Abstract
Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V max 111.5 ± 16.0 pmol/min/mg, K m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V max 101.6 + 12.4 pmol/min/mg, K m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint = V max/K m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLint value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V max/K m, the CLint value of CYP2C19 WT was 785 folds of CYP2C19*23. K m and V max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.
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References
Andersson T, Regårdh CG, Lou YC, Zhang Y, Dahl ML, Bertilsson L (1992) Polymorphic hydroxylation of S-mephenytoin and omeprazole metabolism in Caucasian and Chinese subjects. Pharmacogenetics 2:25–31
Andersson T, Miners JO, Veronese ME, Tassaneeyakul W, Tassaneeyakul W, Meyer UA, Birkett DJ (1993) Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism. Br J Clin Pharmacol 36:521–530
Bertilsson L, Henthorn TK, Sanz E, Tybring G, Säwe J, Villén T (1989) Importance of genetic factors in the regulation of diazepam metabolism: relationship to S-mephenytoin, but not debrisoquin, hydroxylation phenotype. Clin Pharmacol Ther 45:348–355
Blaisdell J, Jorge-Nebert LF, Coulter S, Ferguson SS, Lee S-J, Chanas B, Xi T, Mohrenweiser H, Ghanayem B, Goldstein JA (2004) Discovery of new potentially defective alleles of human CYP2C9. Pharmacogenetics 14:527–537
Brøsen K (2004) Some aspects of genetic polymorphism in the biotransformation of antidepressants. Therapie 59:5–12
Chiba K, Kobayashi K, Manabe K, Tani M, Kamataki T, Ishizaki T (1993) Oxidative metabolism of omeprazole in human liver microsomes: cosegregation with S-mephenytoin 4′-hydroxylation. J Pharmacol Exp Ther 266:52–59
Du H, Wei Z, Yan Y, Xiong Y, Zhang X, Shen L, Ruan Y, Wu X, Xu Q, He L, Qin S (2016) Functional characterization of human CYP2C9 allelic variants in COS-7 cells. Front Pharmacol 7:1–6. doi:10.3389/fphar.2016.00098
Ferguson RJ, De Morais SM, Benhamou S, Bouchardy C, Blaisdell J, Ibeanu G, Wilkinson GR, Sarich TC, Wright JM, Dayer P, Goldstein JA (1998) A new genetic defect in human CYP2C19: mutation of the initiation codon is responsible for poor metabolism of S-mephenytoin. J Pharmacol Exp Ther 284:356–361
Furuta T, Ohashi K, Kamata T, Takashima M, Kosuge K, Kawasaki T, Hanai H, Kubota T, Ishizaki T, Kaneko E (1998) Effect of genetic differences in omeprazole metabolism on cure rates for Helicobacter pylori infection and peptic ulcer. Ann Intern Med 129:1027–1030
Goldstein JA, Faletto MB, Romkes-Sparks M, Sullivan T, Kitareewan S, Raucy JL, Lasker JM, Ghanayem BI (1994) Evidence that CYP2C19 is the major S-mephenytoin 4′-hydroxylase in humans. Biochemistry 33:1743–1752. doi:10.1021/bi00173a017
Hulot J-S, Bura A, Villard E, Azizi M, Remones V, Goyenvalle C, Aiach M, Lechat P, Gaussem P (2006) Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood 108:2244–2247. doi:10.1182/blood-2006-04-013052
Ibeanu GC, Ghanayem BI, Linko P, Li L, Pederson LG, Goldstein JA (1996) Identification of residues 99, 220, and 221 of human cytochrome P450 2C19 as key determinants of omeprazole activity. J Biol Chem 271:12496–12501
Küpfer A, Desmond P, Patwardhan R, Schenker S, Branch RA (1984) Mephenytoin hydroxylation deficiency: kinetics after repeated doses. Clin Pharmacol Ther 35:33–39
Lee S-J, Kim W-Y, Kim H, Shon J-H, Lee SS, Shin J-G (2009) Identification of new CYP2C19 variants exhibiting decreased enzyme activity in the metabolism of S-mephenytoin and omeprazole. Drug Metab Dispos 37:2262–2269. doi:10.1124/dmd.109.028175
Mankowski DC (1999) The role of CYP2C19 in the metabolism of (±) bufuralol, the prototypic substrate of CYP2D6. Drug Metab Dispos 27:1024–1028
Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE (2011) Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization. Protein J 30:581–591. doi:10.1007/s10930-011-9365-6
Pan Y, Mak JW, Ong CE (2013) Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker. Biomed Chromatogr 27:859–865. doi:10.1002/bmc.2872
Seifert A, Tatzel S, Schmid RD, Pleiss J (2006) Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: the molecular basis of substrate binding and regioselectivity toward warfarin. Proteins 64:147–155. doi:10.1002/prot.20951
Sim SC, Risinger C, Dahl M-L, Aklillu E, Christensen M, Bertilsson L, Ingelman-Sundberg M (2006) A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants. Clin Pharmacol Ther 79:103–113. doi:10.1016/j.clpt.2005.10.002
Singh R, Ting JG, Pan Y, Teh LK, Ismail R, Ong CE (2008) Functional role of Ile264 in CYP2C8: mutations affect haem incorporation and catalytic activity. Drug Metab Pharmacokinet 23:165–174
Takahashi M, Saito T, Ito M, Tsukada C, Katono Y, Hosono H, Maekawa M, Shimada M, Mano N, Oda A, Hirasawa N, Hiratsuka M (2015) Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation. Pharmacogenomics J 15:26–32. doi:10.1038/tpj.2014.30
Wang J-F, Wei D-Q, Li L, Zheng S-Y, Li Y-X, Chou K-C (2007) 3D structure modeling of cytochrome P450 2C19 and its implication for personalized drug design. Biochem Biophys Res Commun 355:513–519. doi:10.1016/j.bbrc.2007.01.185
Wang H, An N, Wang H, Gao Y, Liu D, Bian T, Zhu J, Chen C (2011) Evaluation of the Effects of 20 Nonsynonymous Single Nucleotide Polymorphisms of CYP2C19 on S-mephenytoin 4′-hydroxylation. Drug Metab Dispos 17:830–837. doi:10.1124/dmd.110.037549
Ward SA, Helsby NA, Skjelbo E, Brøsen K, Gram LF, Breckenridge AM (1991) The activation of the biguanide antimalarial proguanil co-segregates with the mephenytoin oxidation polymorphism—a panel study. Br J Clin Pharmacol 31:689–692
Wilde MI, McTavish D (1994) Omeprazole. An update of its pharmacology and therapeutic use in acid-related disorders. Drugs 48:91–132
Wilkinson GR, Guengerich FP, Branch RA (1989) Genetic polymorphism of S-mephenytoin hydroxylation. Pharmacol Ther 43:53–76
Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE (2000) Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5:121–131. doi:10.1016/S1097-2765(00)80408-6
Zhou Q, Yu XM, Lin HB, Wang L, Yun QZ, Hu SN, Wang D-M (2009) Genetic polymorphism, linkage disequilibrium, haplotype structure and novel allele analysis of CYP2C19 and CYP2D6 in Han Chinese. Pharmacogenom J 9:380–394. doi:10.1038/tpj.2009.31
Acknowledgments
The authors express gratitude to Professor John Miners (Flinders University, Adelaide, Australia) and Professor Donald Birkett (Johnson and Johnson Research Pty. Ltd., Sydney, Australia) for their kind gifts of vectors pCWori+ and pACYC-OxR. This work was supported by International Medical University internal research funding BS122002-95000.
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Lau, P.S., Leong, K.V.G., Ong, C.E. et al. In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24 . Biochem Genet 55, 48–62 (2017). https://doi.org/10.1007/s10528-016-9771-8
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DOI: https://doi.org/10.1007/s10528-016-9771-8