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Pertti J. Neuvonen - One of the best experts on this subject based on the ideXlab platform.
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Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions
Pharmacological reviews, 2015Co-Authors: Janne T. Backman, Mikko Niemi, Anne M. Filppula, Pertti J. NeuvonenAbstract: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.
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Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates.
British journal of pharmacology, 2012Co-Authors: Anne M. Filppula, Pertti J. Neuvonen, Jouko Laitila, Janne T. BackmanAbstract:BACKGROUND AND PURPOSE Imatinib, a Cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.
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cyp2C8 activity recovers within 96 hours after gemfibrozil dosing estimation of cyp2C8 half life using repaglinide as an in vivo probe
Drug Metabolism and Disposition, 2009Co-Authors: Jt T Backman, Mikko Niemi, Aleksi Tornio, Mikko Neuvonen, Johanna Honkalammi, Kaisa J Kurkinen, Pertti J. NeuvonenAbstract:Gemfibrozil 1-O-beta-glucuronide is a mechanism-based inhibitor of Cytochrome P450 2C8. We studied the recovery of CYP2C8 activity after discontinuation of gemfibrozil treatment using repaglinide as a probe drug, to estimate the in vivo turnover half-life of CYP2C8. In a randomized five-phase crossover study, nine healthy volunteers ingested 0.25 mg of repaglinide alone or after different time intervals after a 3-day treatment with 600 mg of gemfibrozil twice daily. The area under the plasma concentration-time curve (AUC) from time 0 to infinity of repaglinide was 7.6-, 2.9-, 1.4- and 1.0-fold compared with the control phase when it was administered 1, 24, 48, or 96 h after the last gemfibrozil dose, respectively (P < 0.001 versus control for 1, 24, and 48 h after gemfibrozil). Thus, a strong CYP2C8 inhibitory effect persisted even after gemfibrozil and gemfibrozil 1-O-beta-glucuronide concentrations had decreased to less than 1% of their maximum (24-h dosing interval). In addition, the metabolite to repaglinide AUC ratios indicated that significant (P < 0.05) inhibition of repaglinide metabolism continued up to 48 h after gemfibrozil administration. Based on the recovery of repaglinide oral clearance, the in vivo turnover half-life of CYP2C8 was estimated to average 22 +/- 6 h (mean +/- S.D.). In summary, CYP2C8 activity is recovered gradually during days 1 to 4 after gemfibrozil discontinuation, which should be considered when CYP2C8 substrate dosing is planned. The estimated CYP2C8 half-life will be useful for in vitro-in vivo extrapolations of drug-drug interactions involving induction or mechanism-based inhibition of CYP2C8.
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Comparison of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) as inhibitors of Cytochrome P450 2C8.
Basic & clinical pharmacology & toxicology, 2005Co-Authors: Aleksi Tornio, Pertti J. Neuvonen, Jouko Laitila, Marja K. Pasanen, Janne T. BackmanAbstract:Statins are involved in different types of drug interactions. Our objective was to study the effect of statins on Cytochrome P450 (CYP) 2C8-mediated paclitaxel 6 alpha-hydroxylation by incubating paclitaxel and statins (0--100 microM) with pooled human liver microsomes. Simvastatin, lovastatin, atorvastatin and fluvastatin were the most potent inhibitors of CYP2C8 activity with K(i) (IC(50)) values of 7.1 (9.6) muM, 8.4 (15) microM, 16 (38) microM and 19 (37) microM, respectively. Cerivastatin, simvastatin acid and lovastatin acid were less potent inhibitors with K(i) (IC(50)) values ranging from 32 to 55 (30--67) microM. Rosuvastatin and pravastatin showed no appreciable effect on CYP2C8 activity even at 100 microM. In conclusion, all the statins tested, except rosuvastatin and pravastatin, had a significant inhibitory effect on the activity of CYP2C8 in vitro. Because many of the statins accumulate in the liver and because also their metabolites may inhibit CYP2C8 activity, in vivo studies are needed to investigate a possible interaction of simvastatin, lovastatin, atorvastatin and fluvastatin with CYP2C8 substrate drugs.
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Effects of trimethoprim and rifampin on the pharmacokinetics of the Cytochrome P450 2C8 substrate rosiglitazone.
Clinical pharmacology and therapeutics, 2004Co-Authors: Mikko Niemi, Janne T. Backman, Pertti J. NeuvonenAbstract:Background Trimethoprim is a relatively selective inhibitor of the Cytochrome P450 (CYP) 2C8 enzyme in vitro. Rifampin (INN, rifampicin) is a potent inducer of several CYP enzymes, and in vitro studies have suggested that it also induces CYP2C8. Objective Our aims were to investigate possible effects of trimethoprim and rifampin on CYP2C8 activity by use of rosiglitazone, a thiazolidinedione antidiabetic drug metabolized primarily by CYP2C8, as an in vivo probe. Methods Two separate randomized crossover studies with 2 phases were conducted. In study 1, 10 healthy volunteers took 160 mg trimethoprim or placebo orally twice daily for 4 days. On day 3, they ingested a single 4-mg dose of rosiglitazone. In study 2, 10 healthy volunteers took 600 mg rifampin or placebo orally once daily for 5 days. On day 6, they ingested a single 4-mg dose of rosiglitazone. In both studies, plasma rosiglitazone and N-desmethylrosiglitazone concentrations were measured for up to 48 hours. Results In study 1, trimethoprim raised the area under the plasma rosiglitazone concentration–time curve [AUC(0-∞)] by 37% (range, 16% to 51%; P < .0001) and the peak plasma rosiglitazone concentration (Cmax) by 14% (range, −3% to 38%; P = .0014). The elimination half-life (t1/2) of rosiglitazone was prolonged from 3.8 to 4.8 hours (P = .0013). Trimethoprim reduced the formation of N-desmethylrosiglitazone. In study 2, rifampin reduced the AUC(0-∞) and Cmax of rosiglitazone by 54% (range, 46% to 63%; P < .0001) and 28% (range, 2% to 56%; P = .0003), respectively. The t1/2 of rosiglitazone was shortened from 3.8 to 1.9 hours (P < .0001). Rifampin increased the formation of N-desmethylrosiglitazone. Conclusions Trimethoprim raises and rifampin reduces the plasma concentrations of rosiglitazone by inhibiting and inducing, respectively, the CYP2C8-catalyzed biotransformation of rosiglitazone. Clinical Pharmacology & Therapeutics (2004) 76, 239–249; doi: 10.1016/j.clpt.2004.05.001
Frank P. Mockenhaupt - One of the best experts on this subject based on the ideXlab platform.
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Short communication: high prevalence of the Cytochrome P450 2C8*2 mutation in Northern Ghana.
Tropical medicine & international health : TM & IH, 2005Co-Authors: Susanne Röwer, Ulrich Bienzle, Alexander Weise, Ulrike Lambertz, Thomas Forst, Rowland N. Otchwemah, Andreas Pfützner, Frank P. MockenhauptAbstract:Recently, Ghana has changed the first-line treatment of uncomplicated malaria from chloroquine to amodiaquine (AQ) plus artesunate. AQ may cause adverse events such as agranulocytosis and hepatoxicity. The pro-drug AQ is transformed by Cytochrome P450 CYP2C8 to the active metabolite N-desethylaminodiaquine. Several polymorphic variants of CYP2C8 are known, some with reduced activity. In 200 randomly selected children from Northern Ghana, we determined the allele frequencies of the CYP2C8 variants CYP2C8*1 (wild type), CYP2C8*2, CYP2C8*3, and CYP2C8*4. We did not detect CYP2C8*3 and CYP2C8*4, but CYP2C8*2 showed an allele frequency of 0.1675. AQ metabolism in patients with CYP2C8*2 may be impaired, and with an increase of AQ based treatment the risk of severe adverse events may mount.
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short communication high prevalence of the Cytochrome P450 2C8 2 mutation in northern ghana
Tropical Medicine & International Health, 2005Co-Authors: Susanne Röwer, Ulrich Bienzle, Alexander Weise, Ulrike Lambertz, Thomas Forst, Rowland N. Otchwemah, Andreas Pfützner, Frank P. MockenhauptAbstract:Recently, Ghana has changed the first-line treatment of uncomplicated malaria from chloroquine to amodiaquine (AQ) plus artesunate. AQ may cause adverse events such as agranulocytosis and hepatoxicity. The pro-drug AQ is transformed by Cytochrome P450 CYP2C8 to the active metabolite N-desethylaminodiaquine. Several polymorphic variants of CYP2C8 are known, some with reduced activity. In 200 randomly selected children from Northern Ghana, we determined the allele frequencies of the CYP2C8 variants CYP2C8*1 (wild type), CYP2C8*2, CYP2C8*3, and CYP2C8*4. We did not detect CYP2C8*3 and CYP2C8*4, but CYP2C8*2 showed an allele frequency of 0.1675. AQ metabolism in patients with CYP2C8*2 may be impaired, and with an increase of AQ based treatment the risk of severe adverse events may mount.
Hidetaka Kamimura - One of the best experts on this subject based on the ideXlab platform.
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The UDP-glucuronosyltransferase 2B7 isozyme is responsible for gemfibrozil glucuronidation in the human liver.
Drug Metabolism and Disposition, 2007Co-Authors: Yuji Mano, Takashi Usui, Hidetaka KamimuraAbstract:Gemfibrozil, a fibrate hypolipidemic agent, is eliminated in humans by glucuronidation. A gemfibrozil glucuronide has been reported to show time-dependent inhibition of Cytochrome P450 2C8. Comprehensive assessment of the drug interaction between gemfibrozil and Cytochrome P450 2C8 substrates requires a clear understanding of gemfibrozil glucuronidation. However, the primary UDP-glucuronosyltransferase (UGT) isozymes responsible for gemfibrozil glucuronidation remain to be determined. Here, we identified the main UGT isozymes involved in gemfibrozil glucuronidation. Evaluation of 12 recombinant human UGT isozymes shows gemfibrozil glucuronidation activity in UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, with UGT2B7 showing the highest activity. The kinetics of gemfibrozil glucuronidation in pooled human liver microsomes (HLMs) follows Michaelis-Menten kinetics with high and low affinity components. The high affinity K m value was 2.5 μM, which is similar to the K m value of gemfibrozil glucuronidation in recombinant UGT2B7 (2.2 μM). In 16 HLMs, a significant correlation was observed between gemfibrozil glucuronidation and both morphine 3-OH glucuronidation ( r = 0.966, p < 0.0001) and flurbiprofen glucuronidation ( r = 0.937, p < 0.0001), two reactions mainly catalyzed by UGT2B7, whereas no significant correlation was observed between gemfibrozil glucuronidation and either estradiol 3β-glucuronidation and propofol glucuronidation, two reactions catalyzed by UGT1A1 and UGT1A9, respectively. Flurbiprofen and mefenamic acid inhibited gemfibrozil glucuronidation in HLMs with similar IC50 values to those reported in recombinant UGT2B7. These results suggest that UGT2B7 is the main isozyme responsible for gemfibrozil glucuronidation in humans.
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The UDP-glucuronosyltransferase 2B7 isozyme is responsible for gemfibrozil glucuronidation in the human liver.
Drug metabolism and disposition: the biological fate of chemicals, 2007Co-Authors: Yuji Mano, Takashi Usui, Hidetaka KamimuraAbstract:Gemfibrozil, a fibrate hypolipidemic agent, is eliminated in humans by glucuronidation. A gemfibrozil glucuronide has been reported to show time-dependent inhibition of Cytochrome P450 2C8. Comprehensive assessment of the drug interaction between gemfibrozil and Cytochrome P450 2C8 substrates requires a clear understanding of gemfibrozil glucuronidation. However, the primary UDP-glucuronosyltransferase (UGT) isozymes responsible for gemfibrozil glucuronidation remain to be determined. Here, we identified the main UGT isozymes involved in gemfibrozil glucuronidation. Evaluation of 12 recombinant human UGT isozymes shows gemfibrozil glucuronidation activity in UGT1A1, UGT1A3, UGT1A9, UGT2B4, UGT2B7, and UGT2B17, with UGT2B7 showing the highest activity. The kinetics of gemfibrozil glucuronidation in pooled human liver microsomes (HLMs) follows Michaelis-Menten kinetics with high and low affinity components. The high affinity K(m) value was 2.5 microM, which is similar to the K(m) value of gemfibrozil glucuronidation in recombinant UGT2B7 (2.2 microM). In 16 HLMs, a significant correlation was observed between gemfibrozil glucuronidation and both morphine 3-OH glucuronidation (r = 0.966, p < 0.0001) and flurbiprofen glucuronidation (r = 0.937, p < 0.0001), two reactions mainly catalyzed by UGT2B7, whereas no significant correlation was observed between gemfibrozil glucuronidation and either estradiol 3beta-glucuronidation and propofol glucuronidation, two reactions catalyzed by UGT1A1 and UGT1A9, respectively. Flurbiprofen and mefenamic acid inhibited gemfibrozil glucuronidation in HLMs with similar IC(50) values to those reported in recombinant UGT2B7. These results suggest that UGT2B7 is the main isozyme responsible for gemfibrozil glucuronidation in humans.
Jose Pedro Gil - One of the best experts on this subject based on the ideXlab platform.
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Influence of Cytochrome P450 (CYP) 2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine treatment of uncomplicated Plasmodium falciparum malaria in Zanzibar
Malaria journal, 2021Co-Authors: Leyre Pernaute-lau, Mwinyi I. Msellem, Anders Björkman, Ulrika Morris, Andreas Mårtensson, Jose Pedro GilAbstract:BACKGROUND The anti-malarial drug, amodiaquine, a commonly used, long-acting partner drug in artemisinin-based combination therapy, is metabolized to active desethyl-amodiaquine (DEAQ) by Cytochrome P450 2C8 (CYP2C8). The CYP2C8 gene carries several polymorphisms including the more frequent minor alleles, CYP2C8*2 and CYP2C8*3. These minor alleles have been associated with decreased enzymatic activity, slowing the amodiaquine biotransformation towards DEAQ. This study aimed to assess the influence of these CYP2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine (AS-AQ) treatment for uncomplicated Plasmodium falciparum malaria in Zanzibar. METHODS Dried blood spots on filter paper were collected from 618 children enrolled in two randomized clinical trials comparing AS-AQ and artemether-lumefantrine in 2002-2005 in Zanzibar. Study participant were under five years of age with uncomplicated falciparum malaria. Human CYP2C8*2 and CYP2C8*3 genotype frequencies were determined by PCR-restriction fragment length polymorphism. Statistical associations between CYP2C8*2 and/or CYP2C8*3 allele carriers and treatment outcome or occurrence of adverse events were assessed by Fisher's exact test. RESULTS The allele frequencies of CYP2C8*2 and CYP2C8*3 were 17.5 % (95 % CI 15.4-19.7) and 2.7 % (95 % CI 1.8-3.7), respectively. There was no significant difference in the proportion of subjects carrying either CYP2C8*2 or CYP2C8*3 alleles amongst those with re-infections (44.1 %; 95 % CI 33.8-54.8) or those with recrudescent infections (48.3 %; 95 % CI 29.4-67.5), compared to those with an adequate clinical and parasitological response (36.7 %; 95 % CI 30.0-43.9) (P = 0.25 and P = 0.31, respectively). However, patients carrying either CYP2C8*2 or CYP2C8*3 alleles were significantly associated with an increased occurrence of non-serious adverse events, when compared with CYP2C8 *1/*1 wild type homozygotes (44.9 %; 95 % CI 36.1-54.0 vs. 28.1 %; 95 % CI 21.9-35.0, respectively; P = 0.003). CONCLUSIONS CYP2C8 genotypes did not influence treatment efficacy directly, but the tolerability to AS-AQ may be reduced in subjects carrying the CYP2C8*2 and CYP2C8*3 alleles. The importance of this non-negligible association with regard to amodiaquine-based malaria chemotherapy warrants further investigation.
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Influence of Cytochrome P450 (CYP) 2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine treatment of uncomplicated Plasmodium falciparum malaria in Zanzibar
2021Co-Authors: Leyre Pernaute-lau, Anders Björkman, Ulrika Morris, Mwinyi Msellem, Andreas Mårtensson, Jose Pedro GilAbstract:Abstract BackgroundThe antimalarial drug amodiaquine, a commonly used long acting partner drug in artemisinin-based combination therapy, is metabolized to active desethyl-amodiaquine (DEAQ) by Cytochrome P450 2C8 (CYP2C8). The CYP2C8 gene carries several polymorphisms including the more frequent minor alleles CYP2C8*2 and CYP2C8*3. These minor alleles have been associated with decreased enzymatic activity, slowing the amodiaquine biotransformation towards DEAQ. This study aimed to assess the influence of these CYP2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine treatment for uncomplicated Plasmodium falciparum malaria in Zanzibar.MethodsWe analysed data from 618 children under 5 years of age with uncomplicated P. falciparum malaria enrolled in two randomized clinical trials comparing artesunate-amodiaquine and artemether-lumefantrine in 2002-2005 in Zanzibar. CYP2C8*2 and CYP2C8*3 genotype frequencies were determined by PCR-restriction fragment length polymorphism. Statistical associations between CYP2C8*2 and/or CYP2C8*3 allele carriers and treatment outcome or occurrence of adverse events were assessed by Fisher’s Exact test.ResultsThe allele frequencies of CYP2C8*2 and CYP2C8*3 were 17.5% (95% CI 15.4-19.7%) and 2.7% (95% CI 1.8-3.7%), respectively. There was no significant difference in the proportion of subjects carrying either CYP2C8*2 or CYP2C8*3 alleles amongst those with reinfections (44.1%; 95% CI 33.8-54.8) or those with recrudescent infections (48.3%; 95% CI 29.4-67.5), compared to those with an adequate clinical and parasitological response (36.7%; 95% CI 30.0-43.9) (P = 0.25 and P = 0.31, respectively). However, patients carrying either the CYP2C8*2 or CYP2C8*3 alleles were significantly associated with an increased occurrence of non-serious adverse events, when compared with CYP2C8 *1/*1 wildtype homozygotes (44.9%; 95% CI 36.1-54.0 versus 28.1%; 95% CI 21.9-35.0, respectively; P = 0.003). ConclusionsCYP2C8 genotypes did not influence treatment efficacy directly, but the tolerability to AS-AQ may be reduced in subjects carrying the CYP2C8*2 and CYP2C8*3 alleles. The importance of this non-negligible association with regards to amodiaquine-based malaria chemotherapy warrants further investigation.
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Influence of Cytochrome P450 (CYP) 2C8 Polymorphisms on the Efficacy and Tolerability of Artesunate-Amodiaquine Treatment of Uncomplicated Plasmodium Falciparum Malaria in Zanzibar
2020Co-Authors: Leyre Pernaute-lau, Anders Björkman, Ulrika Morris, Mwinyi Msellem, Andreas Mårtensson, Jose Pedro GilAbstract:Abstract Background The antimalarial drug amodiaquine, a commonly used long acting partner drug in artemisinin-based combination therapy, is metabolized to active desethyl-amodiaquine (DEAQ) by Cytochrome P450 2C8 (CYP2C8). The CYP2C8 gene carries several polymorphisms including the more frequent minor alleles CYP2C8*2 and CYP2C8*3. These minor alleles have been associated with decreased enzymatic activity, slowing the amodiaquine biotransformation towards DEAQ. This study aimed to assess the influence of CYP2C8 polymorphisms on the efficacy and tolerability of artesunate-amodiaquine treatment for uncomplicated Plasmodium falciparum malaria in Zanzibar.Methods We analysed data from 618 children <5 years with uncomplicated P. falciparum malaria enrolled in two randomized clinical trials comparing artesunate-amodiaquine and artemether-lumefantrine in 2002-2005 in Zanzibar. CYP2C8*2 and CYP2C8*3 genotypes were determined by PCR-restriction fragment length polymorphism and assessed in relation to clinical data on treatment outcome and tolerance. Results The allele frequencies of CYP2C8*2 and CYP2C8*3 were 17.5% (95% CI 15.4-19.7%) and 2.7% (95% CI 1.8-3.7%), respectively. There was no significant difference in the proportion of subjects carrying either CYP2C8*2 or CYP2C8*3 alleles amongst those with reinfections (44.1 %; 95% CI 33.8-54.8) or those with recrudescent infections (48.3%; 95% CI 29.4-67.5), compared to those with adequate clinical and parasitological response (36.7 %; 95% CI 30.0-43.9) (P = 0.25 and P = 0.31, respectively). However, patients carrying either the CYP2C8*2 or CYP2C8*3 allele were significantly associated with increased occurrence of non-serious adverse events compare with CYP2C8 *1/*1 wildtype homozygotes (44.9%; 95% CI 36.1-54.0 versus 28.1%; 95% CI 21.9-35.0, respectively; P = 0.003). Conclusions CYP2C8 genotypes did not influence treatment efficacy directly, but the tolerability to ASAQ may be reduced in subjects carrying the CYP2C8*3 and CYP2C8*2 alleles. The importance of this non-negligible association with regards to amodiaquine-based malaria chemotherapy warrants further investigation.
Xiangshi Tan - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 2C8 and drug metabolism.
Current topics in medicinal chemistry, 2013Co-Authors: Fangfang Zhong, Xiangshi TanAbstract:CYP 2C8, which carries out the oxidative metabolism of at least 5% of clinical drugs, has attracted increasing attention in recent years. New drugs (substances), inducers and inhibitors of CYP 2C8 have been developed and the drug metabolism has been investigated to understand the clinical role of CYP2C8. The cases of CYP2C8 genetic polymorphisms linked to diseases have increased and have been investigated. Herein, important progress in these areas has been reviewed with an emphasis on drug metabolism. Polymorphisms, diseases related to CYP2C8, some important drugs (substances) and inhibitors are reviewed and discussed.
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Structural and functional insights into CYP2C8.3:A genetic polymorph of Cytochrome P450 2C8
Science China Chemistry, 2010Co-Authors: Hualin Jiang, Lu Sun, Zhong-xian Huang, Xiangshi TanAbstract:The Cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver, and carries out the oxidative metabolism of at least 5% of clinical drugs. Polymorphisms in CYP2C8 have been closely implicated in individualized medication. CYP2C8.3, a common polymorph of CYP2C8 with dual amino acid substitutions R139K and K399R, is found primarily in Caucasians. In this study, CYP2C8.3 and its wild type (WT) CYP2C8 were expressed in E. coli, and their purified proteins were characterized by UV-visible spectroscopy, mass spectrometry, and circular dichroism. Their thermal stability, substrate binding ability, and metabolic activity against paclitaxel were investigated. The electron transfer kinetics during paclitaxel metabolism by WT CYP2C8 or CYP2C8.3 was studied by stopped-flow kinetics. The results revealed that mutations in CYP2C8.3 did not greatly influence the heme active site or protein thermal stability and paclitaxel binding ability, but the metabolic activity against paclitaxel was significantly depressed to just 11% of that of WT CYP2C8. Electron transfer from CYP reductase to CYP2C8.3 was found to be significantly slower than that to WT CYP2C8 during catalysis, and this might be the main reason for the depressed metabolic activity. Since the polymorph CYP2C8.3 is defective in catalyzing substrates of CYP2C8 in vitro, it might be expected to have important clinical and pathophysiological consequences in homozygous individuals, and this study provides valuable information in this aspect.
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Structural and functional insights into polymorphic enzymes of Cytochrome P450 2C8
Amino acids, 2010Co-Authors: Hualin Jiang, Lu Sun, Zhong-xian Huang, Fangfang Zhong, Weiyue Feng, Xiangshi TanAbstract:The Cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.
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Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding
Chinese Journal of Chemistry, 2010Co-Authors: Lu Sun, Zhonghua Wang, Hualin Jiang, Xiangshi Tan, Zhong-xian HuangAbstract:Transitions among various heme coordination/spin states, heme environments and protein conformations of human Cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it’s kind. Our results indicated that the thermal and acid-induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe-S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X-ray structure of P450 2C8 suggested that the specific structure of the β-bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid-induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.
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novel conformational transitions of human Cytochrome P450 2C8 during thermal and acid induced unfolding
Chinese Journal of Chemistry, 2010Co-Authors: Lu Sun, Zhonghua Wang, Hualin Jiang, Xiangshi Tan, Zhong-xian HuangAbstract:Transitions among various heme coordination/spin states, heme environments and protein conformations of human Cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it’s kind. Our results indicated that the thermal and acid-induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe-S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X-ray structure of P450 2C8 suggested that the specific structure of the β-bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid-induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.
