, Jaekwang; Yun, Yeo-Min; Kim, Hahn Young; Yun, Ik Jin; Song, Junghan; Kim, Jin Q

    2011-01-01

    The therapeutic dose of warfarin is dependent upon intrinsic patient characteristics that are highly variable. We assessed the effects of CYP2C9, VKORC1 1173 C/T polymorphisms, and old age on warfarin dosing and sensitivity by measuring plasma S-/R-warfarin levels in Korean patients. INR and the plasma S-/R-warfarin concentrations were determined in 58 patients who had the VKORC1 1173C/T CYP2C9 genotypes, were on a long-term anticoagulation regimen with warfarin, and took a daily dose of warfarin. The pharmacokinetic sensitivity of warfarin was significantly higher in the CYP2C9 *1/*3 genotypes than in the CYP2C9 *1/*1 genotypes [ratio of S-warfarin concentration/dose, 0.53 vs. 0.21; p=0.01]. Pharmacodynamic sensitivity in older patients (≥ 75 years) with the CYP2C9 *1/*1 and VKORC1 1173 TT genotypes was significantly higher as compared to younger patients (<75 years) [Ratio of INR/S-warfarin concentration, 4.88 vs. 3.41; p = 0.026]. The CYP2C9*3 allele and old age (≥ 75 years) with the VKORC1 1173 T allele were also associated with increased risk of over-anticoagulation. The increase of over-anticoagulation risk and warfarin sensitivity is related to the CYP2C9*3 allele and old age with the VKORC1 1173 T allele in Korean patients with thromboembolic disease. These findings suggest that a lower initial and maintenance dose should be considered for the patients with CYP2C9 *3 allele and advanced age in this patient population. However, due to the limited number of patients in the study population, our finding needs to be confirmed by a larger, well-controlled study. PMID:22075505

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

    PubMed

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

    2012-01-01

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

  19. Mechanistic studies of the cationic binding pocket of CYP2C9 in vitro and in silico: metabolism of nonionizable analogs of tienilic acid.

    PubMed

    Tay, Suzanne; Le, Hoa; Ford, Kevin A; Nelson, Sid D; Khojasteh, S Cyrus; Rademacher, Peter M

    2014-11-01

    Tienilic acid (TA) is selectively oxidized at the C-5 position of the thiophene ring by the human liver enzyme cytochrome P450 2C9 (CYP2C9). This oxidation is mediated by the proximal positioning of the thiophene over the heme iron, which is proposed to be coordinated by an interaction of the TA carboxylic acid to a cationic binding pocket in the enzyme active site. In this study, we investigated how chemical modification of TA influences the bioactivation by CYP2C9. For this investigation, nine analogs of TA were chosen with substitutions on either side of the molecule. We tested three parameters, including CYP2C9 inhibition, metabolic profiling, and in silico docking. Of the 10 compounds tested, only two (TA and a noncarboxyl analog) resulted in competitive and time-dependent inhibition of CYP2C9. Metabolic profiling revealed a trend in which substitution of the carboxylate with nonionizable functional groups resulted in metabolic switching from oxidation of the aromatic ring to dealkylation reactions at the opposite side of the structure. The in silico modeling predicted an opposite binding orientation to that of TA for many analogs, including the 3-thenoyl regio-isomer analog, which contradicts previous models. Together these data show that disrupting interactions with the cationic binding pocket of CYP2C9 will impact the sites of metabolism and inhibition of the enzyme.

  20. 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

  1. High resolution melting method to detect single nucleotide polymorphism of VKORC1 and CYP2C9.

    PubMed

    Chen, Chunxia; Li, Siyue; Lu, Xiaojun; Tan, Bin; Huang, Chunyan; Qin, Li

    2014-01-01

    Single nucleotide polymorphisms (SNPs) of VKORC (1173T/C, rs9934438) and CYP2C9 (1075A/C, rs1057910) are major contributory factors on the sensitivity of warfarin in Chinese. Analysis of the two genomic loci could help warfarin treatment individual from bleeding or thrombosis events. An assay with the advantages of simplicity, speed, high sensitivity and low cost for genotyping is calling for clinical laboratories. High resolution method (HRM) meets these callings, but no study with large sample tested its performance in genotyping of rs9934438 and rs1057910. In this study, we identified polymorphisms of rs9934438 and rs1057910 in 255 unrelated Chinese heart valve replacement patients of Han ethnic group from West China Hospital. The two genomic loci were genotyped by HRM using LightCycler® 480 High Resolution Melting Master on LightCycler® 480 Real-Time PCR instruments (Roche Diagnostics), and all amplified PCR products were sent for direct DNA sequencing. The genotyping of rs1057910 between HRM and sequencing showed perfect 100% concordance. While the concordance of rs9934438 between HRM and sequencing was 99.2%. Unexpected mutation interfered genotyping results of HRM when genotyping rs9934438. HRM is a valuable technique for genotype detection of rs9934438 and rs1057910 to assess individual sensitivity to warfarin, where DNA sequencing is added inevitably sometimes.

  2. 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

  3. 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.

  4. 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

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

    PubMed

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

    2016-01-01

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

  6. In vitro and in vivo assessment of CYP2C9-mediated herb–herb interaction of Euphorbiae Pekinensis Radix and Glycyrrhizae Radix

    PubMed Central

    Wang, Xinmin; Peng, Yunru; Jing, Xinyue; Qian, Dawei; Tang, Yuping; Duan, Jin-ao

    2014-01-01

    According to traditional Chinese medicine theories, Euphorbiae Pekinensis Radix and Glycyrrhizae Radix should not be used together in one prescription, because their interaction leads to an unexpected consequence. However, the mechanism remains unclear. The purpose of this study was to find out whether CYP2C9 was involved in this herb–herb interaction by using tolbutamide as a probe substrate in vivo and in vitro. Both Euphorbiae Pekinensis Radix and Glycyrrhizae Radix showed induction activity toward CYP2C9, while the combination of them showed a more potent induction activity toward CYP2C9 in vivo. In vitro study revealed only the combination of the herbs could induce the activity of CYP2C9. Thus, both in vivo and in vitro study indicated combination of Glycyrrhizae Radix and Euphorbiae Pekinensis Radix could induce the activity of CYP2C9 to a high level, which may result in decreased plasma levels of major active ingredients of these two herbs, as well as other herbs in the prescriptions. Further research also appears to be necessary to identify the main enzymes involved in the metabolism of the active ingredients in Glycyrrhizae Radix and Euphorbiae Pekinensis Radix. PMID:25202272

  7. 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.

  8. CYP2C9 genotype vs. metabolic phenotype for individual drug dosing--a correlation analysis using flurbiprofen as probe drug.

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Analysis of genetic variations in CYP2C9, CYP2C19, CYP2D6 and CYP3A5 genes using oligonucleotide microarray

    PubMed Central

    Dong, Yuanyuan; Xiao, Huasheng; Wang, Qi; Zhang, Chunxiu; Liu, Xiuming; Yao, Na; Sheng, Haihui; Li, Haiyan

    2015-01-01

    The cytochrome P450 enzymes play a critical role in the metabolism of many commonly prescribed drugs. Among them, the most important enzymes are highly polymorphic CYP2C9, CYP2C19, CYP2D6 and CYP3A5, which are responsible for about 40% of the metabolism of clinical used drugs. Here we developed a novel CYP450 oligonucleotide microarray that allow for detection of 32 known variations of CYP genes from a single multiplex reaction, including 19 polymorphisms of CYP2D6 gene, 8 polymorphisms of CYP2C9 gene, 4 polymorphisms of CYP2C19 gene and 1 polymorphism of CYP3A5 gene. 229 genomic DNA samples from unrelated Han subjects were analyzed. The microarray results showed to have high call rate and accuracy according to concordance with genotypes identified by independent bidirectional sequencing. Furthermore, we found that the major CYP2C9, CYP2C19, CYP2D6 and CYP3A5 alleles in Chinese Han population were CYP2C9*3 (allelic frequency of 10.7%), CYP2C9*2 (20.31%), CYP2C19*2 (5.68%), CYP2D6*10 (58.52%), CYP2D6*2 (13.76) and CYP3A5*3 (70.69%). With flexible DNA preparation, the microarray can significantly facilitates the process of detecting genetics variations in CYP2C9, CYP2C19, CYP2D6 and CYP3A5 gene and provide safe and effective therapy for individual patients. PMID:26770516

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. Structure, Raman and infrared spectroscopic properties of new nonlinear optical material Na3VO2B6O11

    NASA Astrophysics Data System (ADS)

    Zhang, Ji; Dou, Renqin; Zhang, Deming; Zhang, Qingli; Yin, Shaotang

    2016-08-01

    In this paper we report on the structure of Na3VO2B6O11 (NVBO) single crystals which were investigated by XRD, polarized Raman spectra in the range from 10 to 1600 cm-1 and infrared spectrum (IR) in the range from 100 to 1600 cm-1. Factor group analysis has been used to study the full vibrational representation of the crystal. More than 120 phonon modes have been obtained, which are related to Bsbnd O and Vsbnd O vibration in the trigonal planar BO3 triangle groups and tetrahedral BO4/VO4 groups. The high frequency bands located at 1300-1415 cm-1 are assigned to stretching modes of the trigonal planar BO3 groups. Moreover, intense Raman modes located at 631 cm-1 is related to BO3 bending vibration as well. The weak band at 1158 cm-1 (A1 mode) and strong band at 431 cm-1 (A1 mode) are attributed to asymmetric stretching and bending vibration mode of tetrahedral BO4 groups respectively. The vibrational band at 765-738 cm-1 in the Raman spectra of NVBO crystal maybe related to the breathing vibration of the boroxol ring consisting of two BO4 tetrahedra. In addition, we assigned the intense band 900 (A1 mode) and 831 cm-1 (B2 mode) are relative to the v1 symmetric stretching vibration of VO4 tetrahedra. And the middle intense band at 382 (A1 mode) and 385 (A2 mode) are due to Osbnd Vsbnd O vibration in VO4 tetrahedra.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

    Bogdan, Renee

    2014-01-01

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

  19. Cytochrome P450 2C8 ω3-Long-Chain Polyunsaturated Fatty Acid Metabolites Increase Mouse Retinal Pathologic Neovascularization—Brief Report

    PubMed Central

    Shao, Zhuo; Fu, Zhongjie; Stahl, Andreas; Joyal, Jean-Sébastien; Hatton, Colman; Juan, Aimee; Hurst, Christian; Evans, Lucy; Cui, Zhenghao; Pei, Dorothy; Gong, Yan; Xu, Dan; Tian, Katherine; Bogardus, Hannah; Edin, Matthew L.; Lih, Fred; Sapieha, Przemyslaw; Chen, Jing; Panigrahy, Dipak; Hellstrom, Ann; Zeldin, Darryl C.; Smith, Lois E.H.

    2014-01-01

    Objective Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown. Approach and Results The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8–overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays. Conclusions Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization. PMID:24458713

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

    NASA Astrophysics Data System (ADS)

    Schneider, Elizabeth Ann

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

  1. Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs

    PubMed Central

    Muthiah, Yasotha Devi; Ong, Chin Eng; Sulaiman, Siti Amrah; Ismail, Rusli

    2016-01-01

    Background: In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine. Objective: This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism. Materials and Methods: The selected herbs, such as Eurycoma longifolia Jack (ELJ), Labisia pumila (LP), Echinacea purpurea (EP), Andrographis paniculata (AP), and Ginkgo biloba (GB), were subjected to inhibition studies using an in vitro CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC50), and Ki values were determined to study the potency and mode of inhibition. Results: All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at Ki's of 2 and 4 times the Ki of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a Ki6 times larger than quercetin Ki. AP and EP, on the other hand, showed only weak inhibition. Conclusion: The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. SUMMARY Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their in vitro inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP

  2. Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs

    PubMed Central

    Muthiah, Yasotha Devi; Ong, Chin Eng; Sulaiman, Siti Amrah; Ismail, Rusli

    2016-01-01

    Background: In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine. Objective: This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism. Materials and Methods: The selected herbs, such as Eurycoma longifolia Jack (ELJ), Labisia pumila (LP), Echinacea purpurea (EP), Andrographis paniculata (AP), and Ginkgo biloba (GB), were subjected to inhibition studies using an in vitro CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC50), and Ki values were determined to study the potency and mode of inhibition. Results: All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at Ki's of 2 and 4 times the Ki of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a Ki6 times larger than quercetin Ki. AP and EP, on the other hand, showed only weak inhibition. Conclusion: The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. SUMMARY Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their in vitro inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP

  3. Mechanistic analysis of the inactivation of cytochrome P450 2B6 by phencyclidine: effects on substrate binding, electron transfer, and uncoupling.

    PubMed

    Shebley, Mohamad; Kent, Ute M; Ballou, David P; Hollenberg, Paul F

    2009-04-01

    Phencyclidine (PCP) is a mechanism-based inactivator of cytochrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphetamine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-octylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k(1) = 5.0 s(-1), A(1) = 30%; k(2) = 0.02 s(-1), A(2) = 70%, where A(2) indicates the fractional magnitude of the second phase) compared with the native enzyme (k(1) = 8.0 s(-1), A(1) = 58%; k(2) = 0.3 s(-1), A(2) = 42%). Analysis of benzphetamine metabolism by the inactivated protein using liquid chromatography/electrospray ionization/mass spectrometry showed that the rates of formation of nor-benzphetamine and hydroxylated nor-benzphetamine were decreased by 75 and 69%, respectively, whereas the rates of formation for amphetamine, hydroxybenzphetamine, and methamphetamine showed slight but statistically insignificant decreases after the inactivation. The rate of reduction of P450 2B6 by NADPH and reductase was decreased by 6-fold as a result of the modification by PCP. In addition, the extent of uncoupling of NADPH oxidation from product formation, a process leading to futile production of H(2)O(2), increased significantly during the metabolism of ethylbenzene as a result of the inactivation.

  4. Structural and biophysical characterization of human cytochromes P450 2B6 and 2A6 bound to volatile hydrocarbons: analysis and comparison.

    PubMed

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

    2015-04-01

    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.

  5. 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

  6. CYP2C8-derived epoxyeicosatrienoic acids decrease oxidative stress-induced endothelial apoptosis in development of atherosclerosis: Role of Nrf2 activation.

    PubMed

    Liu, Wan-jun; Wang, Tao; Wang, Bei; Liu, Xin-tian; He, Xing-wei; Liu, Yu-jian; Li, Zhu-xi; Tan, Rong; Zeng, He-song

    2015-10-01

    The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling. PMID:26489615

  7. Allele and genotype frequencies of CYP2C9, CYP2C19 and CYP2D6 in an Italian population.

    PubMed

    Scordo, Maria Gabriella; Caputi, Achille P; D'Arrigo, Concetta; Fava, Giuseppina; Spina, Edoardo

    2004-08-01

    The polymorphic cytochrome P450 isoenzymes (CYPs) 2C9, 2C19 and 2D6 metabolise many important drugs, as well as other xenobiotics. Their polymorphism gives rise to important interindividual and interethnic variability in the metabolism and disposition of several therapeutic agents and may cause differences in the clinical response to these drugs. In this study, we determined the genotype profile of a random Italian population in order to compare the CYP2C9, CYP2C19 and CYP2D6 allele frequencies among Italians with previous findings in other Caucasian populations. Frequencies for the major CYP2C9, CYP2C19 and CYP2D6 mutated alleles and genotypes have been evaluated in 360 unrelated healthy Italian volunteers (210 males and 150 females, aged 19-52 years). Genotyping has been carried out on peripheral leukocytes DNA by molecular biology techniques (PCR, RFLP, long-PCR). CYP2C9, CYP2C19 and CYP2D6 allele and genotype frequencies resulted in equilibrium with the Hardy-Weinberg equation. One hundred and fourteen subjects (31.7%) carried one and 23 subjects (6.4%) carried two CYP2C9 mutated alleles. Sixty-eight (18.9%) volunteers were found to be heterozygous and six (1.7%) homozygous for the CYP2C19*2, while no CYP2C19*3 was detected in the evaluated population. Volunteers could be divided into four CYP2D6 genotypes groups: 192 subjects (53.3%) with no mutated alleles (homozygous extensive metabolisers, EM), 126 (35.0%) with one mutated allele (heterozygous EM), 12 (3.4%) with two mutated alleles (poor metabolisers, PM) and 30 (8.3%) with extracopies of a functional gene (ultrarapid metabolisers, UM). Frequencies of both CYP2C9 and CYP2C19 allelic variants, as well as CYP2D6 detrimental alleles, in Italian subjects were similar to those of other Caucasian populations. Conversely, the prevalence of CYP2D6 gene duplication among Italians resulted very high, confirming the higher frequency of CYP2D6 UM in the Mediterranean area compared to Northern Europe. PMID:15177309

  8. In Vitro and In Vivo Drug-Drug Interaction Studies to Assess the Effect of Abiraterone Acetate, Abiraterone, and Metabolites of Abiraterone on CYP2C8 Activity.

    PubMed

    Monbaliu, Johan; Gonzalez, Martha; Bernard, Apexa; Jiao, James; Sensenhauser, Carlo; Snoeys, Jan; Stieltjes, Hans; Wynant, Inneke; Smit, Johan W; Chien, Caly

    2016-10-01

    Abiraterone acetate, the prodrug of the cytochrome P450 C17 inhibitor abiraterone, plus prednisone is approved for treatment of metastatic castration-resistant prostate cancer. We explored whether abiraterone interacts with drugs metabolized by CYP2C8, an enzyme responsible for the metabolism of many drugs. Abiraterone acetate and abiraterone and its major metabolites, abiraterone sulfate and abiraterone sulfate N-oxide, inhibited CYP2C8 in human liver microsomes, with IC50 values near or below the peak total concentrations observed in patients with metastatic castration-resistant prostate cancer (IC50 values: 1.3-3.0 µM, 1.6-2.9 µM, 0.044-0.15 µM, and 5.4-5.9 µM, respectively). CYP2C8 inhibition was reversible and time-independent. To explore the clinical relevance of the in vitro data, an open-label, single-center study was conducted comprising 16 healthy male subjects who received a single 15-mg dose of the CYP2C8 substrate pioglitazone on day 1 and again 1 hour after the administration of abiraterone acetate 1000 mg on day 8. Plasma concentrations of pioglitazone, its active M-III (keto derivative) and M-IV (hydroxyl derivative) metabolites, and abiraterone were determined for up to 72 hours after each dose. Abiraterone acetate increased exposure to pioglitazone; the geometric mean ratio (day 8/day 1) was 125 [90% confidence interval (CI), 99.9-156] for Cmax and 146 (90% CI, 126-171) for AUClast Exposure to M-III and M-IV was reduced by 10% to 13%. Plasma abiraterone concentrations were consistent with previous studies. These results show that abiraterone only weakly inhibits CYP2C8 in vivo. PMID:27504016

  9. In Vitro and In Vivo Drug-Drug Interaction Studies to Assess the Effect of Abiraterone Acetate, Abiraterone, and Metabolites of Abiraterone on CYP2C8 Activity.

    PubMed

    Monbaliu, Johan; Gonzalez, Martha; Bernard, Apexa; Jiao, James; Sensenhauser, Carlo; Snoeys, Jan; Stieltjes, Hans; Wynant, Inneke; Smit, Johan W; Chien, Caly

    2016-10-01

    Abiraterone acetate, the prodrug of the cytochrome P450 C17 inhibitor abiraterone, plus prednisone is approved for treatment of metastatic castration-resistant prostate cancer. We explored whether abiraterone interacts with drugs metabolized by CYP2C8, an enzyme responsible for the metabolism of many drugs. Abiraterone acetate and abiraterone and its major metabolites, abiraterone sulfate and abiraterone sulfate N-oxide, inhibited CYP2C8 in human liver microsomes, with IC50 values near or below the peak total concentrations observed in patients with metastatic castration-resistant prostate cancer (IC50 values: 1.3-3.0 µM, 1.6-2.9 µM, 0.044-0.15 µM, and 5.4-5.9 µM, respectively). CYP2C8 inhibition was reversible and time-independent. To explore the clinical relevance of the in vitro data, an open-label, single-center study was conducted comprising 16 healthy male subjects who received a single 15-mg dose of the CYP2C8 substrate pioglitazone on day 1 and again 1 hour after the administration of abiraterone acetate 1000 mg on day 8. Plasma concentrations of pioglitazone, its active M-III (keto derivative) and M-IV (hydroxyl derivative) metabolites, and abiraterone were determined for up to 72 hours after each dose. Abiraterone acetate increased exposure to pioglitazone; the geometric mean ratio (day 8/day 1) was 125 [90% confidence interval (CI), 99.9-156] for Cmax and 146 (90% CI, 126-171) for AUClast Exposure to M-III and M-IV was reduced by 10% to 13%. Plasma abiraterone concentrations were consistent with previous studies. These results show that abiraterone only weakly inhibits CYP2C8 in vivo.

  10. Effect of myricetin on cytochrome P450 isoforms CYP1A2, CYP2C9 and CYP3A4 in rats.

    PubMed

    Guo, Yu-Jin; Zheng, Shuang-Li

    2014-04-01

    Myricetin is one of the main ingredients of Chinese bayberry, which is used as a traditional medicine. The purpose of this study was to find out whether myricetin influences the rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated for 14 days with myricetin. 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 corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Our study showed that treatment with multiple doses of myricetin had no effects on rat CYP1A2. However, CYP2C9 and CYP3A4 enzyme activities were inhibited after multiple doses of myricetin. Therefore, caution is needed when myricetin is co-administered with CYP2C9 or CYP3A4 substrates, which may result in herb-drug interactions.

  11. Inhibitory effects of phthalimide derivatives on the activity of the hepatic cytochrome P450 monooxygenases CYP2C9 and CYP2C19.

    PubMed

    Kolukisaoglu, Üner; Wendler, Christian; Goerdes, Dirk; Diener, Annette; Thurow, Kerstin

    2010-12-01

    Affecting hepatic cytochrome (CYP) activity is one of the major concerns in drug-drug interaction. Thus the testing of drug candidates on their impact on these enzymes is an essential step in early drug discovery. We tested a collection of 480 in-house phthalimide derivatives against different CYP450s using a high throughput inhibition assay. In initial tests with the isoform CYP2C19 about 57.5% of the tested phthalimide derivatives showed significantly enhanced inhibitory effects against this enzyme. In addition similar patterns of phthalimide inhibition for CYP2C9 and CYP2C19 were found, whereas the unrelated isoforms CYP2D6 and CYP3A4 were not specifically affected. Also less than 10% of randomly chosen substances inhibited CYP2C9. Analyses of structure-function relationships revealed that the substituent at the nitrogen atom in the isoindole ring is of crucial impact for the activity of CYP2C9/19.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Genome-wide association study of plasma levels of polychlorinated biphenyls disclose an association with the CYP2B6 gene in a population-based sample

    PubMed Central

    Ng, Esther; Salihovic, Samira; Monica Lind, P.; Mahajan, Anubha; Syvänen, Anne-Christine; Axelsson, Tomas; Ingelsson, Erik; Lindgren, Cecilia M.; van Bavel, Bert; Morris, Andrew P.; Lind, Lars

    2015-01-01

    Background Polychlorinated biphenyls (PCBs) are a group of man-made environmental pollutants which accumulate in humans with adverse health effects. To date, very little effort has been devoted to the study of the metabolism of PCBs on a genome-wide level. Objectives Here, we conducted a genome-wide association study (GWAS) to identify genomic regions involved in the metabolism of PCBs. Methods Plasma levels of 16 PCBs ascertained in a cohort of elderly individuals from Sweden (n=1016) were measured using gas chromatography–high resolution mass spectrophotometry (GC-HRMS). DNA samples were genotyped on the Infinium Omni Express bead microarray, and imputed up to reference panels from the 1000 Genomes Project. Association testing was performed in a linear regression framework under an additive model. Results Plasma levels of PCB-99 demonstrated genome-wide significant association with single nucleotide polymorphisms (SNPs) mapping to chromosome 19q13.2. The SNP with the strongest association was rs8109848 (p=3.7×10−13), mapping to an intronic region of CYP2B6. Moreover, when all PCBs were conditioned on PCB-99, further signals were revealed for PCBs -74, -105 and -118, mapping to the same genomic region. The lead SNPs were rs8109848 (p=3.8×10−12) for PCB-118, rs4802104 (p=1.4×10−9) for PCB-74 and rs4803413 (p=2.5×10−9) for PCB-105, all of which map to CYP2B6. Conclusions In our study, we found plasma levels of four lower-chlorinated PCBs to be significantly associated with the genetic region mapping to the CYP2B6 locus. These findings show that CYP2B6 is of importance for the metabolism of PCBs in humans, and may help to identify individuals who may be susceptible to PCB toxicity. PMID:25839716

  18. 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.

  19. Influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to organophosphate pesticides.

    PubMed

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

    2012-01-24

    Previous studies have revealed that organophosphate pesticides (OPs) are primarily metabolized by xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticides-exposed workers. Present study was designed to determine the influence of CYP2C9, GSTM1, GSTT1 and NAT2 genetic polymorphisms on DNA damage in workers occupationally exposed to OPs. We examined 268 subjects including 134 workers occupationally exposed to OPs and an equal number of normal healthy controls. The DNA damage was evaluated using alkaline comet assay and genotyping was done using individual polymerase chain reaction (PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Acetylcholinesterase and paraoxonase activity were found to be significantly lowered in workers as compared to control subjects which were analyzed as biomarkers of toxicity due to OPs exposure (p<0.001). Workers showed significantly higher DNA tail moment (TM) compared to control subjects (14.32±2.17 vs. 6.24±1.37 tail % DNA, p<0.001). GSTM1 null genotype was found to influence DNA TM in workers (p<0.05). DNA TM was also found to be increased with concomitant presence of NAT2 slow acetylation and CYP2C9*3/*3 or GSTM1 null genotypes (p<0.05). DNA TM was found increased in NAT2 slow acetylators with mild and heavy smoking habits in control subjects and workers, respectively (p<0.05). The results of this study suggest that GSTM1 null genotypes, and an association of NAT2 slow acetylation genotypes with CYP2C9*3/*3 or GSTM1 null genotypes may modulate DNA damage in workers occupationally exposed to OPs.

  20. Warfarin pharmacogenetics: polymorphisms of the CYP2C9, CYP4F2, and VKORC1 loci in a genetically admixed Omani population.

    PubMed

    Pathare, Anil V; Al Zadjali, Shoaib; Misquith, Rhea; Alkindi, Salam S; Panjwani, Vinodh; Lapoumeroulie, Claudine; Pravin, Sahaya; Paldi, Andras; Krishnamoorthy, Rajagopal

    2012-02-01

    This is the first study to evaluate the spectrum and prevalence of dose-predictive genetic polymorphisms of the CYP2C9, CYP4F2 and VKORC1 loci together, in a geographically defined, ethnically admixed healthy adult Omani population sharing common lifestyle/environmental factors. Since the present-day Omani population is the result of an admixture of Caucasian, African and Asian ancestries, we compared the pharmacogenetic profile of these three loci in this population. Interestingly, the Omani pharmacogenetic profile, in terms of allele and genotype distribution, has values that are intermediate between Caucasians and African Americans, the African admixture further substantiated by the presence of the CYP2C9*8 allele. However, limitations and usefulness of such comparisons warrant caution, as the data from pharmacogenetic literature do not always represent bona fide population categories. Furthermore, definition of study population based on microgeographical scale would be more appropriate in pharmacogenetic research rather than the flawed racial, ethnic, or social categorizations since pharmacogenetic variation is clinal, and genetic influences will be further altered by lifestyle and environmental factors. PMID:22452429

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

    PubMed

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

    2016-01-01

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

  2. 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.

  3. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    PubMed

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  4. A Genome-Wide Association Study Confirms VKORC1, CYP2C9, and CYP4F2 as Principal Genetic Determinants of Warfarin Dose

    PubMed Central

    Bourgeois, Stephane; Barnes, Chris; Eriksson, Niclas; Soranzo, Nicole; Whittaker, Pamela; Ranganath, Venkatesh; Kumanduri, Vasudev; McLaren, William; Holm, Lennart; Lindh, Jonatan; Rane, Anders; Wadelius, Mia; Deloukas, Panos

    2009-01-01

    We report the first genome-wide association study (GWAS) whose sample size (1,053 Swedish subjects) is sufficiently powered to detect genome-wide significance (p<1.5×10−7) for polymorphisms that modestly alter therapeutic warfarin dose. The anticoagulant drug warfarin is widely prescribed for reducing the risk of stroke, thrombosis, pulmonary embolism, and coronary malfunction. However, Caucasians vary widely (20-fold) in the dose needed for therapeutic anticoagulation, and hence prescribed doses may be too low (risking serious illness) or too high (risking severe bleeding). Prior work established that ∼30% of the dose variance is explained by single nucleotide polymorphisms (SNPs) in the warfarin drug target VKORC1 and another ∼12% by two non-synonymous SNPs (*2, *3) in the cytochrome P450 warfarin-metabolizing gene CYP2C9. We initially tested each of 325,997 GWAS SNPs for association with warfarin dose by univariate regression and found the strongest statistical signals (p<10−78) at SNPs clustering near VKORC1 and the second lowest p-values (p<10−31) emanating from CYP2C9. No other SNPs approached genome-wide significance. To enhance detection of weaker effects, we conducted multiple regression adjusting for known influences on warfarin dose (VKORC1, CYP2C9, age, gender) and identified a single SNP (rs2108622) with genome-wide significance (p = 8.3×10−10) that alters protein coding of the CYP4F2 gene. We confirmed this result in 588 additional Swedish patients (p<0.0029) and, during our investigation, a second group provided independent confirmation from a scan of warfarin-metabolizing genes. We also thoroughly investigated copy number variations, haplotypes, and imputed SNPs, but found no additional highly significant warfarin associations. We present power analysis of our GWAS that is generalizable to other studies, and conclude we had 80% power to detect genome-wide significance for common causative variants or markers explaining at least 1

  5. 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.

  6. 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

  7. Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A

    PubMed Central

    Turpault, Sandrine; Brian, William; Van Horn, Robert; Santoni, Alix; Poitiers, Franck; Donazzolo, Yves; Boulenc, Xavier

    2009-01-01

    AIMS To assess the pharmacokinetics (PK) of selective substrates of CYP1A2 (caffeine), CYP2C9 (S-warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol) and CYP3A (midazolam) when administered orally and concurrently as a cocktail relative to the drugs administered alone. METHODS This was an open-label, single-dose, randomized, six-treatment six-period six-sequence William's design study with a wash-out of 7 or 14 days. Thirty healthy male subjects received 100 mg caffeine, 100 mg metoprolol, 0.03 mg kg−1 midazolam, 20 mg omeprazole and 10 mg warfarin individually and in combination (cocktail). Poor metabolizers of CYP2C9, 2C19 and 2D6 were excluded. Plasma samples were obtained up to 48 h for caffeine, metoprolol and omeprazole, 12 h for midazolam, 312 h for warfarin and the cocktail. Three different validated liquid chromatography tandem mass spectrometry methods were used. Noncompartmental PK parameters were calculated. Log-transformed Cmax, AUClast and AUC for each analyte were analysed with a linear mixed effects model with fixed term for treatment, sequence and period, and random term for subject within sequence. Point estimates (90% CI) for treatment ratios (individual/cocktail) were computed for each analyte Cmax, AUClast and AUC. RESULTS There was no PK interaction between the probe drugs when administered in combination as a cocktail, relative to the probes administered alone, as the 90% CI of the PK parameters was within the prespecified bioequivalence limits of 0.80, 1.25. CONCLUSION The lack of interaction between probes indicates that this cocktail could be used to evaluate the potential for multiple drug–drug interactions in vivo. PMID:20002088

  8. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    PubMed

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  9. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6

    PubMed Central

    Ke, A; Barter, Z; Rowland‐Yeo, K

    2016-01-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug‐drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0–1.7‐fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz. PMID:27435752

  10. Towards a Best Practice Approach in PBPK Modeling: Case Example of Developing a Unified Efavirenz Model Accounting for Induction of CYPs 3A4 and 2B6.

    PubMed

    Ke, A; Barter, Z; Rowland-Yeo, K; Almond, L

    2016-07-01

    In this study, we present efavirenz physiologically based pharmacokinetic (PBPK) model development as an example of our best practice approach that uses a stepwise approach to verify the different components of the model. First, a PBPK model for efavirenz incorporating in vitro and clinical pharmacokinetic (PK) data was developed to predict exposure following multiple dosing (600 mg q.d.). Alfentanil i.v. and p.o. drug-drug interaction (DDI) studies were utilized to evaluate and refine the CYP3A4 induction component in the liver and gut. Next, independent DDI studies with substrates of CYP3A4 (maraviroc, atazanavir, and clarithromycin) and CYP2B6 (bupropion) verified the induction components of the model (area under the curve [AUC] ratios within 1.0-1.7-fold of observed). Finally, the model was refined to incorporate the fractional contribution of enzymes, including CYP2B6, propagating autoinduction into the model (Racc 1.7 vs. 1.7 observed). This validated mechanistic model can now be applied in clinical pharmacology studies to prospectively assess both the victim and perpetrator DDI potential of efavirenz.

  11. Secondary metabolism pathway polymorphisms and plasma efavirenz concentrations in HIV-infected adults with CYP2B6 slow metabolizer genotypes

    PubMed Central

    Haas, David W.; Kwara, Awewura; Richardson, Danielle M.; Baker, Paxton; Papageorgiou, Ioannis; Acosta, Edward P.; Morse, Gene D.; Court, Michael H.

    2014-01-01

    Objectives Efavirenz is widely prescribed for HIV-1 infection, and CYP2B6 polymorphisms 516G→T and 983T→C define efavirenz slow metabolizer genotypes. To identify genetic predictors of higher plasma efavirenz concentrations beyond these two common functional alleles, we characterized associations with mid-dosing interval efavirenz concentrations in 84 HIV-infected adults, all carrying two copies of these major loss-of-function CYP2B6 alleles. Methods Study participants had been randomized to efavirenz-containing regimens in prospective clinical trials and had available plasma efavirenz assay data. Analyses focused on secondary metabolism pathway polymorphisms CYP2A6 -48T→G (rs28399433), UGT2B7 735A→G (rs28365062) and UGT2B7 802T→C (rs7439366). Exploratory analyses also considered 196 polymorphisms and 8 copy number variants in 41 drug metabolism/transport genes. Mid-dosing interval efavirenz concentrations at steady-state were obtained ≥8 h but <19 h post-dose. Linear regression was used to test for associations between polymorphisms and log-transformed efavirenz concentrations. Results Increased efavirenz concentrations were associated with CYP2A6 -48T→G in all subjects (P = 3.8 × 10−4) and in Black subjects (P = 0.027) and White subjects (P = 0.0011) analysed separately; and with UGT2B7 735 G/G homozygosity in all subjects (P = 0.006) and in Black subjects (P = 0.046) and White subjects (P = 0.062) analysed separately. In a multivariable model, CYP2A6 -48T→G and UGT2B7 735 G/G homozygosity remained significant (P < 0.05 for each). No additional polymorphisms or copy number variants were significantly associated with efavirenz concentrations. Conclusions Among individuals with a CYP2B6 slow metabolizer genotype, CYP2A6 and possibly UGT2B7 polymorphisms contribute to even higher efavirenz concentrations. PMID:24729586

  12. Novel conjugated polymers based on dithieno[3,2-b:6,7-b]carbazole for solution processed thin-film transistors.

    PubMed

    Chen, Yagang; Liu, Chengfang; Tian, Hongkun; Bao, Cheng; Zhang, Xiaojie; Yan, Donghang; Geng, Yanhou; Wang, Fosong

    2012-10-26

    Two conjugated polymers (CPs) P-tCzC12 and P-tCzC16 comprising alternating dithieno[3,2-b:6,7-b]carbazole and 4,4'-dihexadecyl-2,2'-bithiophene units have been designed and synthesized. Upon thermal annealing, they can form ordered thin films in which the polymer backbones dominantly adopted an edge-on orientation respective to the substrate with a lamellar spacing of ≈24 Å and a π-stacking distance of ≈3.7 Å. Organic thin-film transistors (OTFTs) were fabricated by solution casting. A hole mobility of 0.39 cm(2) V(-1) s(-1) has been demonstrated with P-tCzC16. This value is the highest among the CPs containing heteroacenes larger than 4 rings.

  13. Evidence of "new hot spots" from determining the nonlinear optical behavior of materials: mechanistic studies of the vanadium borate crystal, Na3VO2B6O11.

    PubMed

    Su, Xin; Yang, Zhihua; Lee, Ming-Hsien; Pan, Shilie; Wang, Ying; Fan, Xiaoyun; Huang, Zhenjun; Zhang, Bingbing

    2015-02-21

    A novel mechanism for the nonlinear optical (NLO) effects of vanadium borate crystals, Na3VO2B6O11 (NVB), with distorted VO4 groups was investigated. A comprehensive analysis of the structure-property relationship was performed by combining the experimental measurements, the electronic structures calculations, the SHG-weighted electron density and the real-space atom-contribution analysis to yield the linear and nonlinear optical properties. The contribution of a (VO4)(3-) anionic group to the second harmonic generation (SHG) response was more pronounced than that of the (BO3)(3-) anionic group, which plays a virtual role in the SHG effects in NVB. The anionic (BO3)(3-) groups make dominant contributions to the birefringence, whereas the contribution of the V(5+) cations to these linear optical effects is negligible.

  14. Evidence of "new hot spots" from determining the nonlinear optical behavior of materials: mechanistic studies of the vanadium borate crystal, Na3VO2B6O11.

    PubMed

    Su, Xin; Yang, Zhihua; Lee, Ming-Hsien; Pan, Shilie; Wang, Ying; Fan, Xiaoyun; Huang, Zhenjun; Zhang, Bingbing

    2015-02-21

    A novel mechanism for the nonlinear optical (NLO) effects of vanadium borate crystals, Na3VO2B6O11 (NVB), with distorted VO4 groups was investigated. A comprehensive analysis of the structure-property relationship was performed by combining the experimental measurements, the electronic structures calculations, the SHG-weighted electron density and the real-space atom-contribution analysis to yield the linear and nonlinear optical properties. The contribution of a (VO4)(3-) anionic group to the second harmonic generation (SHG) response was more pronounced than that of the (BO3)(3-) anionic group, which plays a virtual role in the SHG effects in NVB. The anionic (BO3)(3-) groups make dominant contributions to the birefringence, whereas the contribution of the V(5+) cations to these linear optical effects is negligible. PMID:25609419

  15. 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.

  16. In Silico Predictions of Drug - Drug Interactions Caused by CYP1A2, 2C9 and 3A4 Inhibition - a Comparative Study of Virtual Screening Performance.

    PubMed

    Kaserer, Teresa; Höferl, Martina; Müller, Klara; Elmer, Sebastian; Ganzera, Markus; Jäger, Walter; Schuster, Daniela

    2015-06-01

    The cytochrome P450 (CYP) superfamily represents the major enzyme class responsible for the metabolism of exogenous compounds. Investigation of clearance pathways is therefore an integral part in early drug development, as any alteration of metabolic enzymes may markedly influence the toxicological profile and efficacy of novel compounds. In silico methods are widely applied in drug development to complement experimental approaches. Several different tools are available for that purpose, however, for CYP enzymes they have only been applied retrospectively so far. Within this study, pharmacophore- and shape-based models and a docking protocol were generated for the prediction of CYP1A2, 2C9, and 3A4 inhibition. All theoretically validated models, the validated docking workflow, and additional external bioactivity profiling tools were applied independently and in parallel to predict the CYP inhibition of 29 compounds from synthetic and natural origin. After subsequent experimental assessment of the in silico predictions, we analyzed and compared the prospective performance of all methods, thereby defining the suitability of the applied techniques for CYP enzymes. We observed quite substantial differences in the performances of the applied tools, suggesting that the rational selection of that virtual screening method that proved to perform best can largely improve the success rates when it comes to CYP inhibition prediction. PMID:27490388

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

    PubMed Central

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

    2015-01-01

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

  18. Observed trends in ambient concentrations of C 2-C 8 hydrocarbons in the United Kingdom over the period from 1993 to 2004

    NASA Astrophysics Data System (ADS)

    Dollard, G. J.; Dumitrean, P.; Telling, S.; Dixon, J.; Derwent, R. G.

    Hourly measurements of up to 26 C 2-C 8 hydrocarbons have been made at eight urban background sites, three urban-industrial sites, a kerbside and a rural site in the UK from 1993 onwards up until the end of December 2004. Average annual mean benzene and 1,3-butadiene concentrations at urban background locations have declined at about -20% per year and the observed declines have exactly mimicked the inferred declines in benzene and 1,3-butadiene emissions over the same period. Ninety-day rolling mean concentrations of ethylene, propylene, n- and i-butane, n- and i-pentane, isoprene and propane at urban and rural sites have also declined steadily by between -10% and -30% per year. Rolling mean concentrations of acetylene, 2- and 3-methylpentane, n-hexane, n-heptane, cis- and trans-but-2-ene, cis- and trans-pent-2-ene, toluene, ethylbenzene and o-, m- and p-xylene at a roadside location in London have all declined at between -14% and -21% per year. These declines demonstrate that motor vehicle exhaust catalysts and evaporative canisters have effectively and efficiently controlled vehicular emissions of hydrocarbons in the UK. Urban ethane concentrations arising largely from natural gas leakage have remained largely unchanged over this same period.

  19. Structural and energetic analysis to provide insight residues of CYP2C9, 2C11 and 2E1 involved in valproic acid dehydrogenation selectivity.

    PubMed

    Bello, Martiniano; Mendieta-Wejebe, Jessica E; Correa-Basurto, José

    2014-07-15

    Docking and molecular dynamics (MD) simulation have been two computational techniques used to gain insight about the substrate orientation within protein active sites, allowing to identify potential residues involved in the binding and catalytic mechanisms. In this study, both methods were combined to predict the regioselectivity in the binding mode of valproic acid (VPA) on three cytochrome P-450 (CYP) isoforms CYP2C9, CYP2C11, and CYP2E1, which are involved in the biotransformation of VPA yielding reactive hepatotoxic intermediate 2-n-propyl-4-pentenoic acid (4nVPA). There are experimental data about hydrogen atom abstraction of the C4-position of VPA to yield 4nVPA, however, there are not structural evidence about the binding mode of VPA and 4nVPA on CYPs. Therefore, the complexes between these CYP isoforms and VPA or 4nVPA were studied to explore their differences in binding and energetic stabilization. Docking results showed that VPA and 4nVPA are coupled into CYPs binding site in a similar conformation, but it does not explain the VPA hydrogen atom abstraction. On the other hand, MD simulations showed a set of energetic states that reorient VPA at the first ns, then making it susceptible to a dehydrogenation reaction. For 4nVPA, multiple binding modes were observed in which the different states could favor either undergo other reaction mechanism or ligand expulsion from the binding site. Otherwise, the energetic and entropic contribution point out a similar behavior for the three CYP complexes, showing as expected a more energetically favorable binding free energy for the complexes between CYPs and VPA than with 4nVPA.

  20. Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Clopidogrel: Involvement of Both Covalent Modification of Cysteinyl Residue 475 and Loss of HemeS⃞

    PubMed Central

    Zhang, Haoming; Amunugama, Hemali; Ney, Sarah; Cooper, Nyemade

    2011-01-01

    We have investigated the mechanisms by which clopidogrel inactivates human cytochrome P450 2B6 (CYP2B6) in a reconstituted system. It was found that clopidogrel and its thiolactone metabolite, 2-oxo-clopidogrel, both inactivate CYP2B6 in a time- and concentration-dependent manner. On the basis of kinact/KI ratios, clopidogrel is approximately 5 times more efficient than 2-oxo-clopidogrel in inactivating CYP2B6. Analysis of the molecular mass of the CYP2B6 wild-type (WT) protein that had been inactivated by either clopidogrel or 2-oxo-clopidogrel showed an increase in the mass of the protein by ∼350 Da. This increase in the protein mass corresponds to the addition of the active metabolite of clopidogrel to CYP2B6. It is noteworthy that this adduct can be cleaved from the protein matrix by incubation with dithiothreitol, confirming that the active metabolite is linked to a cysteinyl residue of CYP2B6 via a disulfide bond. Peptide mapping of tryptic digests of the inactivated CYP2B6 using electrospray ionization liquid chromatography-tandem mass spectrometry identified Cys475 as the site of covalent modification by the active metabolite. This was further confirmed by the observation that mutation of Cys475 to a serine residue eliminates the formation of the protein adduct and prevents the C475S variant from mechanism-based inactivation by 2-oxo-clopidogrel. However, this mutation did not prevent the C475S variant from being inactivated by clopidogrel. Furthermore, inactivation of both CYP2B6 WT and C475S by clopidogrel, but not by 2-oxo-clopidogrel, led to the loss of the heme, which accounts for most of the loss of the catalytic activity. Collectively, these results suggest that clopidogrel inactivates CYP2B6 primarily through destruction of the heme, whereas 2-oxo-clopidogrel inactivates CYP2B6 through covalent modification of Cys475. PMID:21862689

  1. 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

  2. Primary role of cytochrome P450 2B6 in the oxidative metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100) to hydroxylated BDEs.

    PubMed

    Gross, Michael S; Butryn, Deena M; McGarrigle, Barbara P; Aga, Diana S; Olson, James R

    2015-04-20

    Human exposure to polybrominated diphenyl ethers (PBDEs) through various routes poses deleterious health effects. PBDEs are biotransformed into hydroxylated metabolites (OH-BDEs) via cytochrome P450s (P450s), which may add to their neurotoxic effects. This study characterizes the in vitro metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), one of the most abundant PBDE congeners found in humans, by recombinant human P450s and pooled human liver microsomes (HLMs). Ten recombinant P450s were individually incubated with BDE-100 to monitor P450-specific metabolism. P450 2B6 was found to be the predominant enzyme responsible for nearly all formation of six mono-OH-pentaBDE and two di-OH-pentaBDE metabolites. Four metabolites were identified as 3-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (3-OH-BDE-100), 5'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (5'-OH-BDE-100), 6'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (6'-OH-BDE-100), and 4'-hydroxy-2,2',4,5',6-pentabromodiphenyl ether (4'-OH-BDE-103) through use of reference standards. The two remaining mono-OH-pentaBDE metabolites were hypothesized using mass spectral fragmentation characteristics of derivatized OH-BDEs, which allowed prediction of an ortho-OH-pentaBDE and a para-OH-pentaBDE positional isomer. Additional information based on theoretical boiling point calculations using COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS) and experimental chromatographic retention times were used to identify the hypothesized metabolites as 2'-hydroxy-2,3',4,4',6-pentabromodiphenyl ether (2'-OH-BDE-119) and 4-hydroxy-2,2',4',5,6-pentabromodiphenyl ether (4-OH-BDE-91), respectively. Kinetic studies of BDE-100 metabolism using P450 2B6 and HLMs revealed Km values ranging from 4.9 to 7.0 μM and 6-10 μM, respectively, suggesting a high affinity toward the formation of OH-BDEs. Compared to the metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99

  3. 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

  4. Content of polyunsaturated fatty acids (PUFAs) in serum and liver of rats fed restricted diets supplemented with vitamins B2, B6 and folic acid.

    PubMed

    Bertrandt, Jerzy; Klos, Anna; Debski, Bogdan

    2004-01-01

    The aim of study was to investigate an influence of nutritional deficiency and dietary addition of vit. B(2), B(6) and folic acid on PUFAs content in rats' serum and liver. Limitation of consumption full value diet to 50% of its previously determined daily consumption, enriched with m/a vitamins, significant decreased of linoleic (LA) and alpha-linolenic (ALA) acids as well as distinctly increased arachidonic (AA) and docosahexaenoic (DHA) acids content in serum in 30th day. In 60th day lower content of AA and DHA fatty acids was found. Nutrition with such diet, lasting 90 days caused decrease of LA content and increase of AA. Diet limitation to its 30% of daily consumption decreased of eicosapentaenoic acid (EPA) and DHA in the 30th day, while AA and DHA content was increased in the 60th day. Distinct decrease of AA content and increase of EPA content were found in the 90th day of experiment. Use of diets, with limited consumption to 50% caused increase of LA and ALA acids content while AA and DHA acids content were significantly decreased in the liver, in 90th day. Limited consumption supplemented diet to 30% caused in liver significant decrease of LA and increase of EPA acids content.

  5. 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. PMID:16608219

  6. 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. PMID:25391641

  7. Toward reduction in animal sacrifice for drugs: molecular modeling of Macaca fascicularis P450 2C20 for virtual screening of Homo sapiens P450 2C8 substrates.

    PubMed

    Rua, Francesco; Di Nardo, Giovanna; Sadeghi, Sheila J; Gilardi, Gianfranco

    2012-01-01

    Macaca fascicularis P450 2C20 shares 92% identity with human cytochrome P450 2C8, which is involved in the metabolism of more than 8% of all prescribed drugs. To date, only paclitaxel and amodiaquine, two substrate markers of the human P450 2C8, have been experimentally confirmed as M. fascicularis P450 2C20 drugs. To bridge the lack of information on the ligands recognized by M. fascicularis P450 2C20, in this study, a three-dimensional homology model of this enzyme was generated on the basis of the available crystal structure of the human homologue P450 2C8 using YASARA. The results indicated that 90.0%, 9.0%, 0.5%, and 0.5% of the residues of the P450 2C20 model were located in the most favorable, allowed, generously allowed, and disallowed regions, respectively. The root-mean-square deviation of the C-alpha superposition of the M. fascicularis P450 2C20 model with the Homo sapiens P450 2C8 was 0.074 Å, indicating a very high similarity of the two structures. Subsequently, the 2C20 model was used for in silico screening of 58 known P450 2C8 substrates and 62 inhibitors. These were also docked in the active site of the crystal structure of the human P450 2C8. The affinity of each compound for the active site of both cytochromes proved to be very similar, meaning that the few key residues that are mutated in the active site of the M. fascicularis P450 do not prevent the P450 2C20 from recognizing the same substrates as the human P450 2C8.

  8. 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

  9. Interactions between CYP2C9 and CYP2C19 in reconstituted binary systems influence their catalytic activity: possible rationale for the inability of CYP2C19 to catalyze methoxychlor demethylation in human liver microsomes.

    PubMed

    Hazai, Eszter; Kupfer, David

    2005-01-01

    Previous studies in our laboratory showed that among cDNA-expressed human cytochrome P450 (P450) supersomes, CYP2C19 was the most active in methoxychlor-O-demethylation. However, based on the lack of inhibition of methoxychlor-O-demethylation by monoclonal anti-CYP2C19 antibodies in human liver microsomes (HLM), CYP2C19 did not seem to catalyze that reaction in HLM. By contrast, CYP2C9, much less active than CYP2C19 in supersomes, was the most active in HLM. The current study examines whether the lack of methoxychlor-O-demethylation by CYP2C19 in HLM was due to CYP2C19 exhibiting inferior competition for the NADPH-cytochrome P450 reductase (CPR) versus CYP2C9 and explores the interactions between CYP2C9 and CYP2C19 in a singular and binary complex of a reconstituted system. When reconstituted with CPR, cytochrome b(5), and lipid, purified CYP2C19 and CYP2C9 catalyzed methoxychlor-O-demethylation. However, whereas equimolar CPR to CYP2C9 supported maximal rates of methoxychlor demethylation and diclofenac hydroxylation, the rate of methoxychlor demethylation by CYP2C19 was not fully saturated, even with a 9-fold molar excess of CPR over CYP2C19. This behavior of CYP2C19 was also observed with S-mephenytoin as the substrate. When a binary reconstitution system was prepared by mixing CYP2C9 and CYP2C19 enzymes, methoxychlor-O-demethylation and S-mephenytoin hydroxylation by CYP2C19 were dramatically inhibited. Inhibition depended on the amount of CPR and substrate used. By contrast, in the incubation containing CYP2C9, diclofenac hydroxylation was activated by the presence of CYP2C19. These results show that interactions among P450 enzymes can modulate their catalytic rates, which depend on the substrate undergoing metabolism.

  10. 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.

  11. Cytochrome P450 (CYP2C9*2,*3) & vitamin-K epoxide reductase complex (VKORC1 -1639G

    PubMed Central

    Kaur, Anupriya; Khan, Farah; Agrawal, Suraksha S.; Kapoor, Aditya; Agarwal, Surendra K.; Phadke, Shubha R.

    2013-01-01

    Background & objectives: Studies have demonstrated the effect of CYP2C9 (cytochrome P450) and VKORC1 (vitamin K epoxide reductase complex) gene polymorphisms on the dose of acenocoumarol. The data from India about these gene polymorphisms and their effects on acenocoumarol dose are scarce. The aim of this study was to determine the occurrence of CYP2C9*2,*3 and VKORC 1 -1639G>A gene polymorphisms and to study their effects on the dose of acenocoumarol required to maintain a target International Normalized Ratio (INR) in patients with mechanical heart valve replacement. Methods: Patients from the anticoagulation clinic of a tertiary care hospital in north India were studied. The anticoagulation profile, INR (International Normalized Ratio) values and administered acenocoumarol dose were obtained from the clinical records of patients. Determination of the CYP2C9*2,*3 and VKORC1 -1639G>A genotypes was done by PCR-RFLP (restriction fragment length polymorphism). Results: A total of 111 patients were studied. The genotype frequencies of CYP2C9 *1/*1,*1/*2,*1/*3 were as 0.883, 0.072, 0.036 and that of VKORC1 -1639G>A for GG, AG, and AA genotypes were 0.883, 0.090, and 0.027, respectively. The percentage of patients carrying any of the variant alleles of CYP2C9 and VKORC1 in heterozygous or homozygous form was 34% among those receiving a low dose of ≤20 mg/wk while it was 13.8 per cent in those receiving >20 mg/wk (P=0.014). A tendency of lower dose requirements was seen among carriers of the studied polymorphisms. There was considerable variability in the dose requirements of patients with and without variant alleles. Interpretation & conclusions: The study findings point towards the role of CYP2C9 and VKORC1 gene polymorphisms in determining the inter-individual dose variability of acenocoumarol in the Indian patients with mechanical heart valve replacement. PMID:23481074

  12. A long-standing mystery solved: the formation of 3-hydroxydesloratadine is catalyzed by CYP2C8 but prior glucuronidation of desloratadine by UDP-glucuronosyltransferase 2B10 is an obligatory requirement.

    PubMed

    Kazmi, Faraz; Barbara, Joanna E; Yerino, Phyllis; Parkinson, Andrew

    2015-04-01

    Desloratadine (Clarinex), the major active metabolite of loratadine (Claritin), is a nonsedating long-lasting antihistamine that is widely used for the treatment of allergic rhinitis and chronic idiopathic urticaria. For over 20 years, it has remained a mystery as to which enzymes are responsible for the formation of 3-hydroxydesloratadine, the major active human metabolite, largely due to the inability of any in vitro system tested thus far to generate this metabolite. In this study, we demonstrated that cryopreserved human hepatocytes (CHHs) form 3-hydroxydesloratadine and its corresponding O-glucuronide. CHHs catalyzed the formation of 3-hydroxydesloratadine with a Km of 1.6 μM and a Vmax of 1.3 pmol/min per million cells. Chemical inhibition of cytochrome P450 (P450) enzymes in CHHs demonstrated that gemfibrozil glucuronide (CYP2C8 inhibitor) and 1-aminobenzotriazole (general P450 inhibitor) inhibited 3-hydroxydesloratadine formation by 91% and 98%, respectively. Other inhibitors of CYP2C8 (gemfibrozil, montelukast, clopidogrel glucuronide, repaglinide, and cerivastatin) also caused extensive inhibition of 3-hydroxydesloratadine formation (73%-100%). Assessment of desloratadine, amodiaquine, and paclitaxel metabolism by a panel of individual CHHs demonstrated that CYP2C8 marker activity robustly correlated with 3-hydroxydesloratadine formation (r(2) of 0.70-0.90). Detailed mechanistic studies with sonicated or saponin-treated CHHs, human liver microsomes, and S9 fractions showed that both NADPH and UDP-glucuronic acid are required for 3-hydroxydesloratadine formation, and studies with recombinant UDP-glucuronosyltransferase (UGT) and P450 enzymes implicated the specific involvement of UGT2B10 in addition to CYP2C8. Overall, our results demonstrate for the first time that desloratadine glucuronidation by UGT2B10 followed by CYP2C8 oxidation and a deconjugation event are responsible for the formation of 3-hydroxydesloratadine. PMID:25595597

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

    PubMed

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

    2013-01-01

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

  14. 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

  15. 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

  16. 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

  17. Effect of CYP2C9, CYP4F2 and VKORC1 genetic polymorphisms on pharmacokinetics and pharmacodynamics of mean daily maintenance dose of warfarin in Chinese patients.

    PubMed

    Zhuang, Wenfang; Wen, Wei; Xuan, Binbin; Chen, Yanhong; Cao, Yanan; Sun, Zhixin; Ma, Jun

    2015-03-01

    In this study, we studied the effects of different genetic variants of CYP2C9, VKORC1 and CYP4F2, and clinical factors on the concentration levels of S-warfarin (WF), R-WF and S, R-7-OH-WF, as well as the mean daily maintenance dose of warfarin in 211 patients on warfarin therapy for at least 3 months. The genotypes of single nucleotide polymorphism (SNP), CYP2C9, VKORC1 1173C>T and CYP4F2 were identified by PCR. Plasma concentrations of S-WF and R-WF and S-7-OH-WF, R-7-OH-WF were determined by high-performance liquid chromatography tandem mass spectrometry on chiral columns. The warfarin dosage requirement correlated negatively with age and was in direct proportion to body weight. VKORC1 1173CC carrier had significantly lower dosage requirements than that with the heterozygous VKORC1 1173CT genotype. The concentration of both 7-OH-S-WF and 7-OH-R-WF, and the warfarin dose showed a significant difference. There were significant differences in the concentrations of S-WF and 7-OH-S-WF among the CYP2C9 variants. The concentration of warfarin, 7-OH-WF and warfarin maintenance dose were not affected by the CYP4F2 V433M variant. In conclusion, VKORC1 1173C>T genotype correlates strongly with a lower daily warfarin dose and the concentration of S-7-OH, R-7-OH warfarin in Han Shanghainese patients. In addition, the results not only demonstrated the effect on pharmacodynamics of warfarin, but also enhanced the enzymatic activity of CYP450 to influence the pharmacokinetic of warfarin. PMID:25304014

  18. Impact of ABCB1 and CYP2B6 Genetic Polymorphisms on Methadone Metabolism, Dose and Treatment Response in Patients with Opioid Addiction: A Systematic Review and Meta-Analysis

    PubMed Central

    Dennis, Brittany B.; Bawor, Monica; Thabane, Lehana; Sohani, Zahra; Samaan, Zainab

    2014-01-01

    Background Genetic variability may influence methadone metabolism, dose requirements, and risk of relapse. Objectives To determine whether the CYP2B6*6 or ABCB1 (rs1045642) polymorphisms are associated with variation in methadone response (plasma concentration, dose, or response to treatment). Methods Two independent reviewers searched Medline, EMBASE, CINAHL, PsycINFO, and Web of Science databases. We included studies that reported methadone plasma concentration, methadone response, or methadone dose in relation to the CYP2B6*6 or ABCB1 polymorphisms. Results We screened 182 articles and extracted 7 articles for inclusion in the meta-analysis. Considerable agreement was observed between the two independent raters on the title (kappa, 0.82), abstract (kappa, 0.43), and full text screening (kappa, 0.43). Trough (R) methadone plasma concentration was significantly higher in CYP2B6*6 homozygous carriers when compared to non-carriers (standardized mean difference [SMD] = 0.53, 95% confidence interval [CI], 0.05–1.00, p = 0.03) with minimal heterogeneity (I2 = 0%). Similarly, trough (S) methadone plasma concentration was higher in homozygous carriers of the *6 haplotype when compared to non-carriers, (SMD = 1.44, 95% CI 0.27–2.61, p = 0.02) however significant heterogeneity was observed (I2 = 69%). Carriers of the CYP2B6*6 haplotype were not found to be significantly different from non-carriers with respect to dose or response to treatment. We found no significant association between the ABCB1 polymorphism and the trough (R), (S) plasma concentrations, methadone dose, or methadone response. Conclusion Although the number of studies included and sample size were modest, this is the first meta analysis to show participants homozygous for the CYP2B6*6 genotype have higher trough (R) and (S) methadone plasma concentrations, suggesting that methadone metabolism is significantly slower in *6 homozygous carriers. PMID:24489693

  19. Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population

    PubMed Central

    Ota, Tomoko; Kamada, Yuka; Hayashida, Mariko; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

    2015-01-01

    The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment. PMID:25552922

  20. Effects of Panax notoginseng saponins on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats in vivo.

    PubMed

    Liu, Rui; Qin, Mengnan; Hang, Pengzhou; Liu, Yan; Zhang, Zhiren; Liu, Gaofeng

    2012-08-01

    The aim of this study was to assess the influence of the Panax notoginseng saponins (PNS) on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP450) 1A2, 2 C9, 2D6 and 3A4 in rats. The activities of CYP1A2, 2 C9, 2D6 and 3A4 were measured using specific probe drugs. After pretreatment for 1 week with PNS or physiological saline (control group), probe drugs caffeine (10 mg/kg; CYP1A2 activity), tolbutamide (15 mg/kg; CYP2C9 activity), metoprolol (20 mg/kg; CYP2D6 activity) and dapsone (10 mg/kg; CYP3A4 activity) were administered to rats by intraperitoneal injection. The blood was then collected at different times for ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis. The data showed that PNS exhibited an induction effect on CYP1A2 by decreasing caffeine C(max) (36.3%, p < 0.01) and AUC(0-∞) (22.77%, p < 0.05) and increasing CL/F (27.03%, p < 0.05) compared with those of the control group. Western blot analysis was used to detect the effect of PNS on the protein level of CYP1A2, and the results showed that PNS could upregulate the protein expression of CYP1A2. However, no significant changes in CYP2C9, 2D6 or 3A4 activities were observed. In conclusion, the results indicate that PNS could induce CYP1A2, which may affect the disposition of medicines primarily dependent on the CYP1A2 pathway. Our work may be the basis of related herb-drug interactions in the clinic.

  1. Biotinylation of glycan chains in β2 glycoprotein I induces dimerization of the molecule and its detection by the human autoimmune anti-cardiolipin antibody EY2C9

    PubMed Central

    Dupuy d'Angeac, Arnaud; Stefas, Ilias; Graafland, Hubert; de Lamotte, Frédéric; Rucheton, Marcel; Palais, Caroline; Eriksson, Anna-Karin; Bosc, Priscille; Rosé, Caroline; Chicheportiche, Robert

    2005-01-01

    Binding of β2GPI (β2 glycoprotein I), a human plasma protein, to AnPLs (anionic phospholipids) plays a key role in the formation of antiphospholipid antibodies involved in autoimmune diseases like antiphospholipid syndrome or systemic lupus erythematosus. We recently showed that binding of β2GPI to AnPLs was enhanced by biotinylation of its glycan chains with biotin-hydrazide. In the present study, we investigated why this chemical modification of β2GPI increased both its affinity for AnPLs and its recognition by anti-cardiolipin antibodies. Electrophoretic analysis showed that: (i) high molecular mass β2GPI (dimers and other oligomers) covalently coupled by imine bonds, were present in variable amounts in oxidized β2GPI and in β2GPI-bh (β2GPI-biotin-hydrazide), but were absent in native β2GPI; (ii) binding of β2GPI-bh to phosphatidylserine-coated microtitre plates generated high molecular mass polymers in a time-dependent manner. Native β2GPI did not polymerize in these conditions. These polymers did not bind more strongly to AnPLs than the monomer β2GPI. However, in solution at 1 μM β2GPI-bh essentially appeared as a dimer as revealed by light-scattering analysis. SPR (surface plasmon resonance) analysis showed that the increased affinity of β2GPI-bh for AnPL monolayers was due to a lower dissociation rate constant compared with native β2GPI. Finally, the monoclonal human aCL (auto-immune anti-cardiolipin antibody) EY2C9 bound to β2GPI-bh but did not bind to monomeric native and oxidized β2GPI. It is likely that the dimeric quaternary structure of β2GPI-bh is in fact responsible for the appearance of the epitopes targeted by the EY2C9 antibody. PMID:16097953

  2. African Lettuce (Launaea taraxacifolia) Displays Possible Anticancer Effects and Herb-Drug Interaction Potential by CYP1A2, CYP2C9, and CYP2C19 Inhibition.

    PubMed

    Thomford, Nicholas E; Mkhize, Buyisile; Dzobo, Kevin; Mpye, Keleabetswe; Rowe, Arielle; Parker, M Iqbal; Wonkam, Ambroise; Skelton, Michelle; September, Alison V; Dandara, Collet

    2016-09-01

    Medicinal plants are part of the healthcare systems worldwide, especially in low- and middle-income countries. African lettuce (Launaea taraxacifolia) is cultivated extensively in Africa, from Senegal in the west to Ethiopia and Tanzania in the east, and in Southern Africa. Potential anticancer effects of L. taraxacifolia have been suggested, but little is known about putative molecular mechanisms or potential for herb-drug interactions through inhibition or induction of drug-metabolizing enzymes. We investigated the effects of crude aqueous extracts of L. taraxacifolia on growth kinetics and cell cycle progression of the WHC01 esophageal cancer cells. Antiproliferative and apoptotic effects were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry, while examining, in parallel, the genes regulating apoptosis and cell cycle in this cell culture model. In addition, we tested the inhibitory and enzyme kinetic effects of the aqueous L. taraxacifolia using recombinant human CYP450 isozyme model systems (CYP1A2, CYP2C9, and CYP2C19). L. taraxacifolia exhibited a significant growth inhibitory effect on the WHC01 cancer cells. Most cell cycle genes were downregulated. Cell cycle analysis showed a G0-G1 cell cycle arrest in WHC01 cells in the presence of L. taraxacifolia extract, accompanied by morphological changes. L. taraxacifolia extract treatment resulted in downregulation of expression levels of CYP1A2 (p < 0.0005) and CYP2C19 (p < 0.003) by 50-70%. L. taraxacifolia extract caused reversible and time-dependent inhibition of the recombinant CYP1A2, CYP2C9, and CYP2C19. This study provides new insights on possible anticancer effects of L. taraxacifolia, a widely used medicinal plant in parts of Africa and across the world especially by patients with cancer. Further mechanistic studies expanding on these observations would be timely and contribute to the field of global precision medicine that requires

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

    PubMed

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

    2011-11-15

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

  4. 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.

  5. 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.

  6. Roles of human CYP2A6 and 2B6 and rat CYP2C11 and 2B1 in the 10-hydroxylation of (-)-verbenone by liver microsomes.

    PubMed

    Miyazawa, Mitsuo; Sugie, Atsushi; Shimada, Tsutomu

    2003-08-01

    (-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

  7. Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms.

    PubMed

    Or, Penelope M Y; Lam, Francis F Y; Kwan, Y W; Cho, C H; Lau, C P; Yu, H; Lin, G; Lau, Clara B S; Fung, K P; Leung, P C; Yeung, John H K

    2012-04-15

    The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. Changes in cytochrome P450s-mediated drug clearance in patients with hepatocellular carcinoma in vitro and in vivo: a bottom-up approach

    PubMed Central

    He, Xiao-Pei; Zhang, Yun-Fei; Gao, Na; Tian, Xin; Fang, Yan; Wen, Qiang; Jia, Lin-Jing; Jin, Han; Qiao, Hai-Ling

    2016-01-01

    Hepatocellular carcinoma (HCC) accompanied by severe liver dysfunction is a serious disease, which results in altered hepatic clearance. Generally, maintenance doses depend upon drug clearance, so individual dosage regimens should be customized for HCC patients based on the condition of patients. Based on clearance of CYP isoform-specific substrates at the microsomal level (CLM), microsomal protein per gram of liver (MPPGL), liver weight, hepatic blood flow, hepatic clearance values (CLH) for 10 CYPs in HCC patients (n=102) were extrapolated using a predictive bottom-up pharmacokinetic model. Compared with controls, the CLM values for CYP2C9, 2D6, 2E1 were significantly increased in HCC patients. Additionally, CYP1A2, 2C8, 2C19 CLM values decreased while the values for CYP2A6, 2B6, 3A4/5 were unchanged. The MPPGL values in HCC tissues were significantly reduced. CLH values of HCC patients for CYP1A2, 2A6, 2B6, 2C8, 2C19, and 3A4/5 were significantly reduced, while this for CYP2E1 were markedly increased and those for CYP2C9 and 2D6 did not change. Moreover, disease (fibrosis and cirrhosis) and polymorphisms of the CYP genes have influenced the CLH for some CYPs. Prediction of the effects of HCC on drug clearance may be helpful for the design of clinical studies and the clinical management of drugs in HCC patients. PMID:27086920

  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.

    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).

  15. 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.

  16. Gas-Phase Reactions of Atomic Gold Cations with Linear Alkanes (C2-C9).

    PubMed

    Zhang, Ting; Li, Zi-Yu; Zhang, Mei-Qi; He, Sheng-Gui

    2016-06-30

    To develop proper ionization methods for alkanes, the reactivity of bare or ligated transition metal ions toward alkanes has attracted increasing interests. In this study, the reactions of the gold cations with linear alkanes from ethane up to nonane (CnH2n+2, n = 2-9) under mild conditions have been characterized by mass spectrometry and density functional theory calculations. When reacting with Au(+), small alkanes (n = 2-6) were confirmed to follow specific reaction channels of dehydrogenation for ethane and hydride transfer for others to generate product ions characteristic of the original alkanes, which indicates that Au(+) can act as a reagent ion to ionize alkanes from ethane to n-hexane. Strong dependence of the chain length of alkanes was observed for the rate constants and reaction efficiencies. Extensive fragmentation took place for larger alkanes (n > 6). Theoretical results show that the fragmentation induced by the hydride transfer occurs after the release of AuH. Moreover, the fragmentation of n-heptane was successfully avoided when the reaction took place in a high-pressure reactor. This implies that Au(+) is a potential reagent ion to ionize linear and even the branched alkanes. PMID:27266670

  17. 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

  18. Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities.

    PubMed

    Doehmer, Johannes; Weiss, Gabriele; McGregor, Gerard P; Appel, Kurt

    2011-02-01

    The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC(50) value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c(max) at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c(max). The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K(i) values were determined. All K(i) values exceeded c(max) by at least a factor of 10-fold. According to FDA regulations 1>c(max)/K(i)>0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.

  19. Age-Dependent Changes in Human Hepatic CYP2C8 and 1A2 Expression

    EPA Science Inventory

    Predicting age-specific metabolism of pyrethroids is important in evaluating age-related sensitivity. Our goal is to use an in vitro to in vivo extrapolation (IVIVE) approach to predict pyrethroid metabolism for different ages incorporating enzyme ontogeny and expressed enzyme ki...

  20. Materials Data on Ce(Ni2B)6 (SG:36) by Materials Project

    DOE Data Explorer

    Kristin Persson

    2016-03-27

    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. Materials Data on Tb(Ni2B)6 (SG:36) by Materials Project

    SciTech Connect

    Kristin Persson

    2015-02-09

    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

  2. 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

  3. 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

  4. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

  5. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

    PubMed Central

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  6. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  7. 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

  8. 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

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

    PubMed

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

    2016-01-01

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

  10. Involvement of multiple cytochrome P450 and UDP-glucuronosyltransferase enzymes in the in vitro metabolism of muraglitazar.

    PubMed

    Zhang, Donglu; Wang, Lifei; Chandrasena, Gamini; Ma, Li; Zhu, Mingshe; Zhang, Hongjian; Davis, Carl D; Humphreys, W Griffith

    2007-01-01

    Muraglitazar (Pargluva), a dual alpha/gamma peroxisome proliferator-activated receptor activator, has both glucose- and lipid-lowering effects in animal models and in patients with diabetes. The human major primary metabolic pathways of muraglitazar include acylglucuronidation, aliphatic/aryl hydroxylation, and O-demethylation. This study describes the identification of human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes involved in the in vitro metabolism of muraglitazar. [(14)C]Muraglitazar was metabolized by cDNA-expressed CYP2C8, 2C9, 2C19, 2D6, and 3A4, but to a very minimal extent by CYP1A2, 2A6, 2B6, 2C18, 2E1, and 3A5. Inhibition of the in vitro metabolism of muraglitazar in human liver microsomes, at a clinically efficacious concentration, by chemical inhibitors and monoclonal antibodies further supported involvement of CYP2C8, 2C9, 2C19, 2D6, and 3A4 in its oxidation. A combination of intrinsic clearance (V(max)/K(m)) and relative concentrations of each P450 enzyme in the human liver was used to predict the contribution of CYP2C8, 2C9, 2C19, 2D6, and 3A4 to the formation of each primary oxidative metabolite and to the overall oxidative metabolism of muraglitazar. Glucuronidation of [(14)C]muraglitazar was catalyzed by cDNA-expressed UGT1A1, 1A3, and 1A9, but not by UGT1A6, 1A8, 1A10, 2B4, 2B7, and 2B15. The K(m) values for muraglitazar glucuronidation by the three active UGT enzymes were similar (2-4 muM). In summary, muraglitazar was metabolized by multiple P450 and UGT enzymes to form multiple metabolites. This characteristic predicts a low potential for the alteration of the pharmacokinetic parameters of muraglitazar via polymorphic drug metabolism enzymes responsible for clearance of the compound or by coadministration of drugs that inhibit or induce relevant metabolic enzymes. PMID:17062778

  11. An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure.

    PubMed

    Spaggiari, Dany; Daali, Youssef; Rudaz, Serge

    2016-07-01

    Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In this work, an extensive cocktail mixture was developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS method were critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLM concentration, (iii) fast incubation (5min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitors with those generated with the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at low concentrations showed a cumulative inhibitory in vitro effect on P450 2B6. PMID:27105555

  12. 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

  13. Pharmacogenetics in Ghana: reviewing the evidence.

    PubMed

    Kudzi, W; Adjei, G O; Ofori-Adjei, D; Dodoo, A N O

    2011-06-01

    Different clinical response of different patients to the same medicine has been recognised and documented since the 1950's. Variability in response of individuals to standard doses of drug therapy is important in clinical practice and can lead to therapeutic failures or adverse drug reactions. Pharmacogenetics seeks to identify individual genetic differences (polymorphisms) in drug absorption, metabolism, distribution and excretion that can affect the activity of a particular drug with the view of improving efficacy and reducing toxicity. Although knowledge of pharmacogenetics is being translated into clinical practice in the developed world, its applicability in the developing countries is low. Several factors account for this including the fact that there is very little pharmacogenetic information available in many indigenous African populations including Ghanaians. A number of genes including Cytochrome P450 (CYP) 2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, MDR1 and TPMT have been genotyped in the Ghanaian population since the completion of the Human genome project. There is however, an urgent need to increase pharmacogenetic research in Ghana to increase availability of data. Introducing Pharmacogenetics into the curriculum of Medical and Pharmacy training institutions will influence translating knowledge of pharmacogenetics into clinical practice. This will also equip health professionals with the skill to integrate genetic information into public health decision making. PMID:21857725

  14. 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. PMID:23232241

  15. 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.

  16. 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. PMID:26599973

  17. 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.

  18. Effect of Single Nucleotide Polymorphisms in Cytochrome P450 Isoenzyme and N-Acetyltransferase 2 Genes on the Metabolism of Artemisinin-Based Combination Therapies in Malaria Patients from Cambodia and Tanzania

    PubMed Central

    Staehli Hodel, Eva Maria; Csajka, Chantal; Ariey, Frédéric; Guidi, Monia; Kabanywanyi, Abdunoor Mulokozi; Duong, Socheat; Decosterd, Laurent Arthur; Olliaro, Piero; Genton, Blaise

    2013-01-01

    The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C→T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C→T and 2850C→T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials. PMID:23229480

  19. In vitro investigations into the roles of drug transporters and metabolizing enzymes in the disposition and drug interactions of dolutegravir, a HIV integrase inhibitor.

    PubMed

    Reese, Melinda J; Savina, Paul M; Generaux, Grant T; Tracey, Helen; Humphreys, Joan E; Kanaoka, Eri; Webster, Lindsey O; Harmon, Kelly A; Clarke, James D; Polli, Joseph W

    2013-02-01

    Dolutegravir (DTG; S/GSK1349572) is a potent HIV-1 integrase inhibitor with a distinct resistance profile and a once-daily dose regimen that does not require pharmacokinetic boosting. This work investigated the in vitro drug transport and metabolism of DTG and assessed the potential for clinical drug-drug interactions. DTG is a substrate for the efflux transporters P-glycoprotein (Pgp) and human breast cancer resistance protein (BCRP). Its high intrinsic membrane permeability limits the impact these transporters have on DTG's intestinal absorption. UDP-glucuronosyltransferase (UGT) 1A1 is the main enzyme responsible for the metabolism of DTG in vivo, with cytochrome P450 (P450) 3A4 being a notable pathway and UGT1A3 and UGT1A9 being only minor pathways. DTG demonstrated little or no inhibition (IC(50) values > 30 μM) in vitro of the transporters Pgp, BCRP, multidrug resistance protein 2, organic anion transporting polypeptide 1B1/3, organic cation transporter (OCT) 1, or the drug metabolizing enzymes CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, UGT1A1, or 2B7. Further, DTG did not induce CYP1A2, 2B6, or 3A4 mRNA in vitro using human hepatocytes. DTG does inhibit the renal OCT2 (IC(50) = 1.9 μM) transporter, which provides a mechanistic basis for the mild increases in serum creatinine observed in clinical studies. These in vitro studies demonstrate a low propensity for DTG to be a perpetrator of clinical drug interactions and provide a basis for predicting when other drugs could result in a drug interaction with DTG. PMID:23132334

  20. Evaluation of the in vitro/in vivo drug interaction potential of BST204, a purified dry extract of ginseng, and its four bioactive ginsenosides through cytochrome P450 inhibition/induction and UDP-glucuronosyltransferase inhibition.

    PubMed

    Zheng, Yu Fen; Bae, Soo Hyeon; Choi, Eu Jin; Park, Jung Bae; Kim, Sun Ok; Jang, Min Jung; Park, Gyu Hwan; Shin, Wan Gyoon; Oh, Euichaul; Bae, Soo Kyung

    2014-06-01

    We evaluated the potential of BST204, a purified dry extract of ginseng, to inhibit or induce human liver cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) in vitro to assess its safety. In vitro drug interactions of four bioactive ginsenosides of BST204, S-Rg3, R-Rg3, S-Rh2, and R-Rh2, were also evaluated. We demonstrated that BST204 slightly inhibited CYP2C8, CYP2D6, CYP2C9, and CYP2B6 activities with IC50 values of 17.4, 26.8, 31.5, and 49.7μg/mL, respectively. BST204 also weakly inhibited UGT1A1, UGT1A9, and UGT2B7 activities with IC50 values of 14.5, 26.6, and 31.5μg/mL, respectively. The potential inhibition by BST204 of the three UGT activities might be attributable to S-Rg3, at least in part, as its inhibitory pattern was similar to that of BST204. However, BST204 showed no time-dependent inactivation of the nine CYPs studied. In addition, BST204 did not induce CYP1A2, 2B6, or 3A4/5. On the basis of an in vivo interaction studies, our data strongly suggest that BST204 is unlikely to cause clinically significant drug-drug interactions mediated via inhibition or induction of most CYPs or UGTs involved in drug metabolism in vivo. Our findings offer a clearer understanding and possibility to predict drug-drug interactions for the safe use of BST204 in clinical practice. PMID:24632066

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

    PubMed

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

    2007-01-01

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

  2. Volumetric Properties of the Mixture Methyl butanoate C5H10O2 + C8H16 Cyclooctane (LB0308, VMSD1212)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Hnědkovský, L.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A `Binary Liquid Systems of Nonelectrolytes' of Volume 23 `Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV `Physical Chemistry'. It corresponds to the data set LB0308 of the ELBT database.

  3. Volumetric Properties of the Mixture Methyl butanoate C5H10O2 + C8H16 Cyclooctane (LB0306, VMSD1111)

    NASA Astrophysics Data System (ADS)

    Cibulka, I.; Hnědkovský, L.; Fontaine, J.-C.; Sosnkowska-Kehiaian, K.; Kehiaian, H. V.

    This document is part of Subvolume A `Binary Liquid Systems of Nonelectrolytes' of Volume 23 `Volumetric Properties of Mixtures and Solutions' of Landolt-Börnstein Group IV `Physical Chemistry'. It corresponds to the data set LB0306 of the ELBT database.

  4. Cerocene Revisited: The Electronic Structure of and Interconversion Between Ce2(C8H8)3 and Ce(C8H8)2

    SciTech Connect

    Walter, Marc D.; Booth, Corwin H.; Lukens, Wayne W.; Andersen, Richard A.

    2009-02-02

    New synthetic procedures for the preparation of Ce(cot)2, cerocene, from [Li(thf)4][Ce(cot)2], and Ce2(cot)3 in high yield and purity are reported. Heating solid Ce(cot)2 yields Ce2(cot)3 and COT while heating Ce2(cot)3 with an excess of COT in C6D6 to 65oC over four months yields Ce(cot)2. The solid state magnetic susceptibility of these three organocerium compounds shows that Ce(cot)2 behaves as a TIP (temperature independent paramagnet) over the temperature range of 5-300 K, while that of Ce2(cot)3 shows that the spin carriers are antiferromagnetically coupled below 10 K; above 10 K, the individual spins are uncorrelated, and [Ce(cot)2]- behaves as an isolated f1 paramagnet. The EPR at 1.5K for Ce2(cot)3 and [Ce(cot)2]- have ground state of MJ= +- 1/2. The LIII edge XANES of Ce(cot)2 (Booth, C.H.; Walter, M.D.; Daniel, M.; Lukens, W.W., Andersen, R.A., Phys. Rev. Lett. 2005, 95, 267202) and 2Ce2(cot)3 over 30-500 K are reported; the Ce(cot)2 XANES spectra show Ce(III) and Ce(IV) signatures up to a temperature of approximately 500 K, whereupon the Ce(IV) signature disappears, consistent with the thermal behavior observed in the melting experiment. The EXAFS of Ce(cot)2 and Ce2(cot)3 are reported at 30 K; the agreement between the molecular parameters for Ce(cot)2 derived from EXAFS and single crystal X-ray diffraction data are excellent. In the case of Ce2(cot)3 no X-ray diffraction data are known to exist, but the EXAFS are consistent with a"triple-decker" sandwich structure. A molecular rationalization is presented for the electronic structure of cerocene having a multiconfiguration ground state that is an admixture of the two configurations Ce(III, 4f1)(cot1.5-)2 and Ce(IV, 4f0)(cot2-)2; the multiconfigurational ground state has profound effects on the magnetic properties and on the nature of the chemical bond in cerocene and, perhaps, other molecules.

  5. In vitro characterization of the metabolic pathways and cytochrome P450 inhibition and induction potential of BMS-690514, an ErbB/vascular endothelial growth factor receptor inhibitor.

    PubMed

    Hong, Haizheng; Su, Hong; Ma, Li; Yao, Ming; Iyer, Ramaswamy A; Humphreys, W Griffith; Christopher, Lisa J

    2011-09-01

    (3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of ErbB human epidermal growth factor receptors (HER1, 2, and 4) and vascular endothelial growth factor receptors 1 to 3 that has been under clinical development for solid tumor malignancies. BMS-690514 is primarily cleared by metabolism with the primary metabolic pathways being direct glucuronidation (M6), hydroxylation (M1, M2, and M37), and O-demethylation (M3). In the current investigation, the metabolic drug-drug interaction potential of BMS-690514 was evaluated in a series of in vitro studies. Reaction phenotyping experiments with cDNA-expressed human cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) enzymes and human liver microsomes (HLM) in the presence of P450 or UGT inhibitors suggested that CYP3A4, CYP2D6, and CYP2C9 were the major enzymes responsible for the oxidative metabolism of BMS-690514, whereas both UGT2B4 and UGT2B7 were responsible for the formation of M6. BMS-690514 did not cause direct or time-dependent inhibition of P450 enzymes (IC(50) values ≥40 μM) in incubations with HLM and probe substrates of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or 3A4. The compound also did not substantially induce CYP1A1, CYP1A2, CYP2B6, CYP3A4, or UGT1A1 at concentrations up to 10 μM in cultured human hepatocytes. Considering the submicromolar plasma C(max) concentration at the anticipated clinical dose of 200 mg, BMS-690514 is unlikely to cause clinically relevant drug-drug interactions when coadministered with other medications. In addition, because multiple enzymatic clearance pathways are available for the compound, inhibition of an individual metabolic pathway either via coadministered drugs or gene polymorphisms is not expected to cause pronounced (>2-fold) increases in BMS-690514 exposure. PMID:21673131

  6. Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

    PubMed

    Hu, Yiding; Kupfer, David

    2002-12-01

    Methoxychlor, a currently used pesticide that in mammals elicits proestrogenic/estrogenic activity and reproductive toxicity, has been classified as a prototype endocrine disruptor. Methoxychlor is prochiral, and its metabolites 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M); 1,1,1-trichloro- 2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M); and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M) are chiral; whereas 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M) is achiral. These metabolites are formed during methoxychlor incubation with liver microsomes or recombinant cytochrome p450s (rp450s). Since methoxychlor-metabolite enantiomers may have different estrogenic/antiestrogenic/antiandrogenic activities than corresponding racemates, the possibility that p450s preferentially generate or use R or S enantiomers, was examined. Indeed, rCYP1A2 and r2A6 mono-demethylated methoxychlor primarily into (R)-mono-OH-M at 91 and 75%, respectively, whereas rCYP1A1, 2B6, 2C8, 2C9, 2C19, and 2D6 formed the (S)-enantiomer at 69, 66, 75, 95, 96, and 80%, respectively. However, rCYP3A4, 3A5, and 2B1(rat) weakly demethylated methoxychlor without enantioselectivity. Human liver microsomes generated (S)-mono-OH-M (77-87%), suggesting that CYP1A2 and 2A6 display only minor catalytic contribution. P450 inhibitors demonstrated that CYP2C9 and possibly 2C19 are major hepatic catalysts forming (S)-mono-OH-M, and CYP1A2 is primarily involved in forming the (R)-mono-OH-M. Demethylation rate of (S)-mono-OH-M versus (R)-mono-OH-M forming achiral bis-OH-M by rCYP1A2 was 97/3, compared with 15/85 and 17/83 for rCYP2C9 and 2C19, respectively, indicating opposite substrate enantioselectivity of rCYP1A2 versus 2C9 and 2C19. Also, rCYP1A2 preferentially O-demethylated (R)-catechol-M into (R)-tris-OH-M (at 80%), contrasting r2C9 and r2C19 that yielded (S)-tris-OH-M at 80 and 77%, respectively. Ortho-hydroxylation of

  7. In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450.

    PubMed

    Koga, Nobuyuki; Ohta, Chiho; Kato, Yoshihisa; Haraguchi, Koichi; Endo, Tetsuya; Ogawa, Kazunori; Ohta, Hideaki; Yano, Masamichi

    2011-11-01

    Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

  8. In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron, a potent and selective β3-adrenoceptor agonist.

    PubMed

    Takusagawa, Shin; Miyashita, Aiji; Iwatsubo, Takafumi; Usui, Takashi

    2012-12-01

    The potential for mirabegron, a β(3)-adrenoceptor agonist for the treatment of overactive bladder, to cause drug-drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro. Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC(50) value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug-drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC(50) values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [C(max)]). Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma C(max)) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.

  9. 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

    2016-08-01

    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.

  10. Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT)

    PubMed Central

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-01-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug–endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  11. Renal drug metabolism in humans: the potential for drug-endobiotic interactions involving cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT).

    PubMed

    Knights, Kathleen M; Rowland, Andrew; Miners, John O

    2013-10-01

    Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis. PMID:23362865

  12. Characterization of 137 Genomic DNA Reference Materials for 28 Pharmacogenetic Genes: A GeT-RM Collaborative Project.

    PubMed

    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

    2016-01-01

    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.

  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. Metabolism of the endocrine disruptor pesticide-methoxychlor by human P450s: pathways involving a novel catechol metabolite.

    PubMed

    Hu, Yiding; Kupfer, David

    2002-09-01

    The metabolism of methoxychlor, a proestrogenic pesticide (endocrine disruptor), was investigated with cDNA expressed human cytochrome P450s and liver microsomes (HLM). In addition to 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M), 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M), and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M), a new metabolite was identified as 1,1,1-trichloro-2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M; previously assumed to be ring-OH-M) and as a key metabolic intermediate. A novel metabolic route was proposed involving methoxychlor O-demethylation to mono-OH-M, followed by bifurcation of the pathway, both leading to the same final product tris-OH-M: pathway a, mono-OH-M is demethylated to bis-OH-M, followed by ortho-hydroxylation forming tris-OH-M and pathway b, mono-OH-M is ortho-hydroxylated forming catechol-M that is O-demethylated forming tris-OH-M. Among the human cDNA-expressed P450s examined, CYP1A2, 2A6, 2C8, 2C9, 2C19, and 2D6 exhibited mainly O-demethylation, with CYP2C19 being the most catalytically competent. CYP3A4, 3A5, and rat 2B1 catalyzed primarily ortho-hydroxylation of mono-OH-M (CYP3A4 being catalytically the most active) but were weak in O-demethylation. CYP1A1, 1B1, 2E1, and 4A11 demonstrated little or no catalytic activity. CYP2B6 appeared unique, catalyzing effectively both O-demethylation and ortho-hydroxylation. Thus, CYP2B6 demethylated methoxychlor to mono-OH-M and ortho-hydroxylated the mono-OH-M forming catechol-M; however, 2B6 did not appreciably demethylate mono-OH-M or ortho-hydroxylate bis-OH-M, suggesting a narrow substrate specificity. CYP2C19-catalyzed demethylation of methoxychlor, mono-OH-M and catechol-M, demonstrating relatively good substrate affinity (K(m) = 0.23 - 0.41 microM). However, the 3A4 ortho-hydroxylation of mono-OH-M and bis-OH-M exhibited lower affinity, K(m) = 12 and 25 microM, respectively. Thus, a

  15. Ellipticine oxidation and DNA adduct formation in human hepatocytes is catalyzed by human cytochromes P450 and enhanced by cytochrome b5.

    PubMed

    Stiborová, Marie; Poljaková, Jitka; Martínková, Eva; Ulrichová, Jitka; Simánek, Vilím; Dvořák, Zdeněk; Frei, Eva

    2012-12-16

    Ellipticine is an antineoplastic agent considered a pro-drug, the pharmacological and genotoxic effects of which are dependent on cytochrome P450 (CYP)- and/or peroxidase-mediated activation to covalent DNA adducts. We investigated whether ellipticine-DNA adducts are formed in human hepatic microsomes and human hepatocytes. We then identified which human CYPs oxidize ellipticine to metabolites forming DNA adducts and the effect of cytochrome b(5) on this oxidation. 13-Hydroxyellipticine, the metabolite forming the major ellipticine-DNA adduct, was generated mainly by CYP3A4 and 1A1, followed by CYP2D6>2C19>1B1>1A2>2E1 and >2C9. Cytochrome b(5) increased formation of this metabolite by human CYPs, predominantly by CYP1A1, 3A4, 1A2 and 2C19. Formation of 12-hydroxyellipticine is generated mainly by CYP2C19, followed by CYP2C9>3A4>2D6>2E1 and >2A6. Other CYPs were less active (CYP2C8 and 2B6) or did not oxidize ellipticine to this metabolite (CYP1A1, 1A2 and 1B1). CYP2D6 was the most efficient enzyme generating ellipticine N(2)-oxide. CYP3A4 and 1A1 in the presence of cytochrome b(5) are mainly responsible for bioactivation of ellipticine to DNA adduct 1 (formed by ellipticine-13-ylium from 13-hydroxyellipticine), while 12-hydroxyellipticine generated during the CYP2C19-mediated ellipticine oxidation is the predominant metabolite forming ellipticine-12-ylium that generates ellipticine-DNA adduct 2. These ellipticine-DNA adducts were also generated by human hepatic microsomes and in primary human hepatocytes exposed to ellipticine. Ellipticine is toxic to these hepatocytes, decreasing their viability; the IC(50) value of ellipticine in these cells was 0.7 μM. In liver CYP3A4 is the predominant ellipticine activating CYP species, which is expected to result in efficient metabolism after oral ingestion of ellipticine in humans.

  16. Metabolism and related human risk factors for hepatic damage by usnic acid containing nutritional supplements.

    PubMed

    Foti, R S; Dickmann, L J; Davis, J A; Greene, R J; Hill, J J; Howard, M L; Pearson, J T; Rock, D A; Tay, J C; Wahlstrom, J L; Slatter, J G

    2008-03-01

    Usnic acid is a component of nutritional supplements promoted for weight loss that have been associated with liver-related adverse events including mild hepatic toxicity, chemical hepatitis, and liver failure requiring transplant. To determine if metabolism factors might have had a role in defining individual susceptibility to hepatotoxicity, in vitro metabolism studies were undertaken using human plasma, hepatocytes, and liver subcellular fractions. Usnic acid was metabolized to form three monohydroxylated metabolites and two regio-isomeric glucuronide conjugates of the parent drug. Oxidative metabolism was mainly by cytochrome P450 (CYP) 1A2 and glucuronidation was carried out by uridine diphosphate-glucuronosyltransferase (UGT) 1A1 and UGT1A3. In human hepatocytes, usnic acid at 20 microM was not an inducer of CYP1A2, CYP2B6, or CYP3A4 relative to positive controls omeprazole, phenobarbital, and rifampicin, respectively. Usnic acid was a relatively weak inhibitor of CYP2D6 and a potent inhibitor of CYP2C19 (the concentration eliciting 50% inhibition (IC(50)) = 9 nM) and CYP2C9 (IC(50) = 94 nM), with less potent inhibition of CYP2C8 (IC(50) = 1.9 microM) and CYP2C18 (IC(50) = 6.3 microM). Pre-incubation of microsomes with usnic acid did not afford any evidence of time-dependent inhibition of CYP2C19, although evidence of slight time-dependent inhibition of CYP2C9 (K(I) = 2.79 microM and K(inact) = 0.022 min(-1)) was obtained. In vitro data were used with SimCYP(R)to model potential drug interactions. Based on usnic acid doses in case reports of 450 mg to >1 g day(-1), these in vitro data indicate that usnic acid has significant potential to interact with other medications. Individual characteristics such as CYP1A induction status, co-administration of CYP1A2 inhibitors, UGT1A1 polymorphisms, and related hyperbilirubinaemias, or co-administration of low therapeutic index CYP2C substrates could work alone or in consort with other idiosyncrasy risk factors to

  17. Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

    PubMed

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

    2015-01-01

    Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity.

  18. Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

    PubMed

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

    2015-01-01

    Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity. PMID:25318994

  19. Evaluation of six proton pump inhibitors as inhibitors of various human cytochromes P450: focus on cytochrome P450 2C19.

    PubMed

    Zvyaga, Tatyana; Chang, Shu-Ying; Chen, Cliff; Yang, Zheng; Vuppugalla, Ragini; Hurley, Jeremy; Thorndike, Denise; Wagner, Andrew; Chimalakonda, Anjaneya; Rodrigues, A David

    2012-09-01

    Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC₅₀ (IC₅₀ ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC₅₀ = 0.73 μM) and esomeprazole (IC₅₀ = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC₅₀ = ∼7.0 μM). Rabeprazole and pantoprazole (IC₅₀ = ≥ 25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC₅₀ ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

  20. 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...

  1. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    PubMed

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (+/- SEM) Km and Vmax of 8.3 +/- 1.3 microM and 454 +/- 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 microM (i.e. about two and six times Km respectively). With 20 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r2 = 0.784-0.797) and then with CYP3A (r2 = 0.434-0.547) isoforms, whereas with 50 microM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r2 = 0.679-0.837) and then with CYP1A2 (r2 = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 micro

  2. Reversible inhibition of three important human liver cytochrome p450 enzymes by tiliroside.

    PubMed

    Sun, Dong-Xue; Lu, Jin-Cai; Fang, Zhong-Ze; Zhang, Yan-Yan; Cao, Yun-Feng; Mao, Yu-Xi; Zhu, Liang-Liang; Yin, Jun; Yang, Ling

    2010-11-01

    Tiliroside, an active flavonoid extensively found in many medicinal plants including Helichrysum italicum, Geranium mexicanum and Helianthemum glomeratum, has been demonstrated to exert multiple biological effects including antiinflammatory, antimicrobial, antioxidant and antitumor activities. Cytochrome P450 (CYP) enzymes play an important role in the Phase I oxidation metabolism of a wide range of xenobiotics and inhibition of CYP isoforms might influence the elimination of drugs and induce serious adverse drug response. The inhibition of seven CYP isoforms (CYP3A4, CYP1A2, CYP2A6, CYP2D6, CYP2C9, CYP2C8 and CYP2E1) by tiliroside was investigated using in vitro human liver microsomal incubation assays. The results showed that tiliroside strongly inhibited the activity of CYP3A4 (IC(50) = 9.0 ± 1.7 μm), CYP2C8 (IC(50) = 12.1 ± 0.9 μm) and CYP2C9 (IC(50) = 10.2 ± 0.9 μm) with other CYP isoforms negligibly influenced. Further kinetic analysis showed that inhibition of these three CYP isoforms by tiliroside is best fit to a competitive way. The K(i) value was calculated to be 5.5 μm, 3.3 μm, 9.4 μm for CYP3A4, CYP2C9 and CYP2C8, respectively. The relatively low K(i) values suggested that tiliroside might induce drug-drug interactions with many clinically used drugs which are mainly metabolized by these three CYP isoforms. Therefore, attention should be given to the probable drug-drug interaction between tiliroside-containing herbs and substrates of CYP3A4, CYP2C9 and CYP2C8.

  3. Inhibition of human cytochrome P450 enzymes by licochalcone A, a naturally occurring constituent of licorice.

    PubMed

    He, Wei; Wu, Jing-Jing; Ning, Jing; Hou, Jie; Xin, Hong; He, Yu-Qi; Ge, Guang-Bo; Xu, Wei

    2015-10-01

    Licochalcone A (LCA) is a major bioactive compound in traditional Chinese herbal liquorice that possesses multiple pharmacological activities. However, the effects of the potential herb-drug interactions (HDIs) between LCA and therapeutic drugs on the inhibition of human cytochrome P450 (CYP) enzymes remain unclear. In the present study, the inhibitory effects of LCA on seven major human CYP isoforms, including CYP1A2, 2D6, 2E1, 2C19, 2C8, 2C9 and 3A4, were investigated in human liver microsomes (HLMs). The results demonstrated that LCA significantly inhibited the activities of CYP1A2, 2C19, 2C8, 2C9 and 3A4 and exhibited weak inhibitory effects on CYP2E1 and CYP2D6. Dixon and Lineweaver-Burk plots revealed that the inhibition types of LCA against CYP1A2, 2C9, 2C19 and 2C8 were best fit as mixed-type inhibitions, while LCA was a competitive inhibitor towards CYP3A4. The inhibition kinetic parameters (K(i)) were calculated to be 1.02 μM, 0.17 μM, 3.89 μM 0.89 μM, and 2.29 μM, for CYP1A2, 2C9, 2C19, 2C8, and 3A4, respectively. Furthermore, the areas under the plasma concentration-time curves (AUCs) of several drugs that are primarily metabolized by CYPs were estimated to increase by 2-398% in the presence of LCA, which suggested that LCA exhibited high HDI potentials via CYP inhibition. These data are significant for the clinical applications of LCA-containing herbs.

  4. 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

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

    PubMed

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

    2016-08-01

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

  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

    2016-01-01

    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

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

    PubMed

    Rendic, S; Nolteernsting, E; Schänzer, W

    1999-11-26

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

  10. Azole antifungal inhibition of buprenorphine, methadone and oxycodone in vitro metabolism.

    PubMed

    Moody, David E; Liu, Fenyun; Fang, Wenfang B

    2015-06-01

    Opioid-related mortality rates have escalated. Drug interactions may increase blood concentrations of the opioid. We therefore used human liver microsomes (HLMs) and cDNA-expressed human cytochrome P450s (rCYPs) to study in vitro inhibition of buprenorphine metabolism to norbuprenorphine (CYP3A4 and 2C8), oxycodone metabolism to noroxycodone (CYP3A4 and 2C18) and oxymorphone (CYP2D6), and methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP; CYP3A4 and 2B6). In this study, we have examined the inhibitory effect of 12 (mostly antifungal) azoles. These compounds have a wide range of solubility; to keep organic solvent ≤1%, there was an equally wide range of highest concentration tested (e.g., itraconazole 5 µM to fluconazole 1000 µM). Inhibitors were first incubated with HLMs at three concentrations with or without preincubation of inhibitor with reducing equivalents to also screen for time-dependent inhibition (TDI). Posaconazole displayed evidence of TDI; metronidazole and albendazole had no significant effect. Azoles were next screened at the highest achievable concentration for non-CYP3A4 pathways. IC50 values (µM) were determined for most CYP3A4 pathways (ranges) and other pathways as dictated by screen results: clotrimazole (0.30 - 0.35; others >30 µM); econazole (2.2 - 4.9; 2B6 R-EDDP - 9.5, S-EDDP - 6.8; 2C8 - 6.0; 2C18 - 1.0; 2D6 - 1.2); fluconazole (7.7 - 66; 2B6 - 313, 361; 2C8 - 1240; 2C18 - 17; 2D6 - 1000); itraconazole (2.5 to >5; others >5); ketoconazole (0.032 - 0.094; 2B6 - 12, 31; 2C8 - 78; 2C18 - 0.98; 2D6 - 182); miconazole (2.3 - 7.6; 2B6 - 2.8, 2.8; 2C8 - 5.3; 2C18 - 3.1; 2D6 - 5.9); posaconazole (3.4 - 20; 2C18 - 3.8; others >30); terconazole (0.48 to >10; 2C18 - 8.1; others >10) and voriconazole (0.40 - 15; 2B6 - 2.4, 2.5; 2C8 - 170; 2C18 - 13; 2D6 >300). Modeling based on estimated Ki values and plasma concentrations from the literature suggest that the orally administered azoles, particularly

  11. 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.

  12. Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat.

    PubMed

    Wu, Lili; Zhong, Wanping; Liu, Junjin; Han, Weichao; Zhong, Shilong; Wei, Qiang; Liu, Shuwen; Tang, Lan

    2015-09-01

    Oxysophocarpine (OSC), an active and toxic quinolizidine alkaloid, is highly valued in Sophora flavescens Ait. and Subprostrate sophora Root. OSC is used to treat inflammation and hepatitis for thousands of years in China. This study aims to investigate the CYP450-mediated reduction responsible for metabolizing OSC and to evaluate the absorption and metabolism of OSC in rat in situ. Four metabolites were identified, with sophocarpine (SC) as the major metabolite. SC formation was rapid in human and rat liver microsomes (HLMs and RLMs, respectively). The reduction rates in the liver are two fold higher than in the intestine, both in humans and rats. In HLMs, inhibitors of CYP2C9, 3A4/5, 2D6, and 2B6 had strong inhibitory effects on SC formation. Meanwhile, inhibitors of CYP3A and CYP2D6 had significant inhibition on SC formation in RLMs. Human recombinant CYP3A4/5, 2B6, 2D6, and 2C9 contributed significantly to SC production. The permeability in rat intestine and the excretion rates of metabolites were highest in the duodenum (p<0.05), and the absorbed amount of OSC in duodenum and jejunum was concentration-dependent. The metabolism could be significantly decreased by CYP3A inhibitor ketoconazole. In conclusion, the liver was the main organ responsible for OSC metabolism. First-pass metabolism via CYP3A4/5, 2B6, 2D6, and 2C9 may be the main reason for the poor OSC bioavailability.

  13. An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents.

    PubMed

    Etheridge, Amy S; Black, Sherry R; Patel, Purvi R; So, James; Mathews, James M

    2007-07-01

    Drug-herb interactions can result from the modulation of the activities of cytochrome P450 (P450) and/or drug transporters. The effect of extracts and individual constituents of goldenseal, Ginkgo biloba (and its hydrolyzate), grape seed, milk thistle, and ginseng on the activities of cytochrome P450 enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes were determined using enzyme-selective probe substrates, and their effect on human P-glycoprotein (Pgp) was determined using a baculovirus expression system by measuring the verapamil-stimulated, vanadate-sensitive ATPase activity. Extracts were analyzed by HPLC to standardize their concentration(s) of constituents associated with the pharmacological activity, and to allow comparison of their effects on P450 and Pgp with literature values. Many of the extracts/constituents exerted > or = 50 % inhibition of P450 activity. These include those from goldenseal (normalized to alkaloid content) inhibiting CYP2C8, CYP2D6, and CYP3A4 at 20 microM, ginkgo inhibiting CYP2C8 at 10 microM, grape seed inhibiting CYP2C9 and CYP3A4 at 10 microM, milk thistle inhibiting CYP2C8 at 10 microM, and ginsenosides F1 and Rh1 (but not ginseng extract) inhibiting CYP3A4 at 10 microM. Goldenseal extracts/constituents (20 microM, particularly hydrastine) and ginsenoside Rh1 stimulated ATPase at about half of the activity of the model substrate, verapamil (20 microM). The data suggest that the clearance of a variety of drugs may be diminished by concomitant use of these herbs via inhibition of P450 enzymes, but less so by Pgp-mediated effects.

  14. [Inhibitory effect of imperatorin and isoimperatorin on activity of cytochrome P450 enzyme in human and rat liver microsomes].

    PubMed

    Cao, Yan; Zhong, Yu-Huan; Yuan, Mei; Li, Hua; Zhao, Chun-Jie

    2013-04-01

    Imperatorin (IM) and isoimperatorin (ISOIM) are major active components of common herbal medicines from Umbelliferae plants, and widely used in clinic. This article studies the inhibitory effect of IM and ISOIM on the activity of cytochrome P450 (CYP) enzyme, and assesses their potential drug-drug interaction. IM and ISOIM were incubated separately with human or rat liver microsomes for 30 min, with phenacetin, bupropion, tolbutamide, S-mephenytoin, dextromethorphan and midazolam as probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS, and IC50 values were calculated to assess the inhibitory effect of the two drugs on human CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 enzymes, as well as on rat CYP1A2, 2B6, 2D2 and 3A1/2, and grade their inhibitory intensity. In human liver microsomes, IM and ISOIM showed different inhibitory effects on all of the six CYP isoenzymes. They were strong inhibitors for 1A2 and 2B6. The IC50 values were 0.05 and 0.20 micromol x L(-1) for 1A2, and 0.18 and 1.07 micromol x L(-1) for 2B6, respectively. They also showed moderate inhibitory effect on 2C19, and weak effect on 2C9, 2D6 and 3A4. In rat liver microsomes, IM and ISOIM were identified as moderate inhibitors for 1A2, with IC50 values of 1.95 and 2.98 micromol x L(-1). They were moderate and weak inhibitors for 2B6, with IC50 values of 6.22 and 21.71 micromol x L(-1), respectively. They also had weaker inhibitory effect on 2D2 and 3A1/2. The results indicated that IM and ISOIM had extensive inhibitory effects on human CYP enzymes. They are strong inhibitors of CYP1 A2 and 2B6 enzymes. However, it is worth noting the interaction arising from the inhibitory effect of CYP enzymes in clinic.

  15. Formation of Polyynes C4H2, C6H2, C8H2, and C10H2 from Reactions of C2H, C4H, C6H, and C8H Radicals with C2H2.

    PubMed

    Sun, Yi-Lun; Huang, Wen-Jian; Lee, Shih-Huang

    2015-10-15

    Some of the polyynes (HC2n+2H, 1 ≤ n ≤ 4) are observable in planetary atmospheres, interstellar space, and flames. Polyynes are proposed to play an important role in synthesis of large carbonaceous molecules. We explore the dynamics of reactions of C2nH (n = 1-4) radicals with C2H2 by interrogating time-of-flight spectra and photoionization efficiency spectra of products C2n+2H2. The reactions of n = 2-4 were investigated for the first time. The translational energy release is biased to low energy but extends to the energetic limit of product HC2n+2H + H, corresponding to a fraction of 0.34-0.36 on translational energy. Product C2n+2H2 has a deconvoluted ionization threshold in good agreement with the ionization energy of polyynes. The quantum chemical calculations support the experimental observations. This work verifies that the title reaction is an important source for formation of polyynes that have been observed in interstellar/circumstellar media and combustion processes. PMID:26722787

  16. Iridium Ziegler-Type Hydrogenation Catalysts Made from [(1,5-COD)Ir( -O2C8H15)]2 and AlEt3: Spectroscopic and Kinetic Evidence for the Irn Species Present and for Nanoparticles as the Fastest Catalyst

    SciTech Connect

    Alley, W.; Hamdemir, I; Wang, Q; Frenkel, A; Li, L; Yang, J; Menard, L; Nuzzo, R; Ozkar, S; Finke, R

    2010-01-01

    Ziegler-type hydrogenation catalysts, those made from a group 8-10 transition metal precatalyst and an AlR{sub 3} cocatalyst, are often used for large scale industrial polymer hydrogenation; note that Ziegler-type hydrogenation catalysts are not the same as Ziegler-Natta polymerization catalysts. A review of prior studies of Ziegler-type hydrogenation catalysts (Alley et al. J. Mol. Catal. A: Chem. 2010, 315, 1-27) reveals that a {approx}50 year old problem is identifying the metal species present before, during, and after Ziegler-type hydrogenation catalysis, and which species are the kinetically best, fastest catalysts-that is, which species are the true hydrogenation catalysts. Also of significant interest is whether what we have termed 'Ziegler nanoclusters' are present and what their relative catalytic activity is. Reported herein is the characterization of an Ir Ziegler-type hydrogenation catalyst, a valuable model (vide infra) for the Co-based industrial Ziegler-type hydrogenation catalyst, made from the crystallographically characterized [(1,5-COD)Ir({mu}-O{sub 2}C{sub 8}H{sub 15})]{sub 2} precatalyst plus AlEt{sub 3}. Characterization of this Ir model system is accomplished before and after catalysis using a battery of physical methods including Z-contrast scanning transmission electron microscopy (STEM), high resolution (HR)TEM, and X-ray absorption fine structure (XAFS) spectroscopy. Kinetic studies plus Hg(0) poisoning experiments are then employed to probe which species are the fastest catalysts. The main findings herein are that (i) a combination of the catalyst precursors [(1,5-COD)Ir({mu}-O{sub 2}C{sub 8}H{sub 15})]{sub 2} and AlEt{sub 3} gives catalytically active solutions containing a broad distribution of Ir{sub n} species ranging from monometallic Ir complexes to nanometer scale, noncrystalline Ir{sub n} nanoclusters (up to Ir{sub {approx}100} by Z-contrast STEM) with the estimated mean Ir species being 0.5-0.7 nm, Ir{sub {approx}4-15} clusters considering the similar, but not identical results from the different analytical methods; furthermore, (ii) the mean Ir{sub n} species are practically the same regardless of the Al/Ir ratio employed, suggesting that the observed changes in catalytic activity at different Al/Ir ratios are primarily the result of changes in the form or function of the Al-derived component (and not due to significant AlEt{sub 3}-induced changes in initial Ir{sub n} nuclearity). However (iii), during hydrogenation, a shift in the population of Ir species toward roughly 1.0-1.6 nm, fcc Ir(0){sub {approx}40-150}, Ziegler nanoclusters occurs with, significantly, (iv) a concomitant increase in catalytic activity. Importantly, and although catalysis by discrete subnanometer Ir species is not ruled out by this study, (v) the increases in activity with increased nanocluster size, plus Hg(0) poisoning studies, provide the best evidence to date that the approximately 1.0-1.6 nm, fcc Ir(0){sub {approx}40-150}, heterogeneous Ziegler nanoclusters are the fastest catalysts in this industrially related catalytic hydrogenation system (and in the simplest, Ockham's Razor interpretation of the data). In addition, (vi) Ziegler nanoclusters are confirmed to be an unusual, hydrocarbon-soluble, highly coordinatively unsaturated, Lewis-acid containing, and highly catalytically active type of nanocluster for use in other catalytic applications and other areas.

  17. In vivo inhibitory effects of puerarin on selected rat cytochrome P450 isoenzymes.

    PubMed

    Guo, Yu-Jin; Liang, Dong-Lou; Xu, Zhi-Sheng; Ye, Qiang

    2014-05-01

    Puerarin, the major bioactive constituent in kudzu root, is used widely in China for the treatment of cardiovascular diseases and diabetes. The purpose of this study was to find out whether puerarin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP2B6, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained bupropion (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (20 mg/kg), was given as oral administration to rats treated with 10 days oral administration of puerarin. 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 results showed that treatment with multiple doses of puerarin had inhibitory effects on rat CYP2B6, CYP2C9 and CYP3A4 enzyme activities. Therefore, caution is needed when puerarin is co-administered with CYP substrates, in view of herb-drug interactions.

  18. 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

  19. The association of cytochrome P450 genetic polymorphisms with sulfolane formation and the efficacy of a busulfan-based conditioning regimen in pediatric patients undergoing hematopoietic stem cell transplantation.

    PubMed

    Uppugunduri, C R S; Rezgui, M A; Diaz, P H; Tyagi, A K; Rousseau, J; Daali, Y; Duval, M; Bittencourt, H; Krajinovic, M; Ansari, M

    2014-06-01

    Cytochrome P450 enzymes (CYPs) and flavin-containing monooxygenases (FMOs) likely have a role in the oxidation of intermediate metabolites of busulfan (Bu). In vitro studies to investigate the involvement of these enzymes are cumbersome because of the volatile nature of the intermediate metabolite tetrahydrothiophene (THT) and the lack of sensitive quantitation methods. This study explored the association between the CYP2C9, CYP2C19, CYP2B6 and FMO3 genotypes and sulfolane (Su, a water soluble metabolite of Bu) plasma levels in children undergoing hematopoietic stem cell transplantation (HSCT). The relationship between these genotypes and the effectiveness of myeloablative conditioning was also analyzed. Sixty-six children receiving an intravenous Bu-based myeloablative conditioning regimen were genotyped for common functional variant alleles in CYP2C9 (*2 and *3), CYP2C19 (*2 and *17), FMO3 (rs2266780, rs2266782 and rs1736557) and CYP2B6 (*5 and *9). The plasma levels of Bu and its metabolite Su were measured after the ninth Bu dose in a subset of 44 patients for whom plasma samples were available. The ratio of Bu to Su was considered the metabolic ratio (MR) and was compared across the genotype groups. Higher MRs were observed in CYP2C9*2 and *3 allele carriers (mean±s.d.: 7.8±3.6 in carriers vs 4.4±2.2 in non-carriers; P=0.003). An increased incidence of graft failure was observed among patients with an MR>5 compared with those with MR values <5 (20% vs 0%; P=0.02). In contrast, a significantly higher incidence of relapse and graft failure (evaluated as event-free survival) was observed in patients with malignant disease who carried CYP2B6 alleles with reduced function on both chromosomes compared with carriers of at least one normal allele (100% vs 40%; P=0.0001). These results suggest that CYP2C9 has a role in the oxidation reactions of THT and indicate that it may be possible to predict the efficacy of Bu-based myeloablative conditioning before HSCT on the

  20. In Vitro Metabolism of Montelukast by Cytochrome P450s and UDP-Glucuronosyltransferases.

    PubMed

    Cardoso, Josiane de Oliveira; Oliveira, Regina Vincenzi; Lu, Jessica Bo Li; Desta, Zeruesenay

    2015-12-01

    Montelukast has been recommended as a selective in vitro and in vivo probe of cytochrome P450 (P450) CYP2C8 activity, but its selectivity toward this enzyme remains unclear. We performed detailed characterization of montelukast metabolism in vitro using human liver microsomes (HLMs), expressed P450s, and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Kinetic and inhibition experiments performed at therapeutically relevant concentrations reveal that CYP2C8 and CYP2C9 are the principal enzymes responsible for montelukast 36-hydroxylation to 1,2-diol. CYP3A4 was the main catalyst of montelukast sulfoxidation and stereoselective 21-hydroxylation, and multiple P450s participated in montelukast 25-hydroxylation. We confirmed direct glucuronidation of montelukast to an acyl-glucuronide. We also identified a novel peak that appears consistent with an ether-glucuronide. Kinetic analysis in HLMs and experiments in expressed UGTs indicate that both metabolites were exclusively formed by UGT1A3. Comparison of in vitro intrinsic clearance in HLMs suggest that direct glucuronidation may play a greater role in the overall metabolism of montelukast than does P450-mediated oxidation, but the in vivo contribution of UGT1A3 needs further testing. In conclusion, our in vitro findings provide new insight toward montelukast metabolism. The utility of montelukast as a probe of CYP2C8 activity may be compromised owing to involvement of multiple P450s and UGT1A3 in its metabolism. PMID:26374173

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

    PubMed

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

    2016-02-01

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

  2. 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

  3. 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.

  4. Evaluation of the impact of Flos Daturae on rat hepatic cytochrome P450 enzymes by cocktail probe drugs.

    PubMed

    Geng, Peiwu; Wang, Shuanghu; Wang, Chunjie; Chen, Jianmiao; Zhang, Lijing; Yang, Suping; Wen, Congcong; Zhou, Yunfang; Zhang, Meiling

    2015-01-01

    Flos Daturae, known as "baimantuoluo" or "yangjinhua" in China, has been used for centuries in Traditional Chinese Medicine for the treatment of asthma, convulsions, pain, and rheumatism. To investigate the influences of Flos Daturae on the activities of rat CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), omeprazole (10 mg/kg), bupropion (10 mg/kg), metoprolol (10 mg/kg) and testosterone (10 mg/kg), was intragastric administered to rats treated with a single low or high dose of Flos Daturae decotion for 7days. Blood samples collected at a series of time-points in plasma were determined by UPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 3.0. The results from the present in vivo study showed that Flos Daturae induce the activity of CYP2D6 enzyme with the decreased Cmax, AUC(0-∞) (P < 0.05) and the increased CL (P < 0.05). However, there were no significant differences of other probe drugs in plasma concentration and pharmacokinetic parameters. There were no significant effects on rat CYP1A2, CYP3A4, CYP2B6, CYP2C9 and CYP2C19 by Flos Daturae. Therefore, the resulting data suggested that caution was needed when Flos Daturae was co-administered with CYP2D6 substrates, which may result in treatment failure and herb-drug interactions. PMID:26885208

  5. 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.

  6. In vitro inhibitory effects of scutellarin on six human/rat cytochrome P450 enzymes and P-glycoprotein.

    PubMed

    Han, Yong-Long; Li, Dan; Yang, Quan-Jun; Zhou, Zhi-Yong; Liu, Li-Ya; Li, Bin; Lu, Jin; Guo, Cheng

    2014-05-05

    Inhibition of cytochrome P450 (CYP) and P-glycoprotein (P-gp) are regarded as the most frequent and clinically important pharmacokinetic causes among the various possible factors for drug-drug interactions. Scutellarin is a flavonoid which is widely used for the treatment of cardiovascular diseases. In this study, the in vitro inhibitory effects of scutellarin on six major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six rat CYPs (CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2) activities were examined by using liquid chromatography-tandem mass spectrometry. Meanwhile, the inhibitory effects of scutellarin on P-gp activity were examined on a human metastatic malignant melanoma cell line WM-266-4 by calcein-AM fluorometry screening assay. Results demonstrated that scutellarin showed negligible inhibitory effects on the six major CYP isoenzymes in human/rat liver microsomes with almost all of the IC50 values exceeding 100 μM, whereas it showed values of 63.8 μM for CYP2C19 in human liver microsomes, and 63.1 and 85.6 μM for CYP2C7 and CYP2C79 in rat liver microsomes, respectively. Scutellarin also showed weak inhibitory effect on P-gp. In conclusion, this study demonstrates that scutellarin is unlikely to cause any clinically significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P-gp.

  7. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols

    PubMed Central

    Nikolić, Dejan; Chen, Shao-Nong; Huang, Ke; Li, Guannan; Pauli, Guido F.; van Breemen, Richard B.

    2014-01-01

    As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug-botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography-tandem mass spectrometry. The hop extract at 5 μg/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4 μg/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2 μM, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1 μM, 1.1 μM and 0.4 μM, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered. PMID:24342125

  8. Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver.

    PubMed

    Qian, Linxi; Zolfaghari, Reza; Ross, A Catharine

    2010-07-01

    Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5'-GGTTCA-(n)5-AGGTCA-3') in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.

  9. 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.

  10. Effect of Radix Sophorae Flavescentis on activity of CYP450 isoforms in rats

    PubMed Central

    Chen, Lianguo; Cai, Jinzhang; Wang, Shuanghu; Hu, Lufeng; Yang, Xuezhi

    2015-01-01

    Kushen (Radix Sophorae Flavescentis) is the dried roots of Sophora Flavescens Ait, alkaloids and flavonoids are the main active constituents of Radix Sophorae Flavescentis. The influence of Radix Sophorae Flavescentis on the activities of CYP450 isoforms CYP2B6, CYP2C19, CYP1A2, CYP2C9, CYP3A4 and CYP2D6 were evaluated by cocktail method. The rats were randomly divided into Radix Sophorae Flavescentis group and control group. The Radix Sophorae Flavescentis group rats were given 5 g/kg Radix Sophorae Flavescentis decoction by intragastric administration. The six probe drugs (bupropion, omeprazole, phenacetin, tolbutamide, midazolam and metroprolol) were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of Radix Sophorae Flavescentis group compared to control group, there were statistical pharmacokinetics difference for omeprazole, phenacetin, tolbutamide and metroprolol. It indicated that the Radix Sophorae Flavescentis may induce the activities of CYP2D6, and inhibit of CYP2C19, CYP1A2 and CYP2C9 of rats. As other drugs are always used after Radix Sophorae Flavescentis, interactions between other drugs and Radix Sophorae Flavescentis undertake the risk of either diminished efficacy or adverse effects. This may give advising for reasonable drug use after Radix Sophorae Flavescentis. PMID:26885078

  11. 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.

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

    PubMed Central

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

    2015-01-01

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

  13. Effects of suberoylanilide hydroxamic acid on rat cytochrome P450 enzyme activities.

    PubMed

    Lin, Kezhi; Zhang, Qingwei; Liu, Zezheng; Yang, Suping; Lin, Yingying; Wen, Congcong; Zheng, Yuancai

    2015-01-01

    Vorinostat (suberoylanilide hydroxamic acid, SAHA) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. The rats were randomly divided into SAHA groups (low, medium and high dosage) and control group. The SAHA group rats were given 12.3, 24.5, and 49 mg/kg SAHA, respectively, by continuous intragastric administration for 7 days. The influence of SAHA on the activities of CYP450 isoforms CYP2B6, CYP1A2, CYP2C19, CYP2D6 and CYP2C9 were evaluated by cocktail method, they were responsed by the changes of pharmacokinetic parameters of bupropion, phenacetin, tolbutamide, metroprolol and omeprazole. The five probe drugs were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of SAHA group compared to control group, there were statistical pharmacokinetics difference for bupropion, phenacetin, tolbutamide and metroprolol. Continuous intragastric administration for 7 days may induce the activities of CYP2C19 of rats, inhibit CYP1A2 and slightly inhibit CYP2B6 and CYP2D6 of rats. This may give advising for reasonable drug use after co-used with SAHA. The results indicated that drug co-administrated with SAHA may need dose adjustment. Furthermore, continuous intragastric administration of SAHA for 7 days, liver cell damaged, causing liver cell edema, in liver metabolism process.

  14. Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

    PubMed

    Jellali, Rachid; Bricks, Thibault; Jacques, Sébastien; Fleury, Marie-José; Paullier, Patrick; Merlier, Franck; Leclerc, Eric

    2016-07-01

    Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

  15. In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

    PubMed

    Hua, Xin; Peng, Xiao; Tan, Shengnan; Li, Chunying; Wang, Wei; Luo, Meng; Fu, Yujie; Zu, Yuangang; Smyth, Hugh

    2014-10-29

    Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA. PMID:25272989

  16. 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. PMID:24890600

  17. 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

  18. Pharmacokinetic Interaction Between Prasugrel and Ritonavir in Healthy Volunteers

    PubMed Central

    Ancrenaz, Virginie; Déglon, Julien; Samer, Caroline; Staub, Christian; Dayer, Pierre; Daali, Youssef; Desmeules, Jules

    2013-01-01

    The new anti-aggregating agent prasugrel is bioactivated by cytochromes P450 (CYP) 3A and 2B6. Ritonavir is a potent CYP3A inhibitor and was shown in vitro as a CYP2B6 inhibitor. The aim of this open-label cross-over study was to assess the effect of ritonavir on prasugrel active metabolite (prasugrel AM) pharmacokinetics in healthy volunteers. Ten healthy male volunteers received 10 mg prasugrel. After at least a week washout, they received 100 mg ritonavir, followed by 10 mg prasugrel 2 hr later. We used dried blood spot sampling method to monitor prasugrel AM pharmacokinetics (Cmax, t1/2, tmax, AUC0–6 hr) at 0, 0.25, 0.5, 1, 1.5, 2, 4 and 6 hr after prasugrel administration. A ‘cocktail’ approach was used to measure CYP2B6, 2C9, 2C19 and 3A activities. In the presence of ritonavir, prasugrel AM Cmax and AUC were decreased by 45% (mean ratio: 0.55, CI 90%: 0.40–0.7, p = 0.007) and 38% (mean ratio: 0.62, CI 90%: 0.54–0.7, p = 0.005), respectively, while t1/2 and tmax were not affected. Midazolam metabolic ratio (MR) dramatically decreased in presence of ritonavir (6.7 ± 2.6 versus 0.13 ± 0.07) reflecting an almost complete inhibition of CYP3A4, whereas omeprazole, flurbiprofen and bupropion MR were not affected. These data demonstrate that ritonavir is able to block prasugrel CYP3A4 bioactivation. This CYP-mediated drug–drug interaction might lead to a significant reduction of prasugrel efficacy in HIV-infected patients with acute coronary syndrome. PMID:22900583

  19. Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen. Role of CYP2C19 and CYP1A2 in O-demethylation.

    PubMed

    Stresser, D M; Kupfer, D

    1998-09-01

    1,1,1-Trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor) is a widely used pesticide that is pro-estrogenic. We have elucidated the human cytochrome P450 enzymes responsible for conversion of methoxychlor into its major metabolite, the mono-O-demethylated derivative (mono-OH-M) that is estrogenic. Incubation of methoxychlor with microsomes from insect cells overexpressing either CYP1A2, CYP2C18, or CYP2C19 yielded mono-OH-M with turnover numbers of 14.9, 15.5, and 39.1 nmol/min/nmol of P450, respectively. CYP2B6 and CYP2C9 were much less active. Incubations with purified CYP2C19 and CYP2C18 resulted in formation of mono-OH-M, and also the bis-demethylated metabolite. Co-incubation of liver microsomes with methoxychlor and various P450 isoform-selective inhibitors suggested involvement of several P450s in mono-O-demethylation, including CYP1A2, CYP2A6, CYP2C9, and CYP2C19. A role for CYP2C19, CYP1A2, and CYP2A6 was also indicated by multivariate regression analysis of the mono-O-demethylase activity in a panel of human liver microsomes characterized for isoform-specific catalytic activities (R2 = 0.96). Based on the totality of the evidence, CYP2C19 appears to be the major catalyst of methoxychlor mono-O-demethylation. However, in individuals lacking functional CYP2C19 (e.g. the "poor metabolizer" phenotype), CYP1A2 may play the predominant role. CYP2A6, CYP2C9, and CYP2B6 probably contribute to a lesser extent. Although CYP2C18 is an efficient methoxychlor demethylase, its expression in liver is reportedly low or absent, suggesting a negligible role for this enzyme in methoxychlor metabolism. Lengthy incubations of liver microsomes with methoxychlor produced other secondary and tertiary metabolites. Efficient conversion of methoxychlor to estrogenic mono-OH-M by liver microsomes suggests that methoxychlor has the potential to be estrogenic in humans, as observed in several animal species.

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

    PubMed

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

    2012-03-01

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

  1. Effect of penicillin-based antibiotics, amoxicillin, ampicillin, and piperacillin, on drug-metabolizing activities of human hepatic cytochromes P450.

    PubMed

    Niwa, Toshiro; Morimoto, Mari; Hirai, Takako; Hata, Tomomi; Hayashi, Misato; Imagawa, Yurie

    2016-02-01

    The effects of three kinds of penicillin-based antibiotics, amoxicillin, ampicillin, and piperacillin, on drug-metabolizing activity of human hepatic cytochrome P450 (P450 or CYP) were investigated. Metabolic activities of P450s expressed in recombinant Escherichia coli at substrate concentrations around the Michaelis constant were compared in the presence or absence of the antibiotics. Amoxicillin, ampicillin, and piperacillin at 0.5 or 1 mM concentrations neither inhibited nor stimulated CYP2C9-mediated tolbutamide methylhydroxylation, CYP2D6-mediated dopamine formation from p-tyramine, or CYP3A4- or CYP3A5-mediated testosterone 6β-hydroxylation. However, amoxicillin and piperacillin inhibited CYP2C8-mediated aminopyrine N-demethylation at 50% inhibitory concentration of 0.83 and 1.14 mM, respectively. These results suggest that piperacillin might inhibit CYP2C8 clinically, although the interactions between these three penicillin-based antibiotics and other drugs that are metabolized by P450s investigated would not be clinically significant.

  2. Human Cytochrome P450 Enzyme Modulation by Gymnema sylvestre: A Predictive Safety Evaluation by LC-MS/MS

    PubMed Central

    Rammohan, Bera; Samit, Karmakar; Chinmoy, Das; Arup, Saha; Amit, Kundu; Ratul, Sarkar; Sanmoy, Karmakar; Dipan, Adhikari; Tuhinadri, Sen

    2016-01-01

    Background: Traditionally GS is used to treat diabetes mellitus. Drug-herb interaction of GS via cytochrome P450 enzyme system by substrate cocktail method using HLM has not been reported. Objective: To evaluate the in-vitro modulatory effects of GS extracts (aqueous, methanol, ethyl acetate, chloroform and n-hexane) and deacylgymnemic acid (DGA) on human CYP1A2, 2C8, 2C9, 2D6 and 3A4 activities in HLM. Material and Methods: Probe substrate-based LCMS/MS method was established for all CYPs. The metabolite formations were examined after incubation of probe substrates with HLM in the presence or absence of extracts and DGA. The inhibitory effects of GS extracts and DGA were characterized with kinetic parameters IC50 and Ki values. Results: GS extracts showed differential effect on CYP activities in the following order of inhibitory potency: ethyl acetate > Chloroform > methanol > n-hexane > aqueous > DGA. This differential effect was observed against CYP1A2, 2C9 and less on CYP3A4 and 2C8 but all CYPs were unaffected by aqueous extract and DGA. The ethyl acetate and chloroform extract exhibited moderate inhibition towards CYP1A2 and 3A4. The aqueous extract and DGA however showed negligible inhibition towards all five major human CYPs with very high IC50 values (>90μg/ml). Conclusion: The results of our study revealed that phytoconstituents contained in GS, particularly in ethyl acetate and chloroform extracts, were able to inhibit CYP1A2, 3A4 and 2C9. The presence of relatively small, lipophillic yet slightly polar compounds within the GS extracts may be attributed for inhibition activities. These suggest that the herb or its extracts should be examined for potential pharmacokinetic drug interactions in vivo. Abbreviations used: GS: Gymnema sylvestre, GSE: Gymnema sylvestre extract, DGA: deacyl gymnemic acid, CYP: cytochrome P450, DMSO: dimethylsulphoxide, HLM: human liver microsomes, LC-MS/MS: liquid chromatography tandem mass spectroscopy, NADPH: reduced

  3. Immunochemical detection of cytochrome P450 enzymes in liver microsomes of 27 cynomolgus monkeys.

    PubMed

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-11-01

    The cynomolgus monkey is widely used as a primate model in preclinical studies because of its evolutionary closeness to humans. Despite their importance in drug metabolism, the content of each cytochrome P450 (P450) enzyme has not been systematically determined in cynomolgus monkey livers. In this study, liver microsomes of 27 cynomolgus monkeys were analyzed by immunoblotting using selective P450 antibodies. The specificity of each antibody was confirmed by analyzing the cross-reactivity against 19 CYP1-3 subfamily enzymes using recombinant proteins. CYP2A, CYP2B6, CYP2C9/19, CYP2C76, CYP2D, CYP2E, CYP3A4, and CYP3A5 were detected in all 27 animals. In contrast, CYP1A, CYP1D, and CYP2J were below detectable levels in all liver samples. The average content of each P450 showed that among the P450s analyzed CYP3A (3A4 and 3A5) was the most abundant (40% of total immunoquantified P450), followed by CYP2A (25%), CYP2C (14%), CYP2B6 (13%), CYP2E1 (11%), and CYP2D (3%). No apparent sex differences were found for any P450. Interanimal variations ranged from 2.6-fold (CYP3A) to 11-fold (CYP2C9/19), and most P450s (CYP2A, CYP2D, CYP2E, CYP3A4, and CYP3A5) varied 3- to 4-fold. To examine the correlations of P450 content with enzyme activities, metabolic assays were performed in 27 cynomolgus monkey livers using 7-ethoxyresorufin, coumarin, pentoxyresorufin, flurbiprofen, bufuralol, dextromethorphan, and midazolam. CYP2D and CYP3A4 contents were significantly correlated with typical reactions of human CYP2D (bufuralol 1'-hydroxylation and dextromethorphan O-deethylation) and CYP3A (midazolam 1'-hydroxylation and 4-hydroxylation). The results presented in this study provide useful information for drug metabolism studies using cynomolgus monkeys.

  4. 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.

  5. Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.

    PubMed

    Hoffmann, Stefan A; Müller-Vieira, Ursula; Biemel, Klaus; Knobeloch, Daniel; Heydel, Sandra; Lübberstedt, Marc; Nüssler, Andreas K; Andersson, Tommy B; Gerlach, Jörg C; Zeilinger, Katrin

    2012-12-01

    Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.

  6. 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

  7. Metabolism of risperidone to 9-hydroxyrisperidone by human cytochromes P450 2D6 and 3A4.

    PubMed

    Fang, J; Bourin, M; Baker, G B

    1999-02-01

    Risperidone is a relatively new antipsychotic drug that has been reported to improve both the positive and the negative symptoms of schizophrenia and produces relatively few extrapyramidal side effects at low doses. Formation of 9-hydroxyrisperidone, an active metabolite, is the most important metabolic pathway of risperidone in human. In the present study, in vitro metabolism of risperidone (100 microM) was investigated using the recombinant human cytochrome P450 (CYP) enzymes CYP1A1, CYP1A2, CYP2C8, CYP2C9-arg144, CYP2C9-cys144, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 supplemented with an NADPH-generating system. 9-Hydroxyrisperidone was determined by a new HPLC method with an Hypersil CN column and a UV detector. Of these enzymes, CYPs 2D6, 3A4 and 3A5 were found to be the ones capable of metabolising risperidone to 9-hydroxyrisperidone, with activities of 7.5, 0.4 and 0.2 pmol pmol(-1) CYP min(-1), respectively. A correlation study using a panel of human liver microsomes showed that the formation of 9-hydroxyrisperidone is highly correlated with CYP2D6 and 3A activities. Thus, both CYP2D6 and 3A4 are involved in the 9-hydroxylation of risperidone at the concentration of risperidone used in this study. This observation is confirmed by the findings that both quinidine (inhibitor of CYP2D6) and ketoconazole (inhibitor of CYP3A4) can inhibit the formation of 9-hydroxyrisperidone. Furthermore, inducers of CYP can significantly increase the formation of 9-hydroxyrisperidone in rat. The formation of 9-hydroxyrisperidone is highly correlated with testosterone 6beta-hydroxylase activities, suggesting that inducible CYP3A contributes significantly to the metabolism of risperidone in rat.

  8. Extrapolation of diclofenac clearance from in vitro microsomal metabolism data: role of acyl glucuronidation and sequential oxidative metabolism of the acyl glucuronide.

    PubMed

    Kumar, Sanjeev; Samuel, Koppara; Subramanian, Ramaswamy; Braun, Matthew P; Stearns, Ralph A; Chiu, Shuet-Hing Lee; Evans, David C; Baillie, Thomas A

    2002-12-01

    Diclofenac is eliminated predominantly (approximately 50%) as its 4'-hydroxylated metabolite in humans, whereas the acyl glucuronide (AG) pathway appears more important in rats (approximately 50%) and dogs (>80-90%). However, previous studies of diclofenac oxidative metabolism in human liver microsomes (HLMs) have yielded pronounced underprediction of human in vivo clearance. We determined the relative quantitative importance of 4'-hydroxy and AG pathways of diclofenac metabolism in rat, dog, and human liver microsomes. Microsomal intrinsic clearance values (CL(int) = V(max)/K(m)) were determined and used to extrapolate the in vivo blood clearance of diclofenac in these species. Clearance of diclofenac was accurately predicted from microsomal data only when both the AG and the 4'-hydroxy pathways were considered. However, the fact that the AG pathway in HLMs accounted for ~75% of the estimated hepatic CL(int) of diclofenac is apparently inconsistent with the 4'-hydroxy diclofenac excretion data in humans. Interestingly, upon incubation with HLMs, significant oxidative metabolism of diclofenac AG, directly to 4'-hydroxy diclofenac AG, was observed. The estimated hepatic CL(int) of this pathway suggested that a significant fraction of the intrahepatically formed diclofenac AG may be converted to its 4'-hydroxy derivative in vivo. Further experiments indicated that this novel oxidative reaction was catalyzed by CYP2C8, as opposed to CYP2C9-catalyzed 4'-hydroxylation of diclofenac. These findings may have general implications in the use of total (free + conjugated) oxidative metabolite excretion for determining primary routes of drug clearance and may question the utility of diclofenac as a probe for phenotyping human CYP2C9 activity in vivo via measurement of its pharmacokinetics and total 4'-hydroxy diclofenac urinary excretion.

  9. Pharmacokinetic interactions with thiazolidinediones.

    PubMed

    Scheen, André J

    2007-01-01

    Type 2 diabetes mellitus is a complex disease combining defects in insulin secretion and insulin action. New compounds called thiazolidinediones or glitazones have been developed for reducing insulin resistance. After the withdrawal of troglitazone because of liver toxicity, two compounds are currently used in clinical practice, rosiglitazone and pioglitazone. These compounds are generally used in combination with other pharmacological agents. Because they are metabolised via cytochrome P450 (CYP), glitazones are exposed to numerous pharmacokinetic interactions. CYP2C8 and CYP3A4 are the main isoenzymes catalysing biotransformation of pioglitazone (as with troglitazone), whereas rosiglitazone is metabolised by CYP2C9 and CYP2C8. For both rosiglitazone and pioglitazone, the most relevant interactions have been described in healthy volunteers with rifampicin (rifampin), which results in a significant decrease of area under the plasma concentration-time curve [AUC] (54-65% for rosiglitazone, p<0.001; 54% for pioglitazone, p<0.001), and with gemfibrozil, which results in a significant increase of AUC (130% for rosiglitazone, p<0.001; 220-240% for pioglitazone, p<0.001). The relevance of such drug-drug interactions in patients with type 2 diabetes remains to be evaluated. However, in the absence of clinical data, it is prudent to reduce the dosage of each glitazone by half in patients treated with gemfibrozil. Conversely, rosiglitazone and pioglitazone do not seem to significantly affect the pharmacokinetics of other compounds. Although some food components have also been shown to potentially interfere with drugs metabolised with the CYP system, no published study deals specifically with these possible CYP-mediated food-drug interactions with glitazones.

  10. Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

    PubMed

    Cui, Ying-Lu; Xu, Fang; Wu, Rongling

    2016-06-29

    The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided. PMID:27302079

  11. Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

    PubMed

    Cui, Ying-Lu; Xu, Fang; Wu, Rongling

    2016-06-29

    The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided.

  12. Pharmacokinetic interaction between prasugrel and ritonavir in healthy volunteers.

    PubMed

    Ancrenaz, Virginie; Déglon, Julien; Samer, Caroline; Staub, Christian; Dayer, Pierre; Daali, Youssef; Desmeules, Jules

    2013-02-01

    The new anti-aggregating agent prasugrel is bioactivated by cytochromes P450 (CYP) 3A and 2B6. Ritonavir is a potent CYP3A inhibitor and was shown in vitro as a CYP2B6 inhibitor. The aim of this open-label cross-over study was to assess the effect of ritonavir on prasugrel active metabolite (prasugrel AM) pharmacokinetics in healthy volunteers. Ten healthy male volunteers received 10 mg prasugrel. After at least a week washout, they received 100 mg ritonavir, followed by 10 mg prasugrel 2 hr later. We used dried blood spot sampling method to monitor prasugrel AM pharmacokinetics (C(max) , t(1/2) , t(max) , AUC(0-6 hr) ) at 0, 0.25, 0.5, 1, 1.5, 2, 4 and 6 hr after prasugrel administration. A 'cocktail' approach was used to measure CYP2B6, 2C9, 2C19 and 3A activities. In the presence of ritonavir, prasugrel AM C(max) and AUC were decreased by 45% (mean ratio: 0.55, CI 90%: 0.40-0.7, p = 0.007) and 38% (mean ratio: 0.62, CI 90%: 0.54-0.7, p = 0.005), respectively, while t(1/2) and t(max) were not affected. Midazolam metabolic ratio (MR) dramatically decreased in presence of ritonavir (6.7 ± 2.6 versus 0.13 ± 0.07) reflecting an almost complete inhibition of CYP3A4, whereas omeprazole, flurbiprofen and bupropion MR were not affected. These data demonstrate that ritonavir is able to block prasugrel CYP3A4 bioactivation. This CYP-mediated drug-drug interaction might lead to a significant reduction of prasugrel efficacy in HIV-infected patients with acute coronary syndrome.

  13. 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

  14. 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. PMID:26111722

  15. Influence of cytochrome P450 polymorphisms on drug therapies: pharmacogenetic, pharmacoepigenetic and clinical aspects.

    PubMed

    Ingelman-Sundberg, Magnus; Sim, Sarah C; Gomez, Alvin; Rodriguez-Antona, Cristina

    2007-12-01

    The polymorphic nature of the cytochrome P450 (CYP) genes affects individual drug response and adverse reactions to a great extent. This variation includes copy number variants (CNV), missense mutations, insertions and deletions, and mutations affecting gene expression and activity of mainly CYP2A6, CYP2B6, CYP2C9, CYP2C19 and CYP2D6, which have been extensively studied and well characterized. CYP1A2 and CYP3A4 expression varies significantly, and the cause has been suggested to be mainly of genetic origin but the exact molecular basis remains unknown. We present a review of the major polymorphic CYP alleles and conclude that this variability is of greatest importance for treatment with several antidepressants, antipsychotics, antiulcer drugs, anti-HIV drugs, anticoagulants, antidiabetics and the anticancer drug tamoxifen. We also present tables illustrating the relative importance of specific common CYP alleles for the extent of enzyme functionality. The field of pharmacoepigenetics has just opened, and we present recent examples wherein gene methylation influences the expression of CYP. In addition microRNA (miRNA) regulation of P450 has been described. Furthermore, this review updates the field with respect to regulatory initiatives and experience of predictive pharmacogenetic investigations in the clinics. It is concluded that the pharmacogenetic knowledge regarding CYP polymorphism now developed to a stage where it can be implemented in drug development and in clinical routine for specific drug treatments, thereby improving the drug response and reducing costs for drug treatment.

  16. 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.

  17. 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

  18. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    PubMed

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-09-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

  19. Geneva Cocktail for Cytochrome P450 and P-Glycoprotein Activity Assessment Using Dried Blood Spots

    PubMed Central

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-01-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography–tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

  20. 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

  1. Effect of bixin and norbixin on the expression of cytochrome P450 in HepG2 cell line.

    PubMed

    Matuo, Míriam Cristina Sakuragui; de Oliveira Takamoto, Rafael Teruiti; Kikuchi, Irene Satiko; de Jesus Andreoli Pinto, Terezinha

    2013-08-01

    Bixin and norbixin are the main components of annatto, which is extracted from Bixa orellana and largely used as natural colorant in the food and pharmaceutical industries. Annatto can enhance CYP1A and CYP2B activity in rats; however, the inducer effect has not been investigated in human cell lines. In this study, the ability of bixin and norbixin to induce the cytochrome P450 (CYP) enzymes was assessed in HepG2 human hepatoma cell line. HepG2 cells were treated with bixin and norbixin, and the expression of the CYP genes quantified by real-time reverse transcription polymerase chain reaction (RT-PCR). Expression of CYP1A1 and CYP1A2 was significantly increased by bixin treatment, while CYP2B6, 2C9, 2E1 and 3A4 were unaffected. Cells were treated with norbixin showed no inducer effect. The results suggest that the inducer potential of annatto is attributed to bixin, but not to norbixin, despite their similarities in molecular structure. PMID:23554079

  2. 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.

  3. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

    PubMed

    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; Braspenning, Joris; Breitkopf-Heinlein, Katja

    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.

  4. Pharmacogenomics of glinides.

    PubMed

    Chen, Miao; Hu, Cheng; Jia, Weiping

    2015-01-01

    Glinides, including repaglinide, nateglinide and mitiglinide, are a type of fasting insulin secretagogue that could help to mimic early-phase insulin release, thus providing improved control of the postprandial glucose levels. Glinides stimulate insulin secretion by inhibiting ATP-sensitive potassium channels in the pancreatic β-cell membrane. Although glinides have been widely used clinically and display excellent safety and efficacy, the response to glinides varies among individuals, which is partially due to genetic factors involved in drug absorption, distribution, metabolism and targeting. Several pharmacogenomic studies have demonstrated that variants of genes involved in the pharmacokinetics or pharmacodynamics of glinides are associated with the drug response. Polymorphisms of genes involved in drug metabolism, such as CYP2C9, CYP2C8 and SLCO1B1, may influence the efficacy of glinides and the incidence of adverse effects. In addition, Type 2 diabetes mellitus susceptibility genes, such as KCNQ1, PAX4 and BETA2, also influence the efficacy of glinides. In this article, we review and discuss current pharmacogenomics researches on glinides, and hopefully provide useful data and proof for clinical application. PMID:25560470

  5. Arachidonic Acid Monooxygenase: Genetic and Biochemical Approaches to Physiological/Pathophysiological Relevance

    PubMed Central

    Capdevila, Jorge H.; Wang, Wenhui; Falck, John R.

    2015-01-01

    Studies with rat genetic models of hypertension pointed to roles for the CYP2C and CYP4A arachidonic acid epoxygenases and ω-hydroxylases in tubular transport, hemodynamics, and blood pressure control. Further progress in defining their physiological functions and significance to human hypertension requires conclusive identifications of the relevant genes and proteins. Here we discuss unequivocal evidence of roles for the murine Cyp4a14, Cyp4a10, and Cyp2c44 genes in the pathophysiology of hypertension by showing that: a) Cyp4a14(-/-) mice develop sexually dimorphic hypertension associated with renal vasoconstriction, and up-regulated expression of Cyp4a12a and pro-hypertensive 20-hydroxyeicosatetraenoic acid (20-HETE) levels, and b) Cyp4a10(-/-) and Cyp2c44(-/-) mice develop salt sensitive hypertension linked to downregulation or lack of the Cyp2c44 epoxygenase, reductions in anti-hypertensive epoxyeicosatrienoic acids (EETs), and increases in distal sodium reabsorption. Based on these studies, the human CYP4A11 and CYPs 2C8 and 2C9 genes and their products are identified as potential candidates for studies of the molecular basis of human hypertension. PMID:25986599

  6. The pre-clinical absorption, distribution, metabolism and excretion properties of IPI-926, an orally bioavailable antagonist of the hedgehog signal transduction pathway.

    PubMed

    Smith, Sherri; Hoyt, Jennifer; Whitebread, Nigel; Manna, Joseph; Peluso, Marisa; Faia, Kerrie; Campbell, Veronica; Tremblay, Martin; Nair, Somarajan; Grogan, Michael; Castro, Alfredo; Campbell, Matthew; Ferguson, Jeanne; Arsenault, Brendan; Nevejans, Jylle; Carter, Bennett; Lee, John; Dunbar, Joi; McGovern, Karen; Read, Margaret; Adams, Julian; Constan, Alexander; Loewen, Gordon; Sydor, Jens; Palombella, Vito; Soglia, John

    2013-10-01

    1. IPI-926 is a novel semisynthetic cyclopamine derivative that is a potent and selective Smoothened inhibitor that blocks the hedgehog signal transduction pathway. 2. The in vivo clearance of IPI-926 is low in mouse and dog and moderate in monkey. The volume of distribution is high across species. Oral bioavailability ranges from moderate in monkey to high in mouse and dog. Predicted human clearance using simple allometry is low (24 L h(-1)), predicted volume of distribution is high (469 L) and predicted half-life is long (20 h). 3. IPI-926 is highly bound to plasma proteins and has minimal interaction with human α-1-acid glycoprotein. 4. In vitro metabolic stability ranges from stable to moderately stable. Twelve oxidative metabolites were detected in mouse, rat, dog, monkey and human liver microsome incubations and none were unique to human. 5. IPI-926 is not a potent reversible inhibitor of CYP1A2, 2C8, 2C9 or 3A4 (testosterone). IPI-926 is a moderate inhibitor of CYP2C19, 2D6 and 3A4 (midazolam) with KI values of 19, 16 and 4.5 µM, respectively. IPI-926 is both a substrate and inhibitor (IC50 = 1.9 µM) of P-glycoprotein. 6. In summary, IPI-926 has desirable pre-clinical absorption, distribution, metabolism and excretion properties. PMID:23527529

  7. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

  8. Pharmacogenomics of glinides.

    PubMed

    Chen, Miao; Hu, Cheng; Jia, Weiping

    2015-01-01

    Glinides, including repaglinide, nateglinide and mitiglinide, are a type of fasting insulin secretagogue that could help to mimic early-phase insulin release, thus providing improved control of the postprandial glucose levels. Glinides stimulate insulin secretion by inhibiting ATP-sensitive potassium channels in the pancreatic β-cell membrane. Although glinides have been widely used clinically and display excellent safety and efficacy, the response to glinides varies among individuals, which is partially due to genetic factors involved in drug absorption, distribution, metabolism and targeting. Several pharmacogenomic studies have demonstrated that variants of genes involved in the pharmacokinetics or pharmacodynamics of glinides are associated with the drug response. Polymorphisms of genes involved in drug metabolism, such as CYP2C9, CYP2C8 and SLCO1B1, may influence the efficacy of glinides and the incidence of adverse effects. In addition, Type 2 diabetes mellitus susceptibility genes, such as KCNQ1, PAX4 and BETA2, also influence the efficacy of glinides. In this article, we review and discuss current pharmacogenomics researches on glinides, and hopefully provide useful data and proof for clinical application.

  9. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery. PMID:16859410

  10. Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding.

    PubMed

    Gomaa, Mohamed Sayed; Yee, Sook Wah; Milbourne, Ceri Elizabeth; Barbera, Maria Chiara; Simons, Claire; Brancale, Andrea

    2006-08-01

    Homology models of cytochrome P450 RA1 (CYP26A1) were constructed using three human P450 structures, CYP2C8, CYP2C9 and CYP3A4 as templates for the model building. Using MOE software the lowest energy CYP26A1 model was then assessed for stereochemical quality and side chain environment. Further active site optimisation of the CYP26A1 model built using the CYP3A4 template was performed by molecular dynamics to generate a final CYP26A1 model. The natural substrate, all-trans-retinoic acid (atRA), and inhibitor R 15866, were docked into the model allowing further validation of the active site architecture. Using the docking studies structurally and functionally important residues were identified with subsequent characterisation of secondary structure. Multiple hydrophobic interactions, including the side chains of TRP112, PHE299, PHE222, PHE84, PHE374 and PRO371, are important for binding of atRA and R115866. Additional hydrogen bonding interactions were noted as follows: atRA-- C==O of the atRA carboxylate group and ARG86; R115866--benzothiazole nitrogen and the backbone NH of SER115.

  11. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  12. Insights into drug metabolism from modelling studies of cytochrome P450-drug interactions.

    PubMed

    Maréchal, Jean-Didier; Sutcliffe, Michael J

    2006-01-01

    The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolising CYPs provide crucial protection from the harmful effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Elucidating the structural features of CYPs that contribute to their metabolism of structurally diverse substrates impacts on the rational design of improved therapeutic drugs and specific inhibitors. Models capable of predicting the possible involvement of CYPs in the metabolism of drugs or drug candidates are thus important tools in drug discovery and development. Ideally, functional information would be obtained from crystal structures of all the CYPs of interest. Initially only crystal structures of distantly related bacterial CYPs were available - comparative modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain some useful functional information. A significant step forward in the reliability of these models came six years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of five human enzymes, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. The evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism, is presented as a case study. PMID:16918473

  13. Effect of cefixime and cefdinir, oral cephalosporins, on cytochrome P450 activities in human hepatic microsomes.

    PubMed

    Niwa, Toshiro; Shiraga, Toshifumi; Hashimoto, Tomoko; Kagayama, Akira

    2004-01-01

    The effects of two kinds of oral cephalosporins, cefixime and cefdinir, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Both cefixime and cefdinir at 2 mM concentration neither inhibited nor stimulated CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, or CYP3A4-mediated testosterone 6beta-hydroxylation. The free fractions of cefixime and cefdinir in the incubation mixture, which were measured by ultracentrifugation, were 86.1-93.8% and 94.1-97.8%, respectively. These results suggest that both cefixime and cefdinir would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.

  14. 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

  15. Drug-drug and food-drug pharmacokinetic interactions with new insulinotropic agents repaglinide and nateglinide.

    PubMed

    Scheen, André J

    2007-01-01

    This review describes the current knowledge on drug-drug and food-drug interactions with repaglinide and nateglinide. These two meglitinide derivatives, commonly called glinides, have been developed for improving insulin secretion of patients with type 2 diabetes mellitus. They are increasingly used either in monotherapy or in combination with other oral antihyperglycaemic agents for the treatment of type 2 diabetes. Compared with sulfonylureas, glinides have been shown to (i) provide a better control of postprandial hyperglycaemia, (ii) overcome some adverse effects, such as hypoglycaemia, and (iii) have a more favourable safety profile, especially in patients with renal failure. The meal-related timing of administration of glinides and the potential influence of food and meal composition on their bioavailability may be important. In addition, some food components (e.g. grapefruit juice) may cause pharmacokinetic interactions. Because glinides are metabolised via cytochrome P450 (CYP) 3A4 isoenzyme, they are indeed exposed to pharmacokinetic interactions. In addition to CYP3A4, repaglinide is metabolised via CYP2C8, while nateglinide metabolism also involves CYP2C9. Furthermore, both compounds and their metabolites may undergo specialised transport/uptake in the intestine, another source of pharmacokinetic interactions. Clinically relevant drug-drug interactions are those that occur when glinides are administered together with other glucose-lowering agents or compounds widely coadministered to diabetic patients (e.g. lipid-lowering agents), with drugs that are known to induce (risk of lower glinide plasma levels and thus of deterioration of glucose control) or inhibit (risk of higher glinide plasma levels leading to hypoglycaemia) CYP isoenzymes concerned in their metabolism, or with drugs that have a narrow efficacy : toxicity ratio. Pharmacokinetic interactions reported in the literature appear to be more frequent and more important with repaglinide than with

  16. Different in vitro metabolism of paclitaxel and docetaxel in humans, rats, pigs, and minipigs.

    PubMed

    Vaclavikova, Radka; Soucek, Pavel; Svobodova, Lenka; Anzenbacher, Pavel; Simek, Petr; Guengerich, F Peter; Gut, Ivan

    2004-06-01

    We investigated cytochrome P450 (P450)-catalyzed metabolism of the important cancer drugs paclitaxel and docetaxel in rat, pig, minipig, and human liver microsomes and cDNA-expressed P450 enzymes. In rat microsomes, paclitaxel was metabolized mainly to C3'-hydroxypaclitaxel (C3'-OHP) and to a lesser extent to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP), and another unknown monohydroxylated paclitaxel. In pig and minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3'-OHP was a minor product. In minipigs, C2-OHP was the next minor product. In human liver microsomes, 6 alpha-hydroxypaclitaxel (6 alpha-OHP) was the main metabolite, followed by C3'-OHP and C2-OHP. Among different cDNA-expressed human P450 enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1, and 3A4), only CYP3A4 enzyme formed C3'-OHP and C2-OHP. Docetaxel was metabolized in pig, minipig, rat, and human liver microsomes mainly to hydroxydocetaxel (OHDTX), whereas CYP3A-induced rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from 10 different individuals formed OHDTX at different rates correlated with CYP3A4 content. Troleandomycin as a selective inhibitor of CYP3A inhibited the formation of C3'-OHP, C2-OHP, and di-OHP, as well as the unknown OHP produced in rat, minipig, and pig microsomes. In human liver microsomes, troleandomycin inhibited C3'-OHP and C2-OHP formation, and a suitable inhibitor of human CYP2C8, fisetin, strongly inhibited the formation of 6 alpha-OHP, known to be catalyzed by human CYP2C8. In conclusion, the metabolism of docetaxel is the same in all four species, but metabolism of paclitaxel is different, and 6 alpha-OHP remains a uniquely human metabolite. Pigs and minipigs compared with each other formed the same metabolites of paclitaxel.

  17. In vitro inhibition and induction of human liver cytochrome P450 enzymes by gentiopicroside: potent effect on CYP2A6.

    PubMed

    Deng, Yating; Wang, Lu; Yang, Yong; Sun, Wenji; Xie, Renming; Liu, Xueying; Wang, Qingwei

    2013-01-01

    Gentiopicroside (GE), a naturally occurring iridoid glycoside, has been developed into a Novel Traditional Chinese Drug named gentiopicroside injection, and it was approved for the treatment of acute jaundice and chronic active hepatitis by SFDA. However, the inhibitory and inducible effects of GE on the activity of cytochrome P450 (CYP450) are unclear. The purpose of this study was to evaluate the ability of GE to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, GE inhibited CYP2A6 and CYP2E1 in a concentration-dependent manner, with IC₅₀ values of 21.8 µg/ml and 594 µg/ml, respectively, and the IC₅₀ of CYP2A6 was close to the C(max) value observed clinically. GE was a non-competitive inhibitor of CYP2A6 at lower concentrations and a competitive inhibitor at higher concentrations. GE did not produce inhibition of CYP2C9, CYP2D6, CYP1A2 or CYP3A4 activities. However, a significant increase of CYP1A2 and CYP3A4 activity was observed at high concentrations. In cultured human hepatocytes no significant induction of CYP1A2, CYP3A4 or CYP2B6 was observed. Given these results, the in vivo potential inhibition of GE on CYP2A6 deserves further investigation, and it seems that the hepatoprotective effect of GE is irrelevant to its effect on P450s.

  18. Serum-free culture of primary human hepatocytes in a miniaturized hollow-fibre membrane bioreactor for pharmacological in vitro studies.

    PubMed

    Lübberstedt, Marc; Müller-Vieira, Ursula; Biemel, Klaus M; Darnell, Malin; Hoffmann, Stefan A; Knöspel, Fanny; Wönne, Eva C; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B; Zeilinger, Katrin

    2015-09-01

    Primary human hepatocytes represent an important cell source for in vitro investigation of hepatic drug metabolism and disposition. In this study, a multi-compartment capillary membrane-based bioreactor technology for three-dimensional (3D) perfusion culture was further developed and miniaturized to a volume of less than 0.5 ml to reduce demand for cells. The miniaturized bioreactor was composed of two capillary layers, each made of alternately arranged oxygen and medium capillaries serving as a 3D culture for the cells. Metabolic activity and stability of primary human hepatocytes was studied in this bioreactor in the presence of 2.5% fetal calf serum (FCS) under serum-free conditions over a culture period of 10 days. The miniaturized bioreactor showed functions comparable to previously reported data for larger variants. Glucose and lactate metabolism, urea production, albumin synthesis and release of intracellular enzymes (AST, ALT, GLDH) showed no significant differences between serum-free and serum-supplemented bioreactors. Activities of human-relevant cytochrome P450 (CYP) isoenzymes (CYP1A2, CYP3A4/5, CYP2C9, CYP2D6, CYP2B6) analyzed by determination of product formation rates from selective probe substrates were also comparable in both groups. Gene expression analysis showed moderately higher expression in the majority of CYP enzymes, transport proteins and enzymes of Phase II metabolism in the serum-free bioreactors compared to those maintained with FCS. In conclusion, the miniaturized bioreactor maintained stable function over the investigated period and thus provides a suitable system for pharmacological studies on primary human hepatocytes under defined serum-free conditions.

  19. 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.

  20. 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.

  1. Metabolism of phenytoin by the gingiva of normal humans: the possible role of reactive metabolites of phenytoin in the initiation of gingival hyperplasia.

    PubMed

    Zhou, L X; Pihlstrom, B; Hardwick, J P; Park, S S; Wrighton, S A; Holtzman, J L

    1996-08-01

    Gingival hyperplasia is a well-known complication of therapy with cyclosporine, calcium channel blockers, and phenytoin. It is characterized by the presence of inflammation and a marked fibrotic response. The mechanism of this adverse reaction is unknown. We propose that it may be initiated by the metabolic activation of these drugs to form reactive metabolites. These then cause cellular injury and lead to the gingival hyperplasia. To evaluate this hypothesis we examined phenytoin metabolism and the cytochrome P450 contents of gingival tissues from 10 patients undergoing surgery for various periodontal conditions. We found that microsomes obtained from the gingiva show significant phenytoin hydroxylase activity as determined by the production of 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) (range, 12.8 pmol HPPH/min.mg microsomal protein to 276.9 pmol HPPH/min.mg microsomal protein; rat control, 133.7 +/- 11.5 pmol HPPH/min.mg microsomal protein). We also found that CYP1A1, CYP1A2, CYP2C9, CYP2E1, and CYP3A4 were present in these microsomes. We detected no CYP2B6 or CYP2D6. We believe that these data support our hypothesis that the proliferative inflammation observed with drugs such as phenytoin, nifedipine, and cyclosporine may be initiated by the formation of reactive metabolites and that the formation of these metabolites may be catalyzed by one or more CYPs found in the gingiva. These metabolites may then cause cellular injury and induce a reactive inflammatory response, followed by fibroblastic proliferation. This proliferation leads to the excess collagen deposition observed with gingival hyperplasia.

  2. Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

    PubMed

    Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

    2016-06-01

    We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes.

  3. Sequences associated with human iris pigmentation.

    PubMed Central

    Frudakis, Tony; Thomas, Matthew; Gaskin, Zach; Venkateswarlu, K; Chandra, K Suresh; Ginjupalli, Siva; Gunturi, Sitaram; Natrajan, Sivamani; Ponnuswamy, Viswanathan K; Ponnuswamy, K N

    2003-01-01

    To determine whether and how common polymorphisms are associated with natural distributions of iris colors, we surveyed 851 individuals of mainly European descent at 335 SNP loci in 13 pigmentation genes and 419 other SNPs distributed throughout the genome and known or thought to be informative for certain elements of population structure. We identified numerous SNPs, haplotypes, and diplotypes (diploid pairs of haplotypes) within the OCA2, MYO5A, TYRP1, AIM, DCT, and TYR genes and the CYP1A2-15q22-ter, CYP1B1-2p21, CYP2C8-10q23, CYP2C9-10q24, and MAOA-Xp11.4 regions as significantly associated with iris colors. Half of the associated SNPs were located on chromosome 15, which corresponds with results that others have previously obtained from linkage analysis. We identified 5 additional genes (ASIP, MC1R, POMC, and SILV) and one additional region (GSTT2-22q11.23) with haplotype and/or diplotypes, but not individual SNP alleles associated with iris colors. For most of the genes, multilocus gene-wise genotype sequences were more strongly associated with iris colors than were haplotypes or SNP alleles. Diplotypes for these genes explain 15% of iris color variation. Apart from representing the first comprehensive candidate gene study for variable iris pigmentation and constituting a first step toward developing a classification model for the inference of iris color from DNA, our results suggest that cryptic population structure might serve as a leverage tool for complex trait gene mapping if genomes are screened with the appropriate ancestry informative markers. PMID:14704187

  4. Identifying a selective substrate and inhibitor pair for the evaluation of CYP2J2 activity.

    PubMed

    Lee, Caroline A; Jones, J P; Katayama, Jonathan; Kaspera, Rüdiger; Jiang, Ying; Freiwald, Sascha; Smith, Evan; Walker, Gregory S; Totah, Rheem A

    2012-05-01

    CYP2J2, an arachidonic acid epoxygenase, is recognized for its role in the first-pass metabolism of astemizole and ebastine. To fully assess the role of CYP2J2 in drug metabolism, a selective substrate and potent specific chemical inhibitor are essential. In this study, we report amiodarone 4-hydoxylation as a specific CYP2J2-catalyzed reaction with no CYP3A4, or other drug-metabolizing enzyme, involvement. Amiodarone 4-hydroxylation enabled the determination of liver relative activity factor and intersystem extrapolation factor for CYP2J2. Amiodarone 4-hydroxylation correlated with astemizole O-demethylation but not with CYP2J2 protein content in a sample of human liver microsomes. To identify a specific CYP2J2 inhibitor, 138 drugs were screened using terfenadine and astemizole as probe substrates with recombinant CYP2J2. Forty-two drugs inhibited CYP2J2 activity by ≥50% at 30 μM, but inhibition was substrate-dependent. Of these, danazol was a potent inhibitor of both hydroxylation of terfenadine (IC(50) = 77 nM) and O-demethylation of astemizole (K(i) = 20 nM), and inhibition was mostly competitive. Danazol inhibited CYP2C9, CYP2C8, and CYP2D6 with IC(50) values of 1.44, 1.95, and 2.74 μM, respectively. Amiodarone or astemizole were included in a seven-probe cocktail for cytochrome P450 (P450) drug-interaction screening potential, and astemizole demonstrated a better profile because it did not appreciably interact with other P450 probes. Thus, danazol, amiodarone, and astemizole will facilitate the ability to determine the metabolic role of CYP2J2 in hepatic and extrahepatic tissues. PMID:22328583

  5. Molecules in Laboratory and in Interstellar Space?

    NASA Astrophysics Data System (ADS)

    Thimmakondu, Venkatesan S.

    2016-06-01

    In this talk, the quantum chemistry of astronomically relevant molecules will be outlined with an emphasis on the structures and energetics of C_7H_2 isomers, which are yet to be identified in space. Although more than 100's of isomers are possible for C_7H_2, to date only 6 isomers had been identified in the laboratory. The equilibrium geometries of heptatriynylidene (1), cyclohepta-1,2,3,4-tetraen-6-yne (2), and heptahexaenylidene (3), which we had investigated theoretically will be discussed briefly. While 1 and 3 are observed in the laboratory, 2 is a hypothetical molecule. The theoretical data may be useful for the laboratory detection of 2 and astronomical detection of 2 and 3. THIS WORK IS SUPPORTED BY A RESEARCH GRANT (YSS/2015/00099) FROM SERB, DST, GOVERNMENT OF INDIA. Apponi, A. P.; McCarthy, M. C.; Gottlieb, C. A.; Thaddeus, P. Laboratory Detection of Four New Cumulene Carbenes: H_2C_7, H_2C_8, H_2C_9, and D_2C10, Astrophys. J. 2000, 530, 357-361 Ball, C. D; McCarthy, M. C.; Thaddeus, P. Cavity Ringdown Spectroscopy of the Linear Carbon Chains HC_7H, HC_9H, HC11H, and HC13H. J. Chem. Phys. 2000, 112, 10149-10155 Dua, S.; Blanksby, S. J.; Bowie, J. H. Formation of Neutral C_7H_2 Isomers from Four Isomeric C_7H_2 Radical Anion Precursors in the Gas Phase. J. Phys. Chem. A, 2000, 104, 77-85. Thimmakondu, V. S. The equilibrium geometries of heptatriynylidene, cyclohepta-1,2,3,4-tetraen-6-yne, and heptahexaenylidene, Comput. Theoret. Chem. 2016, 1079, 1-10

  6. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    PubMed

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. PMID:27271369

  7. Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions.

    PubMed

    Maréchal, J-D; Kemp, C A; Roberts, G C K; Paine, M J I; Wolf, C R; Sutcliffe, M J

    2008-03-01

    The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes. PMID:18026129

  8. 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.

  9. 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

  10. 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.

  11. 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. PMID:25138712

  12. 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). PMID:26851241

  13. Epoxyeicosatrienoic Acids Affect Electrolyte Transport in Renal Tubular Epithelial Cells: Dependence on Cyclooxygenase and Cell Polarity

    PubMed Central

    Nüsing, Rolf M.; Schweer, Horst; Fleming, Ingrid; Zeldin, Darryl C.; Wegmann, Markus

    2007-01-01

    We investigated the effects of epoxyeicosatrienoic acids (EETs) on ion transport in the polarized renal distal tubular cell line, MDCK C7. Of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) studied, only apical, but not basolateral, application of 5,6-EET increased short circuit current (Isc) with kinetics similar to those of arachidonic acid. The ion transport was blocked by preincubation with the cyclooxygenase inhibitor indomethacin or with the chloride channel blocker NPPB. Further, both a Cl−-free bath solution and the Ca2+ antagonist verapamil blocked 5,6-EET-induced ion transport. Although the presence of the PGE2 receptors EP2, EP3, and EP4 was demonstrated, apically added PGE2 was ineffective and basolaterally added PGE2 caused a different kinetics in ion transport compared to 5,6-EET. Moreover, PGE2 sythesis in MDCK C7 cells was unaffected by 5,6-EET treatment. GC/MS/MS analysis of cell supernatants revealed the presence of the biologically inactive 5,6-dihydroxy-PGE1 in 5,6-EET-treated cells, but not in control cells. Indomethacin suppressed the formation of 5,6-dihydroxy-PGE1. 5,6-epoxy-PGE1 the precursor of 5,6-dihydroxy-PGE1, caused a similar ion transport as 5,6-EET. Cytochrome P450 enzymes homolog to human CYP2C8, CYP2C9, and CYP2J2 protein were detected immunologically in the MDCK C7 cells. Our findings suggest that 5,6-EET affects Cl-transport in renal distal tubular cells independent of PGE2 but by a mechanism, dependent on its conversion to 5,6-epoxy-PGE1 by cyclooxygenase. We suggest a role for this P450 epoxygenase product in the regulation of electrolyte transport, especially as a saluretic compound acting from the luminal side of tubular cells in the mammalian kidney. PMID:17494091

  14. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

    PubMed

    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

  15. Pomegranate juice and pomegranate extract do not impair oral clearance of flurbiprofen in human volunteers: divergence from in vitro results.

    PubMed

    Hanley, M J; Masse, G; Harmatz, J S; Court, M H; Greenblatt, D J

    2012-11-01

    Nutrient interactions with prescription drugs are a topic of ongoing basic and clinical research. Pomegranate juice and a 1-g capsule containing pomegranate extract were evaluated in vitro and in vivo as inhibitors of cytochrome P450 2C9 (CYP2C9), with flurbiprofen serving as the index substrate. Fluconazole was the positive control inhibitor. The in vitro 50% inhibitory concentration (IC(50)) values for pomegranate juice and extract were below 1% (vol/vol), with no evidence of mechanism-based (irreversible) inhibition. In clinical studies, flurbiprofen pharmacokinetics were unchanged by pomegranate juice or extract as compared to a low-polyphenol placebo control beverage. However, fluconazole significantly reduced the oral clearance of flurbiprofen. Despite inhibition of CYP2C9 in vitro, pomegranate juice and extract had no effect on CYP2C9 activity in human subjects, and can be consumed by patients taking CYP2C9 substrate drugs with negligible risk of a pharmacokinetic interaction.

  16. Effect of Host Genetic Variation on the Pharmacokinetics and Clinical Response of Non-nucleoside Reverse Transcriptase Inhibitors.

    PubMed

    Saitoh, Akihiko; Spector, Stephen A

    2008-01-01

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been used widely for treating human immunodeficiency virus type 1 (HIV-1) infected patients as a component of highly active antiretroviral therapy (HAART) and for the prevention of mother-to-child transmission (MTCT). Cytochrome P450 (CYP) 2B6 is an important hepatic isoenzyme responsible for the metabolism of NNRTIs including efavirenz and nevirapine. Recent pharmacogenetic studies have shown that CYP2B6 genetic variants alter hepatic CYP2B6 protein expression and function, and the pharmacokinetics of several CYP2B6 substrates. In particular, the CYP2B6-G516T polymorphism in exon 4 affects the pharmacokinetics of efavirenz. Other studies have shown associations of the CYP2B6-G516T genotype with nevirapine pharmacokinetics and central nervous system adverse effects related to efavirenz use. In total, CYP2B6 genetic variants are important determinants of efavirenz and nevirapine pharmacokinetics . Further studies are needed to identify the associations of CYP2B6 genetic variants with the development of NNRTI resistant viruses.

  17. [Pharmacogenetics of oral anticoagulants: individualized drug treatment for more efficacy and safety].

    PubMed

    Loriot, Marie-Anne; Beaune, Philippe

    2007-06-30

    Oral anti-vitamin K (AVK) anticoagulants constitute the first cause of iatrogenic accidents in France because of narrow therapeutic index and bleeding risk. The wide interindividual variation in AVK response is partly genetically determined. The main enzyme responsible for the metabolism of AVK is the hepatic cytochrome P450 2C9 (CYP2C9). Vitamine K epoxide reductase complex subunit I (VKORC1) is a key enzyme in the vitamin K cycle, cofactor required for the activation of vitamin K-dependent clotting factors, and is the target enzyme of AVK inhibition. Genetic variations affecting both CYP2C9 and VKORC1 are associated with variability in drug response and may explain differences in dose requirements. Genotyping for CYP2C9 and VKORC1 variants before initiation of treatment may allow clinicians to develop dosing protocols and identify the patients at a higher risk for bleeding complications.

  18. The Use of Immobilized Cytochrome P4502C9 in PMMA-Based Plug-Flow Bioreactors for the Production of Drug Metabolites

    PubMed Central

    Wollenberg, Lance A.; Kabulski, Jarod L.; Powell, Matthew J.; Chen, Jifeng; Flora, Darcy R.; Tracy, Timothy S.; Gannett, Peter M.

    2013-01-01

    Cytochrome P450 enzymes play a key role in the metabolism of pharmaceutical agents. To determine metabolite toxicity, it is necessary to obtain P450 metabolites from various pharmaceutical agents. Here, we describe a bioreactor that is made by immobilizing cytochrome P450 2C9 (CYP2C9) to a poly (methyl methacrylate) surface and, as an alternative to traditional chemical synthesis, can be used to biosynthesize P450 metabolites in a plug-flow bioreactor. As part of the development of the CYP2C9 bioreactor, we have studied two different methods of attachment: 1) coupling via the N-terminus using N-hydroxysulfosuccinimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and 2) using the Ni(II) chelator 1-acetato-4-benzyl-triazacyclononane to coordinate the enzyme to the surface using a C-terminal histidine tag. Additionally, the propensity for metabolite production of the CYP2C9 proof-of-concept bioreactors as a function of enzyme attachment conditions (e.g., time and enzyme concentration) was examined. Our results show that the immobilization of CYP2C9 enzymes to a PMMA surface represents a viable and alternative approach to the preparation of CYP2C9 metabolites for toxicity testing. Furthermore, the basic approach can be adapted to any cytochrome P450 enzyme and in a high-throughput, automated process. PMID:24166101

  19. Clinical pharmacology of axitinib.

    PubMed

    Chen, Ying; Tortorici, Michael A; Garrett, May; Hee, Brian; Klamerus, Karen J; Pithavala, Yazdi K

    2013-09-01

    -fold increase in AUC from time zero to infinity (AUC∞) following a single 5-mg dose in subjects with moderate hepatic impairment. In the presence of ketoconazole, a strong CYP3A4/5 inhibitor, axitinib C max and AUC∞ increased by 1.5- and 2-fold, respectively, whereas co-administration of rifampin, a strong CYP3A4/5 inducer, resulted in a 71 and 79 % decrease in the C max and AUC∞, respectively. Axitinib does not inhibit CYP3A4/5, CYP1A2, CYP2C8, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, or UGT1A1 at concentrations obtained with the clinical doses and is not expected to have major interactions with drugs that are metabolized by these enzymes. Axitinib is an inhibitor of the efflux transporter P-glycoprotein (P-gp) in vitro, but is not expected to inhibit P-gp at therapeutic plasma concentrations. A two-compartment population pharmacokinetic model with first-order absorption and lag time was used to describe axitinib pharmacokinetics. No clinically relevant effects of age, sex, body weight, race, renal function, UGT1A1 genotype, or CYP2C19 inferred phenotype on the clearance of axitinib were identified. PMID:23677771

  20. Multiple genetic variants predict steady-state nevirapine clearance in HIV-infected Cambodians

    PubMed Central

    Bertrand, Julie; Chou, Monidarin; Richardson, Danielle M.; Verstuyft, Céline; Leger, Paul D.; Mentré, France; Taburet, Anne-Marie; Haas, David W.

    2013-01-01

    Objective In a previous analysis involving protocol ANRS 12154, interindividual variability in steady-state nevirapine clearance among HIV-infected Cambodians was partially explained by CYP2B6 516G→T (CYP2B6*6). Here, we examine whether additional genetic variants predict nevirapine clearance in this cohort. Methods Analyses included Phnom Penh ESTHER (Ensemble pour une Solidarité Thérapeutique Hospitalière en Réseau) cohort participants who had consented for genetic testing. All participants were receiving nevirapine plus two nucleoside analogs. The mean individual nevirapine clearance estimates were derived from a population model developed on nevirapine concentrations at 18 and 36 months of therapy. Polymorphisms were assayed in ABCB1, CYP2A6, CYP2B6, CYP2C19, CYP3A4, CYP3A5, and NR1I2. Results Of 198 assayed loci, 130 were polymorphic. Among 129 individuals with evaluable genetic data, nevirapine clearance ranged from 1.06 to 5.00 l/h in 128 individuals and was 7.81 l/h in one individual. In bivariate linear regression, CYP2B6 516G→T (CYP2B6*6) was associated with lower nevirapine clearances (P = 3.5 × 10–6). In a multivariate linear regression model conditioned on CYP2B6 516G→T, independent associations were identified with CYP2B6 rs7251950, CYP2B6 rs2279343, and CYP3A4 rs2687116. The CYP3A4 association disappeared after censoring the outlier clearance value. A model that included CYP2B6 516G→T (P = 1.0 × 10–9), rs7251950 (P = 4.8 × 10–5), and rs2279343 (P = 7.1 × 10–5) explained 11% of interindividual variability in nevirapine clearance. Conclusion Among HIV-infected Cambodians, several CYP2B6 polymorphisms were associated independently with steady-state nevirapine clearance. The prediction of nevirapine clearance was improved by considering several polymorphisms in combination. PMID:23104099

  1. A genome-wide scan for common genetic variants with a large influence on warfarin maintenance dose

    PubMed Central

    Cooper, Gregory M.; Johnson, Julie A.; Langaee, Taimour Y.; Feng, Hua; Stanaway, Ian B.; Schwarz, Ute I.; Ritchie, Marylyn D.; Stein, C. Michael; Roden, Dan M.; Smith, Joshua D.; Veenstra, David L.; Rettie, Allan E.

    2008-01-01

    Warfarin dosing is correlated with polymorphisms in vitamin K epoxide reductase complex 1 (VKORC1) and the cytochrome P450 2C9 (CYP2C9) genes. Recently, the FDA revised warfarin labeling to raise physician awareness about these genetic effects. Randomized clinical trials are underway to test genetically based dosing algorithms. It is thus important to determine whether common single nucleotide polymorphisms (SNPs) in other gene(s) have a large effect on warfarin dosing. A retrospective genome-wide association study was designed to identify polymorphisms that could explain a large fraction of the dose variance. White patients from an index warfarin population (n = 181) and 2 independent replication patient populations (n = 374) were studied. From the approximately 550 000 polymorphisms tested, the most significant independent effect was associated with VKORC1 polymorphisms (P = 6.2 × 10−13) in the index patients. CYP2C9 (rs1057910 CYP2C9*3) and rs4917639) was associated with dose at moderate significance levels (P ∼ 10−4). Replication polymorphisms (355 SNPs) from the index study did not show any significant effects in the replication patient sets. We conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside of the CYP2C9 and VKORC1 genes. Randomized clinical trials that account for these 2 genes should therefore produce results that are definitive and broadly applicable. PMID:18535201

  2. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    PubMed

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone. PMID:27126613

  3. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    PubMed

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

  4. YouScript IMPACT Registry

    ClinicalTrials.gov

    2015-09-10

    Adverse Drug Events; Adverse Drug Reactions; Drug Interaction Potentiation; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; Cytochrome P450 Enzyme Deficiency; Cytochrome P450 CYP2D6 Enzyme Deficiency; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Cytochrome P450 CYP3A Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2C9 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2C19 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  5. Pharmacogenetic Testing Among Home Health Patients

    ClinicalTrials.gov

    2016-09-20

    Adverse Drug Events; Adverse Drug Reactions; Drug Interaction Potentiation; Drug Metabolism, Poor, CYP2D6-RELATED; Drug Metabolism, Poor, CYP2C19-RELATED; Cytochrome P450 Enzyme Deficiency; Cytochrome P450 CYP2D6 Enzyme Deficiency; Cytochrome P450 CYP2C9 Enzyme Deficiency; Cytochrome P450 CYP2C19 Enzyme Deficiency; Cytochrome P450 CYP3A Enzyme Deficiency; Poor Metabolizer Due to Cytochrome P450 CYP2C9 Variant; Poor Metabolizer Due to Cytochrome p450 CYP2C19 Variant; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  6. Potentially significant versus clinically significant drug interactions: pomegranate juice as a case in point.

    PubMed

    Andrade, Chittaranjan

    2014-04-01

    In vitro and in vivo laboratory data show that pomegranate juice consistently inhibits intestinal CYP2C9 and CYP3A4 enzymes. Pomegranate juice may therefore increase the bioavailability of drugs that are metabolized by these enzymes. However, studies in humans find that pomegranate juice does not increase exposure to either CYP2C9 or CYP3A4 substrates. These contradictory findings suggest that potential drug interactions identified in the laboratory may not necessarily translate into clinically significant drug interactions in humans, and hence that laboratory data are insufficient grounds upon which clinical decisions may be based.

  7. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  8. Metabolism of profenofos to 4-bromo-2-chlorophenol, a specific and sensitive exposure biomarker

    PubMed Central

    Dadson, Oswald A.; Ellison, Corie A.; Singleton, Steven T.; Chi, Lai-Har; McGarrigle, Barbara P.; Lein, Pamela J.; Farahat, Fayssal M.; Farahat, Taghreed; Olson, James R.

    2016-01-01

    Profenofos is a direct acting phosphorothioate organophosphorus (OP) pesticide capable of inhibiting β-esterases such as acetylcholinesterase, butyrylcholinesterase, and carboxylesterase. Profenofos is known to be detoxified to the biologically inactive metabolite, 4-bromo-2-chlorophenol (BCP); however, limited data are available regarding the use of urinary BCP as an exposure biomarker in humans. A pilot study conducted in Egyptian agriculture workers, demonstrated that urinary BCP levels prior to application (3.3–30.0 μg/g creatinine) were elevated to 34.5-3566 μg/g creatinine during the time workers were applying profenofos to cotton fields. Subsequently, the in vitro enzymatic formation of BCP was examined using pooled human liver microsomes and recombinant human cytochrome P-450s (CYPs) incubated with profenofos. Of the nine human CYPs studied, only CYPs 3A4, 2B6, and 2C19 were able to metabolize profenofos to BCP. Kinetic studies indicated that CYP 2C19 has the lowest Km, 0.516 μM followed by 2B6 (Km = 1.02 μM) and 3A4 (Km = 18.9 μM). The Vmax for BCP formation was 47.9, 25.1, and 19.2 nmol/min/nmol CYP for CYP2B6, 2C19, and 3A4, respectively. Intrinsic clearance (Vmax/Km) values of 48.8, 46.9, and 1.02 ml/min/nmol CYP 2C19, 2B6, and 3A4, respectively, indicate that CYP2C19 and CYP2B6 are primarily responsible for the detoxification of profenofos. These findings support the use of urinary BCP as a biomarker of exposure to profenofos in humans and suggest polymorphisms in CYP 2C19 and CYP 2B6 as potential biomarkers of susceptibility. PMID:23415833

  9. Metabolism of profenofos to 4-bromo-2-chlorophenol, a specific and sensitive exposure biomarker.

    PubMed

    Dadson, Oswald A; Ellison, Corie A; Singleton, Steven T; Chi, Lai-Har; McGarrigle, Barbara P; Lein, Pamela J; Farahat, Fayssal M; Farahat, Taghreed; Olson, James R

    2013-04-01

    Profenofos is a direct acting phosphorothioate organophosphorus (OP) pesticide capable of inhibiting β-esterases such as acetylcholinesterase, butyrylcholinesterase, and carboxylesterase. Profenofos is known to be detoxified to the biologically inactive metabolite, 4-bromo-2-chlorophenol (BCP); however, limited data are available regarding the use of urinary BCP as an exposure biomarker in humans. A pilot study conducted in Egyptian agriculture workers, demonstrated that urinary BCP levels prior to application (3.3-30.0 μg/g creatinine) were elevated to 34.5-3,566 μg/g creatinine during the time workers were applying profenofos to cotton fields. Subsequently, the in vitro enzymatic formation of BCP was examined using pooled human liver microsomes and recombinant human cytochrome P-450s (CYPs) incubated with profenofos. Of the nine human CYPs studied, only CYPs 3A4, 2B6, and 2C19 were able to metabolize profenofos to BCP. Kinetic studies indicated that CYP 2C19 has the lowest Km, 0.516 μM followed by 2B6 (Km=1.02 μM) and 3A4 (Km=18.9μM). The Vmax for BCP formation was 47.9, 25.1, and 19.2 nmol/min/nmol CYP for CYP2B6, 2C19, and 3A4, respectively. Intrinsic clearance (Vmax/Km) values of 48.8, 46.9, and 1.02 ml/min/nmol CYP 2C19, 2B6, and 3A4, respectively, indicate that CYP2C19 and CYP2B6 are primarily responsible for the detoxification of profenofos. These findings support the use of urinary BCP as a biomarker of exposure to profenofos in humans and suggest polymorphisms in CYP 2C19 and CYP 2B6 as potential biomarkers of susceptibility. PMID:23415833

  10. Amodiaquine pharmacogenetics.

    PubMed

    Gil, Jose Pedro

    2008-10-01

    Amodiaquine is a central drug in the new global strategy of combination therapies for the control of malaria. Amodiaquine is mainly metabolized hepatically towards its major active metabolite desethylamodiaquine, by the polymorphic P450 isoform CYP2C8. Amodiaquine is associated with rare but serious side effects, as well as with relatively frequent mild ones. These are expected to be at least partially related to CYP2C8 alleles. Pharmacogenetic knowledge of amodiaquine exposed populations is important for pharmacovigilance issues and in being a first step for future realistic applications from a personal medicine perspective.

  11. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-01-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

  12. Warfarin pharmacogenomics.

    PubMed

    Li, Jiayi; Wang, Shan; Barone, Joseph; Malone, Brian

    2009-08-01

    Warfarin, an anticoagulant, is used to prevent and treat thromboembolic disease. One of the drawbacks of this agent, also known as Coumadin (Bristol-Myers Squibb), is that it is difficult to administer at the correct dose as a result of its narrow therapeutic index, its tendency to cause bleeding, and the individual variability in patient response. Achieving safe and effective doses of warfarin therapy is both an urgent and important concern for many clinicians.Recent research has focused on single-nucleotide polymorphisms (SNPs) of genes that encode two proteins: the cytochrome P450 2C9 enzyme and VKORC1 (vitamin K epoxide reductase complex). Studies suggest that CYP 2C9 influences warfarin metabolism, whereas VKORC1 plays a role in the pharmacodynamic response in expression of the enzymatic target of warfarin. Patients who carry CYP 2C9*2 and CYP 2C9*3 alleles tend to require lower warfarin maintenance doses because of their slowed metabolism compared with patients who carry the "wild-type" allele. Patients who carry the VKORC1 A haplotype tend to require lower wafarin maintenance doses as a result of a decreased expression of messenger RNA (mRNA), which produces the proteins necessary for the formation of VKORC1. PMID:20140106

  13. Warfarin Pharmacogenomics

    PubMed Central

    Li, Jiayi; Wang, Shan; Barone, Joseph; Malone, Brian

    2009-01-01

    Warfarin, an anticoagulant, is used to prevent and treat thromboembolic disease. One of the drawbacks of this agent, also known as Coumadin (Bristol-Myers Squibb), is that it is difficult to administer at the correct dose as a result of its narrow therapeutic index, its tendency to cause bleeding, and the individual variability in patient response. Achieving safe and effective doses of warfarin therapy is both an urgent and important concern for many clinicians. Recent research has focused on single-nucleotide polymorphisms (SNPs) of genes that encode two proteins: the cytochrome P450 2C9 enzyme and VKORC1 (vitamin K epoxide reductase complex). Studies suggest that CYP 2C9 influences warfarin metabolism, whereas VKORC1 plays a role in the pharmacodynamic response in expression of the enzymatic target of warfarin. Patients who carry CYP 2C9*2 and CYP 2C9*3 alleles tend to require lower warfarin maintenance doses because of their slowed metabolism compared with patients who carry the “wild-type” allele. Patients who carry the VKORC1 A haplotype tend to require lower wafarin maintenance doses as a result of a decreased expression of messenger RNA (mRNA), which produces the proteins necessary for the formation of VKORC1. PMID:20140106

  14. Non-aromatic A-ring replacement in the triaryl bis-sulfone CB2 receptor inhibitors.

    PubMed

    Gilbert, Eric J; Zhou, Guowei; Wong, Michael K C; Tong, Ling; Shankar, Bandarpalle B; Huang, Chunli; Kelly, Joseph; Lavey, Brian J; McCombie, Stuart W; Chen, Lei; Rizvi, Razia; Dong, Youhao; Shu, Youheng; Kozlowski, Joseph A; Shih, Neng-Yang; Hipkin, R William; Gonsiorek, Waldemar; Malikzay, Asra; Lunn, Charles A; Favreau, Len; Lundell, Daniel J

    2010-01-15

    The triaryl bis-sulfone 1 was modified by converting the aryl A-ring to a piperidine ring. The piperidine ring was further elaborated to a spirocyclopropyl piperidine moiety. The effect on CB2 binding potency, rat calcium channel affinity, and CYP 2C9 inhibition is described.

  15. Modulatory effects of extracts of vinegar-baked Radix Bupleuri and saikosaponins on the activity of cytochrome P450 enzymes in vitro.

    PubMed

    Yu, Tongya; Chen, Xianzhi; Wang, Yinjie; Zhao, Ruizhi; Mao, Shirui

    2014-10-01

    1. In this article, the modulatory effects of extracts from vinegar-baked Radix Bupleuri (VBRB) and saikosaponins on the activity of CYP1A2, CYP2C9 and CYP3A4 were investigated in vitro. 2. Microsomal in vitro incubation method was utilized to simulate metabolic reaction under physiological environment by incubating the marker with liver microsomes in the absence or presence of VBRB and saikosaponins. The contents of 4-acetamidophenol, 6β-hydroxyltestosterone and 4-hydroxydiclofenac, the metabolites of phenacetin, testosterone and diclofenac, which were selected as specific probe drugs of CYP1A2, CYP2C9 and CYP3A4, respectively, were analyzed by high-performance liquid chromatography. 3. The production of the metabolites was incubation time dependent. The modulatory effects of different VBRB extracts and saikosaponins on CYP isoforms increased with concentration. Among all the extracts studied, BC1 has a strong inhibition effect compared to the three CYP isoforms tested, while the others have only significant inhibition on the activity of CYP2C9. 4. This in vitro study demonstrated that various extracts of VBRB tested in this study have negligible potential to interfere with CYP1A2- and CYP3A4-metabolized drugs; risk of herb-drug interaction might occur when VBRB is concurrently taken with CYP2C9 substrates.

  16. Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules

    EPA Science Inventory

    The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

  17. Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

    SciTech Connect

    Sparks, Steven M.; Banker, Pierette; Bickett, David M.; Carter, H. Luke; Clancy, Daphne C.; Dickerson, Scott H.; Dwornik, Kate A.; Garrido, Dulce M.; Golden, Pamela L.; Nolte, Robert T.; Peat, Andrew J.; Sheckler, Lauren R.; Tavares, Francis X.; Thomson, Stephen A.; Wang, Liping; Weiel, James E.

    2009-05-15

    Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.

  18. Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

    PubMed

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

    2015-12-01

    Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality.

  19. 45 CFR 1610.7 - Transfers of LSC funds.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... involvement activities (PAI) pursuant to 45 CFR part 1614, the prohibitions or requirements of this part shall.... (b)(1) In regard to the requirement in § 1610.2(b)(5) on priorities, persons or entities receiving a... CFR part 1620; (2) In regard to the requirement in § 1610.2(b)(6) on timekeeping, persons or...

  20. 45 CFR 1610.7 - Transfers of LSC funds.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... involvement activities (PAI) pursuant to 45 CFR part 1614, the prohibitions or requirements of this part shall.... (b)(1) In regard to the requirement in § 1610.2(b)(5) on priorities, persons or entities receiving a... CFR part 1620; (2) In regard to the requirement in § 1610.2(b)(6) on timekeeping, persons or...

  1. 45 CFR 1610.7 - Transfers of LSC funds.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... involvement activities (PAI) pursuant to 45 CFR part 1614, the prohibitions or requirements of this part shall.... (b)(1) In regard to the requirement in § 1610.2(b)(5) on priorities, persons or entities receiving a... CFR part 1620; (2) In regard to the requirement in § 1610.2(b)(6) on timekeeping, persons or...

  2. 45 CFR 1610.7 - Transfers of LSC funds.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... involvement activities (PAI) pursuant to 45 CFR part 1614, the prohibitions or requirements of this part shall.... (b)(1) In regard to the requirement in § 1610.2(b)(5) on priorities, persons or entities receiving a... CFR part 1620; (2) In regard to the requirement in § 1610.2(b)(6) on timekeeping, persons or...

  3. 45 CFR 1610.7 - Transfers of LSC funds.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... involvement activities (PAI) pursuant to 45 CFR part 1614, the prohibitions or requirements of this part shall.... (b)(1) In regard to the requirement in § 1610.2(b)(5) on priorities, persons or entities receiving a... CFR part 1620; (2) In regard to the requirement in § 1610.2(b)(6) on timekeeping, persons or...

  4. Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  5. 78 FR 67218 - Agency Information Collection Activities: Company-Run Annual Stress Test Reporting Template and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-08

    ... confidential treatment (5 U.S.C. 552(b)(4)). \\5\\ 12 U.S.C. 5365(i)(2)(B). \\6\\ 77 FR 61238 (October 9, 2012). In 2012, the OCC first implemented the reporting templates referenced in the final rule. See 77 FR 49485 (August 16, 2012) and 77 FR 66663 (November 6, 2012). The OCC is now revising them as described below....

  6. Promoter methylation of E-cadherin, p16, and RAR-beta(2) genes in breast tumors and dietary intake of nutrients important in one-carbon metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aberrant DNA methylation plays a critical role in carcinogenesis, and the availability of dietary factors involved in 1-carbon metabolism may contribute to aberrant DNA methylation. We investigated the association of intake of folate, vitamins B(2), B(6), B(12), and methionine with promoter methylat...

  7. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  8. 77 FR 66663 - Agency Information Collection Activities: Proposed Information Collection; Submission for OMB Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-06

    ... stress test requirement (77 FR 3408). The OCC intends to use the data collected through these templates.... 5365(i)(2)(B). \\6\\ 77 FR 61238, October 9, 2012--Prior to issuance of the final rule, the OCC published... reports using CCAR reporting form FR Y-14A.\\7\\ Therefore, the OCC based its reporting requirements...

  9. 78 FR 51272 - Agency Information Collection Activities: Revision of an Approved Information Collection; Comment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-20

    .... 5365(i)(2)(B). \\6\\ 77 FR 61238 (October 9, 2012). In 2012, the OCC first implemented the reporting templates referenced in the final rule. See 77 FR 49485 (August 16, 2012) and 77 FR 66663 (November 6, 2012... reports using CCAR reporting form FR Y-14A.\\7\\ The OCC also recognizes the Board has a proposal to...

  10. 78 FR 16263 - Agency Information Collection Activities; Proposed Information Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-14

    ...)(2)(A). \\3\\ 12 U.S.C. 5301(12). \\4\\ 12 U.S.C. 5365(i)(2)(C). \\5\\ 12 U.S.C. 5365(i)(2)(B). \\6\\ 77 FR... template for larger banks with total consolidated assets of $50 billion or more. \\7\\ See 77 FR 52718 for... is soliciting comment concerning its information collection titled, ``Annual Stress Test...

  11. 78 FR 15403 - Agency Information Collection Activities; Proposed Collection; Comment Request; Company-Run...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-11

    ...). \\5\\ 12 U.S.C. 5365(i)(2)(B). \\6\\ 77 FR 61238, October 9, 2012. The OCC intends to use the data... institutions with total consolidated assets of $50 billion or more.\\7\\ \\7\\ See 77 FR 49485 for the Paperwork...; Comment Request; Company-Run Annual Stress Test Reporting Template and Documentation for...

  12. 77 FR 49485 - Agency Information Collection Activities: Proposed Information Collection; Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-16

    ..., OMB Supporting Statement for the Capital Assessments and Stress Testing information collection (FR Y....C. 5365(i)(2)(B). \\6\\ 77 FR 3408, Jan. 24, 2012. The OCC intends to use the data collected through... billion or more are required to submit reports using CCAR reporting form FR Y-14A.\\7\\ Therefore, the...

  13. 12 CFR Appendix B to Part 707 - Model Clauses and Sample Forms

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... to member confusion. As terms are usually construed against the drafter, credit unions should be very... instead to each credit union's board of directors. 12 CFR 202.4(c)(2). B-6Sample Form (Regular Share... federal credit union, pursuant to section 117 of the FCU Act, 12 U.S.C. 117. It also states the...

  14. Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw; Chi, Chin-Wen; Ho, Li-Kang

    2004-08-01

    Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH.

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

    PubMed Central

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

    2016-01-01

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

  16. Effect of blueberry juice on clearance of buspirone and flurbiprofen in human volunteers

    PubMed Central

    Hanley, Michael J; Masse, Gina; Harmatz, Jerold S; Cancalon, Paul F; Dolnikowski, Gregory G; Court, Michael H; Greenblatt, David J

    2013-01-01

    Aim The present study evaluated the possibility of drug interactions involving blueberry juice (BBJ) and substrate drugs whose clearance is dependent on cytochromes P4503A (CYP3A) and P4502C9 (CYP2C9). Methods A 50:50 mixture of lowbush and highbush BBJ was evaluated in vitro as an inhibitor of CYP3A activity (hydroxylation of triazolam and dealkylation of buspirone) and of CYP2C9 activity (flurbiprofen hydroxylation) using human liver microsomes. In clinical studies, clearance of oral buspirone and oral flurbiprofen was studied in healthy volunteers with and without co-treatment with BBJ. Results BBJ inhibited CYP3A and CYP2C9 activity in vitro, with 50% inhibitory concentrations (IC50) of less than 2%, but without evidence of mechanism-based (irreversible) inhibition. Grapefruit juice (GFJ) also inhibited CYP3A activity, but inhibitory potency was increased by pre-incubation, consistent with mechanism-based inhibition. In clinical studies, GFJ significantly increased area under the plasma concentration−time curve (AUC) for the CYP3A substrate buspirone. The geometric mean ratio (GMR = AUC with GFJ divided by AUC with water) was 2.12. In contrast, the effect of BBJ (GMR = 1.39) was not significant. In the study of flurbiprofen (CYP2C9 substrate), the positive control inhibitor fluconazole significantly increased flurbiprofen AUC (GMR = 1.71), but BBJ had no significant effect (GMR = 1.03). Conclusion The increased buspirone AUC associated with BBJ is quantitatively small and could have occurred by chance. BBJ has no effect on flurbiprofen AUC. The studies provide no evidence for concern about clinically important pharmacokinetic drug interactions of BBJ with substrate drugs metabolized by CYP3A or CYP2C9. PMID:22943633

  17. Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review.

    PubMed

    Stout, Stephen M; Cimino, Nina M

    2014-02-01

    Exogenous cannabinoids are structurally and pharmacologically diverse compounds that are widely used. The purpose of this systematic review is to summarize the data characterizing the potential for these compounds to act as substrates, inhibitors, or inducers of human drug metabolizing enzymes, with the aim of clarifying the significance of these properties in clinical care and drug interactions. In vitro data were identified that characterize cytochrome P-450 (CYP-450) enzymes as potential significant contributors to the primary metabolism of several exogenous cannabinoids: tetrahydrocannabinol (THC; CYPs 2C9, 3A4); cannabidiol (CBD; CYPs 2C19, 3A4); cannabinol (CBN; CYPs 2C9, 3A4); JWH-018 (CYPs 1A2, 2C9); and AM2201 (CYPs 1A2, 2C9). CYP-450 enzymes may also contribute to the secondary metabolism of THC, and UDP-glucuronosyltransferases have been identified as capable of catalyzing both primary (CBD, CBN) and secondary (THC, JWH-018, JWH-073) cannabinoid metabolism. Clinical pharmacogenetic data further support CYP2C9 as a significant contributor to THC metabolism, and a pharmacokinetic interaction study using ketoconazole with oromucosal cannabis extract further supports CYP3A4 as a significant metabolic pathway for THC and CBD. However, the absence of interaction between CBD from oromucosal cannabis extract with omeprazole suggests a less significant role of CYP2C19 in CBD metabolism. Studies of THC, CBD, and CBN inhibition and induction of major human CYP-450 isoforms generally reflect a low risk of clinically significant drug interactions with most use, but specific human data are lacking. Smoked cannabis herb (marijuana) likely induces CYP1A2 mediated theophylline metabolism, although the role of cannabinoids specifically in eliciting this effect is questionable.

  18. In Vivo Information-Guided Prediction Approach for Assessing the Risks of Drug-Drug Interactions Associated with Circulating Inhibitory Metabolites

    PubMed Central

    Parker, Robert B.; Laizure, S. Casey

    2012-01-01

    The in vivo drug-drug interaction (DDI) risks associated with cytochrome P450 inhibitors that have circulating inhibitory metabolites cannot be accurately predicted by conventional in vitro-based methods. A novel approach, in vivo information-guided prediction (IVIP), was recently introduced for CYP3A- and CYP2D6-mediated DDIs. This technique should be applicable to the prediction of DDIs involving other important cytochrome P450 metabolic pathways. Therefore, the aims of this study were to extend the IVIP approach to CYP2C9-mediated DDIs and evaluate the IVIP approach for predicting DDIs associated with inhibitory metabolites. The analysis was based on data from reported DDIs in the literature. The IVIP approach was modified and extended to CYP2C9-mediated DDIs. Thereafter, the IVIP approach was evaluated for predicting the DDI risks of various inhibitors with inhibitory metabolites. Although the data on CYP2C9-mediated DDIs were limited compared with those for CYP3A- and CYP2D6-mediated DDIs, the modified IVIP approach successfully predicted CYP2C9-mediated DDIs. For the external validation set, the prediction accuracy for area under the plasma concentration-time curve (AUC) ratios ranged from 70 to 125%. The accuracy (75–128%) of the IVIP approach in predicting DDI risks of inhibitors with circulating inhibitory metabolites was more accurate than in vitro-based methods (28–805%). The IVIP model accommodates important confounding factors in the prediction of DDIs, which are difficult to handle using in vitro-based methods. In conclusion, the IVIP approach could be used to predict CYP2C9-mediated DDIs and is easily modified to incorporate the additive effect of circulating inhibitory metabolites. PMID:22563046

  19. Effect of crude extract of Eugenia jambolana Lam. on human cytochrome P450 enzymes.

    PubMed

    Chinni, Santhivardhan; Dubala, Anil; Kosaraju, Jayasankar; Khatwal, Rizwan Basha; Satish Kumar, M N; Kannan, Elango

    2014-11-01

    The fruit of Eugenia jambolana Lam. is very popular for its anti-diabetic property. Previous studies on the crude extract of E. jambolana (EJE) have successfully explored the scientific basis for some of its traditional medicinal uses. Considering its wide use and consumption as a seasonal fruit, the present study investigates the ability of E. jambolana to interact with cytochrome P450 enzymes. The standardized EJE was incubated with pooled human liver microsomes to assess the CYP2C9-, CYP2D6-, and CYP3A4-mediated metabolism of diclofenac, dextromethorphan, and testosterone, respectively. The metabolites formed after the enzymatic reactions were quantified by high performance liquid chromatography. EJE showed differential effect on cytochrome P450 activities with an order of inhibitory potential as CYP2C9 > CYP3A4 > CYP2D6 having IC50 of 76.69, 359.02, and 493.05 µg/mL, respectively. The selectivity of EJE for CYP2C9 rather than CYP3A4 and CYP2D6 led to perform the enzyme kinetics to explicate the mechanism underlying the inhibition of CYP2C9-mediated diclofenac 4'-hydroxylation. EJE was notably potent in inhibiting the reaction in a non-competitive manner with Ki of 84.85 ± 5.27 µg/mL. The results revealed the CYP2C9 inhibitory potential of EJE with lower Ki value suggesting that EJE should be examined for its potential pharmacokinetic and pharmacodynamic interactions when concomitantly administered with other drugs. PMID:24590863

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

    PubMed

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

    2016-01-01

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

  1. Metabolism of bupropion by carbonyl reductases in liver and intestine.

    PubMed

    Connarn, Jamie N; Zhang, Xinyuan; Babiskin, Andrew; Sun, Duxin

    2015-07-01

    Bupropion's metabolism and the formation of hydroxybupropion in the liver by cytochrome P450 2B6 (CYP2B6) has been extensively studied; however, the metabolism and formation of erythro/threohydrobupropion in the liver and intestine by carbonyl reductases (CR) has not been well characterized. The purpose of this investigation was to compare the relative contribution of the two metabolism pathways of bupropion (by CYP2B6 and CR) in the subcellular fractions of liver and intestine and to identify the CRs responsible for erythro/threohydrobupropion formation in the liver and the intestine. The results showed that the liver microsome generated the highest amount of hydroxybupropion (Vmax = 131 pmol/min per milligram, Km = 87 μM). In addition, liver microsome and S9 fractions formed similar levels of threohydrobupropion by CR (Vmax = 98-99 pmol/min per milligram and Km = 186-265 μM). Interestingly, the liver has similar capability to form hydroxybupropion (by CYP2B6) and threohydrobupropion (by CR). In contrast, none of the intestinal fractions generate hydroxybupropion, suggesting that the intestine does not have CYP2B6 available for metabolism of bupropion. However, intestinal S9 fraction formed threohydrobupropion to the extent of 25% of the amount of threohydrobupropion formed by liver S9 fraction. Enzyme inhibition and Western blots identified that 11β-dehydrogenase isozyme 1 in the liver microsome fraction is mainly responsible for the formation of threohydrobupropion, and in the intestine AKR7 may be responsible for the same metabolite formation. These quantitative comparisons of bupropion metabolism by CR in the liver and intestine may provide new insight into its efficacy and side effects with respect to these metabolites.

  2. 24 CFR 3500.15 - Affiliated business arrangements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... service except where title is being held for the beneficial owner. (6) Franchise is defined in 16 CFR 436.2(a). (7) Franchisor is defined in 16 CFR 436.2(c). (8) Franchisee is defined in 16 CFR 436.2(d). (9... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Affiliated business...

  3. Troglitazone thiol adduct formation in human liver microsomes: enzyme kinetics and reaction phenotyping.

    PubMed

    Gan, Jinping; Qu, Qinling; He, Bing; Shyu, Wen C; Rodrigues, A David; He, Kan

    2008-08-01

    Troglitazone (TGZ) induced hepatotoxicity has been linked to cytochrome P450 (CYP)-catalyzed reactive metabolite formation. Therefore, the kinetics and CYP specificity of reactive metabolite formation were studied using dansyl glutathione (dGSH) as a trapping agent after incubation of TGZ with human liver microsomes (HLM) and recombinant human CYP proteins. CYP2C8 exhibited the highest rate of TGZ adduct (TGZ-dGS) formation, followed by CYP3A4, CYP3A5, and CYP2C19. The involvement of CYP2C8 and CYP3A4 was confirmed with CYP form-selective chemical inhibitors. The impact of TGZ concentration on the rate of TGZ-dGS formation was also evaluated. In this instance, two distinctly different profiles were observed with recombinant CYP3A4 and CYP2C8. It is concluded that both CYP3A4/5 and CYP2C8 play a major role in the formation of TGZ adduct in HLM. However, the contribution of these CYPs varies depending on their relative expression and the concentration of TGZ. PMID:19356091

  4. 24 CFR 3500.15 - Affiliated business arrangements.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... service except where title is being held for the beneficial owner. (6) Franchise is defined in 16 CFR 436.2(a). (7) Franchisor is defined in 16 CFR 436.2(c). (8) Franchisee is defined in 16 CFR 436.2(d). (9... unintentional and the result of a bona fide error. An error of legal judgment with respect to a...

  5. Leukocyte chemoattractant activity of diacylglycerol

    SciTech Connect

    Wright, T.M.; Hoffman, R.D.; Nishijima, J.; Shin, H.S.

    1986-03-05

    Phosphatidylinositol breakdown with the generation of 1,2-diacylglycerol (1,2-DG) and inositol phosphates occurs in response to receptor mediated stimulation of lymphocytes and polymorphonuclear neutrophils (PMN). In the authors attempt to demonstrate the direct role of 1,2-DG in cell migration, they have found 1,2 dioctanoyl glycerol (1,2-C8DG) to be a chemoattractant for 6C3HED, a mouse thymic lymphoma, and human peripheral blood PMN's. The chemoattractant activity for both cell types was observed at concentrations from 0.5 to 10mM in an under agarose assay. The maximum effect of 1,2-C8DG on 6C3HED cells was similar to that of 1mM lysophosphatidylcholine and the maximum effect of 1,2-C8DG on PMN's was similar to that of 10/sup -7/M f-met-leu-phe. Other 1,2-DG's with acyl chains ranging from 6 to 18 carbons in length and 1-oleoyl-2-acetyl-glycerol were also chemoattractants for 6C3HED, although their activities were less than 1,2-C8DG. In addition, phorbol myristate acetate (PMA), another activator of protein kinase C, was a chemoattractant for 6C3HED and human PMN's. PMA was more potent than 1,2-C8DG for both 6C3HED and PMN's with chemoattractant activity in the range of 30nM to 1..mu..M. These studies support the direct role of 1,2-DG in the transduction of chemotactic stimuli in leukocytes and further suggest that the formation of diacylglycerol represents a common step in the migratory responses of lymphoid and myeloid cells.

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

    PubMed Central

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

    2013-01-01

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

  7. Effects of cilostazol on the pharmacokinetics of carvedilol after oral and intravenous administration in rats.

    PubMed

    Lim, T H; Cho, Y A; Choi, D H

    2015-08-01

    This study was designed to investigate the effects of cilostazol on the pharmacokinetics of carvedilol following oral or intravenous administration of carvedilol in rats. Clinically carvedilol and cilostazol can be prescribed for treatment of cardiovascular diseases. Carvedilol and cilostazol are all substrates of CYP2C9 enzymes. Carvedilol was administered orally or intravenously without or with oral administration of cilostazol to rats. The effects of cilostazol on cytochrome P450 (CYP) 2C9 activity and P-gp activity were also evaluated. Cilostazol inhibited CYP2C9 activity in a concentration-dependent manner with 50% inhibitory concentration (IC(50)) of 8.7 μM. Compared with the control group, the area under the plasma concentration-time curve (AUC) of carvedilol was significantly (P < 0.05) increased by 38.0%. The peak concentration (C(max)) was significantly (P < 0.05) increased by 49.2% in the presence of cilostazol after oral administration of carvedilol. Consequently, the relative bioavailability (R.B.) of carvedilol was increased by 1.15 - 1.38-fold, and the absolute bioavailability (A.B.) of carvedilol in the presence of cilostazol was significantly (P < 0.05) higher than that of the control. After intravenous administration, the AUC of carvedilol was significantly (P < 0.05) increased by 19.2% compared to that in the control by cilostazol. These results suggest that cilostazol effectively inhibited the metabolism of carvedilol. The increased oral bioavailability of carvedilol might be due to the inhibition of CYP2C9-mediated metabolism of carvedilol in the liver by cilostazol.

  8. Automated Classification and Cluster Visualization of Genotypes Derived from High Resolution Melt Curves

    PubMed Central

    Kanderian, Sami; Jiang, Lingxia; Knight, Ivor

    2015-01-01

    Introduction High Resolution Melting (HRM) following PCR has been used to identify DNA genotypes. Fluorescent dyes bounded to double strand DNA lose their fluorescence with increasing temperature, yielding different signatures for different genotypes. Recent software tools have been made available to aid in the distinction of different genotypes, but they are not fully automated, used only for research purposes, or require some level of interaction or confirmation from an analyst. Materials and Methods We describe a fully automated machine learning software algorithm that classifies unknown genotypes. Dynamic melt curves are transformed to multidimensional clusters of points whereby a training set is used to establish the distribution of genotype clusters. Subsequently, probabilistic and statistical methods were used to classify the genotypes of unknown DNA samples on 4 different assays (40 VKORC1, CYP2C9*2, CYP2C9*3 samples in triplicate, and 49 MTHFR c.665C>T samples in triplicate) run on the Roche LC480. Melt curves of each of the triplicates were genotyped separately. Results Automated genotyping called 100% of VKORC1, CYP2C9*3 and MTHFR c.665C>T samples correctly. 97.5% of CYP2C9*2 melt curves were genotyped correctly with the remaining 2.5% given a no call due to the inability to decipher 3 melt curves in close proximity as either homozygous mutant or wild-type with greater than 99.5% posterior probability. Conclusions We demonstrate the ability to fully automate DNA genotyping from HRM curves systematically and accurately without requiring any user interpretation or interaction with the data. Visualization of genotype clusters and quantification of the expected misclassification rate is also available to provide feedback to assay scientists and engineers as changes are made to the assay or instrument. PMID:26605797

  9. Factors influencing quality of anticoagulation control and warfarin dosage in patients after aortic valve replacement within the 3 months of follow up.

    PubMed

    Wypasek, E; Mazur, P; Bochenek, M; Awsiuk, M; Grudzien, G; Plincer, D; Undas, A

    2016-06-01

    Warfarin dosage estimation using the pharmacogenetic algorithms has been shown to improve the quality of anticoagulation control in patients with atrial fibrillation. We sought to assess the genetic, demographic and clinical factors that determine the quality of anticoagulation in patients following aortic valve replacement (AVR). We studied 200 consecutive patients (130 men) aged 63 ± 12.3 years, undergoing AVR, in whom warfarin dose was established using a pharmacogenetic algorithm. The quality of anticoagulation within the first 3 months since surgery was expressed as the time of international normalized ratio (INR) in the therapeutic range (TTR). The median TTR in the entire cohort was 59.6% (interquartile range, 38.7 - 82.7). Ninety-nine (49.5%) patients with TTR ≥ 60% did not differ from those with poor anticoagulation control (TTR < 60%) with regard to demographic and cardiovascular risk factors. Coronary artery disease (n = 84, 42%) and previous stroke (n = 5, 2.5%) predicted higher TTR, while possession of CYP2C9*2 variant allele (n = 49, 25%) was associated with lower TTR (P = 0.01). In turn, VKORC1 c.-1639A, CYP2C9*2 and *3 variants were independently associated with actual warfarin dose (P < 0.0001). In AVR patients better anticoagulation control is observed in patients with coronary artery disease and history of stroke, which might result in part from previous lifestyle modification and therapy. Possession of CYP2C9*2 and/or CYP2C9*3 allele variants is associated with lower TTR values and warfarin dose variations in AVR patients, the latter affected also by VKORC1 c.-1693G>A polymorphism. PMID:27511999

  10. Pharmacogenetic typing for oral anti-coagulant response among factor V Leiden mutation carriers

    PubMed Central

    Nahar, Risha; Saxena, Renu; Deb, Roumi; Verma, Ishwar C.

    2012-01-01

    CONTEXT: Factor V Leiden mutation is the most common inherited predisposition for hypercoagulability and thereby a common genetic cause for initiation of oral anti-coagulation therapy. There is a dearth of knowledge of coumarin response profile in such thrombophilic population. AIMS: The current pilot study aims to estimate coumarin sensitivity in an Indian cohort with an inherited thrombophilia risk factor (Factor V Leiden mutation carriers) based on the observed frequency of CYP2C9 *2, *3 and VKORC1-1639G >A genotype combinations. SETTINGS AND DESIGN: A retrospective study carried out in a tertiary health care center in India. MATERIALS AND METHODS: Carriers of FVL mutation were genotyped for CYP2C9 (*2, F*3) and VKORC1 (-1639G >A) variants by PCR-RFLP technique. STATISTICAL ANALYSIS USED: Chi-square test to analyze difference in expected and observed genotype frequency. RESULTS: Sixty-one (n = 61) unrelated carriers of FVL mutation were observed in the 13 years study period. The allele frequency of CYP2C9 *2, CYP2C9 *3, and VKORC1-1639A in this cohort was 0.06, 0.11, and 0.16, respectively. Six (9.7%) individuals had two of the three variant alleles (heterozygous or homozygous), and 28 (45.9%) were heterozygous for at least one polymorphism. CONCLUSIONS: Pre-prescription genotyping for coumarin drugs, if introduced in Indians with inherited thrombophilia (in whom oral anti-coagulant therapy may be necessary), is likely to identify 9.7% (hypersensitive) subjects in whom the optimum anti-coagulation may be achieved with reduced dosages, 44.3% (normal sensitivity) who may require higher dose and also 55.6% (hyper and moderate sensitivity) subjects who are likely to experience bleeding episodes. PMID:23716941

  11. Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450.

    PubMed

    Chiangsom, Abhiruj; Lawanprasert, Somsong; Oda, Shingo; Kulthong, Kornphimol; Luechapudiporn, Rataya; Yokoi, Tsuyoshi; Maniratanachote, Rawiwan

    2016-06-01

    Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 μg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 μg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 μg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 μg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes. PMID:27212065

  12. [Effect of Fuzheng Huayu recipe on CYP450 isozymes in normal and liver fibrosis rats].

    PubMed

    Zheng, Tian-hui; Liu, Wei; Li, Shu-ping; Yang, Tao; Wang, Chang-hong; Liu, Cheng-hai

    2015-03-01

    To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats. PMID:26226765

  13. CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis.

    PubMed

    Ramesh, M; Bharatam, Prasad V

    2012-02-01

    Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms. PMID:21562823

  14. CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis.

    PubMed

    Ramesh, M; Bharatam, Prasad V

    2012-02-01

    Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms.

  15. Race influences warfarin dose changes associated with genetic factors.

    PubMed

    Limdi, Nita A; Brown, Todd M; Yan, Qi; Thigpen, Jonathan L; Shendre, Aditi; Liu, Nianjun; Hill, Charles E; Arnett, Donna K; Beasley, T Mark

    2015-07-23

    Warfarin dosing algorithms adjust for race, assigning a fixed effect size to each predictor, thereby attenuating the differential effect by race. Attenuation likely occurs in both race groups but may be more pronounced in the less-represented race group. Therefore, we evaluated whether the effect of clinical (age, body surface area [BSA], chronic kidney disease [CKD], and amiodarone use) and genetic factors (CYP2C9*2, *3, *5, *6, *11, rs12777823, VKORC1, and CYP4F2) on warfarin dose differs by race using regression analyses among 1357 patients enrolled in a prospective cohort study and compared predictive ability of race-combined vs race-stratified models. Differential effect of predictors by race was assessed using predictor-race interactions in race-combined analyses. Warfarin dose was influenced by age, BSA, CKD, amiodarone use, and CYP2C9*3 and VKORC1 variants in both races, by CYP2C9*2 and CYP4F2 variants in European Americans, and by rs12777823 in African Americans. CYP2C9*2 was associated with a lower dose only among European Americans (20.6% vs 3.0%, P < .001) and rs12777823 only among African Americans (12.3% vs 2.3%, P = .006). Although VKORC1 was associated with dose decrease in both races, the proportional decrease was higher among European Americans (28.9% vs 19.9%, P = .003) compared with African Americans. Race-stratified analysis improved dose prediction in both race groups compared with race-combined analysis. We demonstrate that the effect of predictors on warfarin dose differs by race, which may explain divergent findings reported by recent warfarin pharmacogenetic trials. We recommend that warfarin dosing algorithms should be stratified by race rather than adjusted for race.

  16. Genetics-Based Pediatric Warfarin Dosage Regimen Derived Using Pharmacometric Bridging

    PubMed Central

    Lala, Mallika; Burckart, Gilbert J.; Takao, Cheryl M.; Pravica, Vera; Momper, Jeremiah D.; Gobburu, Jogarao V.S.

    2013-01-01

    BACKGROUND Warfarin dosage regimens using CYP2C9 and VKORC1 polymorphisms have been extensively studied in adults and is included in US Food and Drug Administration-approved warfarin labeling. However, no dosage algorithm is available for pediatric patients. OBJECTIVE To derive a genetics-based pediatric dosge regimen for warfarin, including starting dose and titration scheme. METHODS A model-based approach was developed based on a previously validated warfarin dosage model in adults, with subsequent comparison to pediatric data from pediatric warfarin dose, genotyping, and international normalized ratio (INR) results. The adult model was based on a previously established model from the CROWN (CReating an Optimal Warfarin dosing Nomogram) trial. Pediatric warfarin data were obtained from a study conducted at the Children’s Hospital of Los Angeles with 26 subjects. Variant alleles of CYP2C9 (rs1799853 or *2, and rs1057910 or *3) and the VKORC1 single nucleotide polymorphism (SNP) rs9923231 (−1639 G>A) were assessed, where the rs numbers are reference SNP identification tags assigned by the National Center for Biotechnology Information. RESULTS A pediatric warfarin model was derived using the previously validated model and clinical pharmacology considerations. The model was validated, and clinical trial simulation and stochastic modeling were used to optimize pediatric dosage and titration. The final dosage regimen was optimized based on simulations targeting a high (≥60%) proportion of INRs within the therapeutic range by week 2 of warfarin therapy while minimizing INRs >3.5 or <2. CONCLUSIONS The proposed pediatric warfarin dosage scheme based on individual CYP2C9 (alleles *1,*2,*3) and VKORC1 rs9923231 (-1639 G>A) genotypes may offer improved dosage compared to current treatment strategies, especially in patients with variant CYP2C9 and VKORC1 alleles. This pilot study provides the foundation for a larger prospective evaluation of genetics-based warfarin

  17. On the Feasibility of Characterizing Free-floating Planets with Current and Future Space-based Microlensing Surveys

    NASA Astrophysics Data System (ADS)

    Henderson, Calen B.; Shvartzvald, Yossi

    2016-10-01

    Simultaneous space- and ground-based microlensing surveys, such as K2's Campaign 9 (K2C9) and WFIRST, facilitate measuring the masses and distances of free-floating planet (FFP) candidates, which are identified as single-lens events with timescales that are of the order of 1 day. Measuring the mass and distance of an FFP lens requires determining the size of the source star ρ, measuring the microlens parallax {π }{{E}}, and using high-resolution imaging to search for the lens flux {F}{\\ell } from a possible host star. Here we investigate the accessible parameter space for each of these components considering different satellites for a range of FFP masses, Galactic distances, and source star properties. We find that at the beginning of K2C9, when its projected separation {D}\\perp from the Earth is ≲0.2 au, it will be able to measure {π }{{E}} for Jupiter-mass FFP candidates at distances larger than ∼2 kpc and to Earth-mass lenses at ∼8 kpc. At the end of K2C9, when {D}\\perp = 0.81 au, it is sensitive to planetary-mass lenses for distances ≳3.5 kpc, and even then only to those with mass ≳M Jup. From lens flux constraints we find that it will be possible to exclude hosts down to the deuterium-burning limit for events within ∼2 kpc. This indicates that the ability to characterize FFPs detected during K2C9 is optimized for events occurring toward the beginning of the campaign. WFIRST, on the other hand, will be able to detect and characterize FFP masses down to or below super-Earths throughout the Galaxy during its entire microlensing survey.

  18. Effects of Eleutheroside B and Eleutheroside E on activity of cytochrome P450 in rat liver microsomes

    PubMed Central

    2014-01-01

    Background Chemicals of herbal products may cause unexpected toxicity or adverse effect by the potential for alteration of the activity of CYP450 when co-administered with other drugs. Eleutherococcus senticosus (ES), has been widely used as a traditional herbal medicine and popular herbal dietary supplements, and often co-administered with many other drugs. The main bioactive constituents of ES were considered to be eleutherosides including eleutheroside B (EB) and eleutheroside E (EE). This study was to investigate the effects of EB and EE on CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in rat liver microsomes in vitro. Method Probe drugs of tolbutamide (TB), dextromethorphan (DM), chlorzoxazone (CLZ) and testosterone (TS) as well as eleutherosides of different concentrations were added to incubation systems of rat liver microsomes in vitro. After incubation, validated HPLC methods were used to quantify relevant metabolites. Results The results suggested that EB and EE exhibited weak inhibition against the activity of CYP2C9 and CYP2E1, but no effects on CYP2D6 and CYP3A4 activity. The IC50 values for EB and EE were calculated to be 193.20 μM and 188.36 μM for CYP2E1, 595.66 μM and 261.82 μM for CYP2C9, respectively. Kinetic analysis showed that inhibitions of CYP2E1 by EB and EE were best fit to mixed-type with Ki value of 183.95 μM and 171.63 μM, respectively. Conclusions These results indicate that EB and EE may inhibit the metabolism of drugs metabolized via CYP2C9 and CYP2E1, and have the potential to increase the toxicity of the drugs. PMID:24383621

  19. Surface Engineering of Porous Silicon Microparticles for Intravitreal Sustained Delivery of Rapamycin

    PubMed Central

    Nieto, Alejandra; Hou, Huiyuan; Moon, Sang Woong; Sailor, Michael J.; Freeman, William R.; Cheng, Lingyun

    2015-01-01

    Purpose. To understand the relationship between rapamycin loading/release and surface chemistries of porous silicon (pSi) to optimize pSi-based intravitreal delivery system. Methods. Three types of surface chemical modifications were studied: (1) pSi-COOH, containing 10-carbon aliphatic chains with terminal carboxyl groups grafted via hydrosilylation of undecylenic acid; (2) pSi-C12, containing 12-carbon aliphatic chains grafted via hydrosilylation of 1-dodecene; and (3) pSiO2-C8, prepared by mild oxidation of the pSi particles followed by grafting of 8-hydrocarbon chains to the resulting porous silica surface via a silanization. Results. The efficiency of rapamycin loading follows the order (micrograms of drug/milligrams of carrier): pSiO2-C8 (105 ± 18) > pSi-COOH (68 ± 8) > pSi-C12 (36 ± 6). Powder X-ray diffraction data showed that loaded rapamycin was amorphous and dynamic drug-release study showed that the availability of the free drug was increased by 6-fold (compared with crystalline rapamycin) by using pSiO2-C8 formulation (P = 0.0039). Of the three formulations in this study, pSiO2-C8-RAP showed optimal performance in terms of simultaneous release of the active drug and carrier degradation, and drug-loading capacity. Released rapamycin was confirmed with the fingerprints of the mass spectrometry and biologically functional as the control of commercial crystalline rapamycin. Single intravitreal injections of 2.9 ± 0.37 mg pSiO2-C8-RAP into rabbit eyes resulted in more than 8 weeks of residence in the vitreous while maintaining clear optical media and normal histology of the retina in comparison to the controls. Conclusions. Porous silicon–based rapamycin delivery system using the pSiO2-C8 formulation demonstrated good ocular compatibility and may provide sustained drug release for retina. PMID:25613937

  20. Pharmacogenetics of Oral Antidiabetic Drugs

    PubMed Central

    Becker, Matthijs L.; Pearson, Ewan R.; Tkáč, Ivan

    2013-01-01

    Oral antidiabetic drugs (OADs) are used for more than a half-century in the treatment of type 2 diabetes. Only in the last five years, intensive research has been conducted in the pharmacogenetics of these drugs based mainly on the retrospective register studies, but only a handful of associations detected in these studies were replicated. The gene variants in CYP2C9, ABCC8/KCNJ11, and TCF7L2 were associated with the effect of sulfonylureas. CYP2C9 encodes sulfonylurea metabolizing cytochrome P450 isoenzyme 2C9, ABCC8 and KCNJ11 genes encode proteins constituting ATP-sensitive K+ channel which is a therapeutic target for sulfonylureas, and TCF7L2 is a gene with the strongest association with type 2 diabetes. SLC22A1, SLC47A1, and ATM gene variants were repeatedly associated with the response to metformin. SLC22A1 and SLC47A1 encode metformin transporters OCT1 and MATE1, respectively. The function of a gene variant near ATM gene identified by a genome-wide association study is not elucidated so far. The first variant associated with the response to gliptins is a polymorphism in the proximity of CTRB1/2 gene which encodes chymotrypsinogen. Establishment of diabetes pharmacogenetics consortia and reduction in costs of genomics might lead to some significant clinical breakthroughs in this field in a near future. PMID:24324494

  1. A pharmacogenetics-based warfarin maintenance dosing algorithm from Northern Chinese patients.

    PubMed

    Chen, Jinxing; Shao, Liying; Gong, Ling; Luo, Fang; Wang, Jin'e; Shi, Yi; Tan, Yu; Chen, Qianlong; Zhang, Yu; Hui, Rutai; Wang, Yibo

    2014-01-01

    Inconsistent associations with warfarin dose were observed in genetic variants except VKORC1 haplotype and CYP2C9*3 in Chinese people, and few studies on warfarin dose algorithm was performed in a large Chinese Han population lived in Northern China. Of 787 consenting patients with heart-valve replacements who were receiving long-term warfarin maintenance therapy, 20 related Single nucleotide polymorphisms were genotyped. Only VKORC1 and CYP2C9 SNPs were observed to be significantly associated with warfarin dose. In the derivation cohort (n = 551), warfarin dose variability was influenced, in decreasing order, by VKORC1 rs7294 (27.3%), CYP2C9*3(7.0%), body surface area(4.2%), age(2.7%), target INR(1.4%), CYP4F2 rs2108622 (0.7%), amiodarone use(0.6%), diabetes mellitus(0.6%), and digoxin use(0.5%), which account for 45.1% of the warfarin dose variability. In the validation cohort (n = 236), the actual maintenance dose was significantly correlated with predicted dose (r = 0.609, P<0.001). Our algorithm could improve the personalized management of warfarin use in Northern Chinese patients. PMID:25126975

  2. Is pomegranate juice a potential perpetrator of clinical drug-drug interactions? Review of the in vitro, preclinical and clinical evidence.

    PubMed

    Srinivas, Nuggehally R

    2013-12-01

    The area of fruit juice-drug interaction has received wide attention with numerous scientific and clinical investigations performed and reported for scores of drugs metabolized by CYP3A4/CYP2C9. While grapefruit juice has been extensively studied with respect to its drug-drug interaction potential, numerous other fruit juices such as cranberry juice, orange juice, grape juice, pineapple juice and pomegranate juice have also been investigated for its potential to show drug-drug interaction of any clinical relevance. This review focuses on establishing any relevance for clinical drug-drug interaction potential with pomegranate juice, which has been shown to produce therapeutic benefits over a wide range of disease areas. The review collates and evaluates relevant published in vitro, preclinical and clinical evidence of the potential of pomegranate juice to be a perpetrator in drug-drug interactions mediated by CYP3A4 and CYP2C9. In vitro and animal pharmacokinetic data support the possibility of CYP3A4/CYP2C9 inhibition by pomegranate juice; however, the human relevance for drug-drug interaction was not established based on the limited case studies.

  3. Effects of icaritin on cytochrome P450 enzymes in rats.

    PubMed

    Liang, Dong-Lou; Zheng, Shuang-Li

    2014-04-01

    The purpose of this study was to find out whether icaritin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated with multiple doses of icaritin. 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 corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Treatment with multiple doses of icaritin had inhibitive effects on rat CYP1A2, CYP2C9 and CYP3A4 enzyme activities. However, icaritin has no inductive or inhibitory effect on the activity of CYP2E1. Therefore, caution is needed when icaritin is co-administered with some CYP1A2, CYP2C9 or CYP3A4 substrates, which may result in treatment failure and herb-drug interactions.

  4. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method

    PubMed Central

    Kong, Lingti; Song, Chunli; Ye, Linhu; Guo, Daohua; Yu, Meiling; Xing, Rong

    2016-01-01

    Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment. PMID:27006677

  5. Validation of Clinical Testing for Warfarin Sensitivity

    PubMed Central

    Langley, Michael R.; Booker, Jessica K.; Evans, James P.; McLeod, Howard L.; Weck, Karen E.

    2009-01-01

    Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 −1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses. PMID:19324988

  6. Metabolic profiling of praziquantel enantiomers

    PubMed Central

    Wang, Haina; Fang, Zhong-Ze; Zheng, Yang; Zhou, Kun; Hu, Changyan; Krausz, Kristopher W; Sun, Dequn; Idle, Jeffrey R.; Gonzalez, Frank J

    2014-01-01

    Praziquantel (PZQ), prescribed as a racemic mixture, is the most readily available drug to treat schistosomiasis. In the present study, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based metabolomics was employed to decipher the metabolic pathways and enantioselective metabolic differences of PZQ. Many phase I and four new phase II metabolites were found in urine and feces samples of mice 24h after dosing indicating that the major metabolic reaction encompassed oxidation, dehydrogenation, and glucuronidation. Differences in the formation of all these metabolites were observed between (R)-PZQ and (S)-PZQ. In an in vitro phase I incubation system, the major involvement of CYP3A, CYP2C9, and CYP2C19 in the metabolism of PZQ, and CYP3A, CYP2C9, and CYP2C19 exhibited different catalytic activity towards the PZQ enantiomers. Apparent Km and Vmax differences were observed in the catalytic formation of three mono-oxidized metabolites by CYP2C9 and CYP3A4 further supporting the metabolic differences for PZQ enantiomers. Molecular docking showed that chirality resulted in differences in location and conformation, which likely accounts for the metabolic differences. In conclusion, in silico, in vitro, and in vivo methods revealed the enantioselective metabolic profile of praziquantel. PMID:24821110

  7. Sulfaphenazole and α-naphthoflavone attenuate the metabolism of the synthetic cannabinoids JWH-018 and AM2201 found in K2/spice.

    PubMed

    Chimalakonda, Krishna C; James, Laura P; Radominska-Pandya, Anna; Moran, Jeffery H

    2013-03-01

    "K2" or "Spice" is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 - 50 μM) and ANF (0.1 - 5.0 μM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes.

  8. Sulfaphenazole and α-naphthoflavone attenuate the metabolism of the synthetic cannabinoids JWH-018 and AM2201 found in K2/Spice

    PubMed Central

    Chimalakonda, Krishna C.; James, Laura P.; Radominska-Pandya, Anna; Moran, Jeffery H.

    2014-01-01

    “K2” or “Spice” is an emerging drug of abuse that is laced with psychoactive synthetic cannabinoids JWH-018 and AM2201. Previous studies have identified hydroxylated (OH) and carboxylated (COOH) species as primary human metabolites, and kinetic studies have implicated CYP2C9 and -1A2 as major hepatic P450s involved in JWH-018 and AM2201 oxidation. The present study extends these findings by testing the hypothesis that CYP2C9- and 1A2-selective chemical inhibitors, sulfaphenazole (SFZ) and α-naphthoflavone (ANF), block oxidation of JWH-018 and AM2201 in human liver microsomes (HLM). A concentration-dependent inhibition of JWH-018 and AM2201 oxidation was observed in the presence of increasing concentration of SFZ (0.5 – 50 μM) and ANF (0.1 – 5.0 μM). No metabolic inhibition was observed with omeprazole, quinidine, and ketoconazole. The results presented herein further demonstrate the importance of CYP2C9- and 1A2-mediated oxidation of JWH-018 and AM2201 and the likelihood of adverse toxicity in populations with polymorphic alleles of these enzymes. PMID:24329780

  9. [A preliminary study on ingredient of secretion from fungi of orchid mycorrhiza].

    PubMed

    Wu, Jingping; Qian, Ji; Zheng, Shizhang

    2002-07-01

    Fusarium sp., which was not reported before in orchid mycorrhiza research, was isolated from the roots of Dendrobium desiflorum from Fujian Province by routine work of purification and identification. The results showed that the secretion contained Vitamin B2, B6, and folic acid, and mycelia contained Vitamin B2 and B6. It was also found that the mycelia of orchid mycorrhizal fungi contained and secreted gibberellin.

  10. Gene variants in CYP2C19 are associated with altered in vivo bupropion pharmacokinetics but not bupropion-assisted smoking cessation outcomes.

    PubMed

    Zhu, Andy Z X; Zhou, Qian; Cox, Lisa Sanderson; Ahluwalia, Jasjit S; Benowitz, Neal L; Tyndale, Rachel F

    2014-11-01

    Bupropion is used clinically to treat depression and to promote smoking cessation. It is metabolized by CYP2B6 to its active metabolite hydroxybupropion, yet alterations in CYP2B6 activity have little impact on bupropion plasma levels. Furthermore, less than 10% of a bupropion dose is excreted as urinary bupropion and its characterized metabolites hydroxybupropion, threohydrobupropion, and erythrohydrobupropion, suggesting that alternative metabolic pathways may exist. In vitro data suggested CYP2C19 could metabolize bupropion. The current study investigated the impact of functional CYP2C19 genetic variants on bupropion pharmacokinetics and treatment outcomes. In 42 healthy volunteers, CYP2C19*2 (a reduced activity allele) was associated with higher bupropion area under the plasma concentration-time curve (AUC), but similar hydroxybupropion AUC. The mean bupropion AUC was 771 versus 670 hours⋅ng/ml in individuals with and without CYP2C19*2, respectively (P = 0.017). CYP2C19*2 was also associated with higher threohydrobupropion and erythrohydrobupropion AUC (P < 0.005). Adjusting for CYP2B6 genotype did not alter these associations, and CYP2C19 variants did not alter the utility of the hydroxybupropion/bupropion ratio as a measure of CYP2B6 activity. Finally, in a clinical trial of 540 smokers, CYP2C19 genotype was not associated with smoking cessation outcomes, supporting the hypothesis that bupropion response is mediated by hydroxybupropion, which is not altered by CYP2C19. In conclusion, our study reports the first in vivo evidence that reduced CYP2C19 activity significantly increases the steady-state exposure to bupropion and its reductive metabolites threohydrobupropion and erythrohydrobupropion. These pharmacokinetic changes were not associated with differences in bupropion's ability to promote smoking cessation in smokers, but may influence the side effects and toxicity associated with bupropion.

  11. Liquid-liquid distribution of B group vitamins in polyethylene glycol-based systems

    NASA Astrophysics Data System (ADS)

    Korenman, Ya. I.; Zykov, A. V.; Mokshina, N. Ya.

    2011-05-01

    General regularities of the liquid-liquid distribution of B1, B2, B6, and B12 vitamins in aqueous polyethylene glycol (PEG-2000, PEG-5000) solution-aqueous salt solution systems are studied. The influence of the salting-out agent, the concentration of the polymer, and its molecular weight on the distribution coefficients and recovery factors of the vitamins are considered. Equations relating the distribution coefficients (log D) to the polymer concentration are derived.

  12. Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome.

    PubMed

    Liu, Xu; Luo, Yu; Lai, Yan; Yao, Yian; Li, Jimin; Wang, Yunkai; Zheng, S Lilly; Xu, Jianfeng; Liu, Xuebo

    2016-06-01

    Polymorphisms of CYP2C19 are associated with platelet response to clopidogrel. This study was conducted to evaluate the contribution of the previously identified polymorphisms to the response of clopidogrel in a cohort of Chinese Han patients. A total of 222 acute coronary syndrome patients undergoing percutaneous coronary intervention treated with clopidogrel were enrolled from September 2012 to June 2013. Residual platelet aggregations for all patients were measured by the Verify Now P2Y12 system. Sixteen single-nucleotide polymorphisms among nine genes were genotyped including CYP2C19, ABCB1 and PON1. In this study, CYP2C19*2 and CYP2C19*17 were strongly associated with higher platelet aggregation and lower platelet aggregation to clopidogrel treatment, respectively (P <0.001). Patients with CYP2C19*2 allele had a higher risk of high on-treatment platelet reactivity than non carriers (adjusted OR, 5.434; 95% CI, 1.918-15.399, P =0.01). The coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and the coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were also associated with poor response to clopidogrel. No significant relation of CYP2C19*3 and other polymorphisms to the platelet aggregation was found. In conclusion, CYP2C19*2, CYP2C19*17 coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were identified to be associated with response to clopidogrel treatment in Chinese Han patients. PMID:27350664

  13. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes.

    PubMed

    Price, R J; Scott, M P; Giddings, A M; Walters, D G; Stierum, R H; Meredith, C; Lake, B G

    2008-06-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.