Abstract
The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile]), and verapamil N-demethylation (formation of norverapamil [2,8-bis(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (111) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18.
In human liver microsomes (n=4), the intrinsic clearance (CLint), as derived from the ratio of V max/Km, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9±1.0 vs 2.4±0.6 ml*min-1 *g-1 mean±SD; p<0.05), S-Verapamil (16.8±3.3 vs 2.2±1.2 ml* mini*g-1, p<0.05) and R-verapamil (12.1±2.9 vs 3.6 ±1.3 ml*min-1 * g-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CLint of D-703 formation in human liver microsomes showed a modest but significant degree of stereo selectivity (p<0.05) with a S/R-ratio of 1.41±0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5±4.5% and 45%, that of D-702 by 52.7±7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703.
In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.
Similar content being viewed by others
References
Abernathy DR, Egan JM, Dickinson TH, Carrum G (1988) Substrateselective inhibition by verapamil and diltiazem: differential disposition of antipyrine and teophylline in humans. J Pharmacol Exp Ther 244:994–999
Andersson T, Miners JO, Veronese ME, Tassaneeyakul W, Meyer UA, Birkett DJ (1993) Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism. Br J Clin Pharmacol 36:521–530
Barbarash RA, Baumann JL, Fischer JH, Kondos GT, Batenhorst RL (1988) Near-total reduction in verapamil bioavailability by rifampicin. Chest 94:954–959
Beaune PH, Dansette PM, Mansuy D, Kiffel L, Finck M, Amar C, Leroux JP, Homberg JC (1987) Human anti-endoplasmatic reticulum autoantibodies appearing in a drug-induced hepatitis are directed against a human liver cytochrome P-450 that hydroxylates the drug. Proc Natl Acad Sci USA 84:551–555
Botsch S, Gautier JC, Beaune P, Eichelbaum M, Kroemer HK (1993) Identification and characterization of the cytochrome P450 enzymes involved in N-dealkylation of propafenone: molecular base for interaction potential and variable disposition of active metabolites. Mol Pharmacol 43:120–126
Brian WR, Srivastava PK, Umbenhauer DR, Lloyd RS, Guengerich FP (1989) Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae. Biochemistry 28:4993–4999
Eichelbaum M, Ende M, Remberg G, Schomerus M, Dengler H (1978) The metabolism of DL-[14C]-verapamil in man. Drug Metab Dispos 7:145–148
Engel G, Hofmann U, Heidemann H, Eichelbaum M (1994) Identification of cytochrome P450 enzymes involved in the metabolism of antipyrin: potential and limitations of different in vitro techniques. Naunyn-Schmiedeberg's Arch Pharmacol 349 [Suppl]:R529
Fuhr U, Woodcock BG, Siewert M (1992) Verapamil and drug metabolism by cytochrome P450 isoform CYP1A2. Eur J Clin Pharmacol 42:463–464
Goldstein JA, de Morais SMF (1994) Biochemistry and molecular biology of the human CYP2C subfamily. Pharmacogenetics 4:285–299
Goldstein J, Faletto MB, Romkes-Sparks M, Sullivan T, Kitareewan S, Rancy L, Laker JM, Ghanayem BI (1994) Evidence that CYP2C 19 is the major (S)mephenytoin 4′-hydroxylase in humans. Biochemistry 33:1743–1752
Knodell RG, Dubey RK, Wilkinson GR, Guengerich FP (1988) Oxidative metabolism of hexobarbital in human liver: relationship to polymorphic S-mephenytoin 4′-hydroxylation. J Pharmacol Exp Ther 245:845–849
Kroemer HK, Echizen H, Heidemann H, Eichelbaum M (1992) Predictability of the in vivo metabolism of verapamil from in vitro data: contribution of individual metabolic pathways and stereoselective aspects. J Pharmacol Exp Ther 260:1052–1057
Kroemer HK, Gautier JC, Beaune P, Henderson C, Wolf CR, Eichelbaum M (1993) Identification of P450 enzymes involved in metabolism of verapamil in humans. Naunyn-Schmiedeberg's Arch Pharmacol 348:332–337
Lecoeur S, Bonierbale E, Challine D, Gautier JC, Valadon P, Dansette PM, Catinot R, Ballet F, Mansuy D, Beaune PH (1994) Specificity of in vitro covalent binding of tienilic acid metabolites to human liver microsomes in relationship to the type of hepatotoxicity: comparison with two directly hepatotoxic drugs. Chem Res Toxicol 7:434–442
Leemann T, Transon C, Dayer P (1993) Cytochrome P450TB (CYP2C): a major monooxygenase catalyzing diclofenac 4′-hydroxylation in human liver. Life Sci 52:29–34
Leo MA, Lasker JM, Raucy JL, Kim C-I, Black M, Lieber CS (1989) Metabolism of retinol and retinoic acid by human liver cytochrome P450IIC8. Arch Biochem Biophys 269:305–312
Lindholm A, Henrisson S (1987) Verapamil inhibits cyclosporine metabolism. Lancet 1:1262–1263
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with folin phenol reagent. J Biol Chem 193:265–275
Meier UT, Meyer UA (1987) Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4′-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency. Biochemistry 26:8466–8474
Mikus G, Eichelbaum M, Fischer C, Gumulka S, Klotz U, Kroemer HK (1990) Interaction of verapamil and cimetidine: stereochemical aspects of drug metabolism, drug disposition and drug action. J Pharmacol Exp Ther 253:1042–1048
Miners JO, Smith KJ, Robson RA, McManus ME, Veronese ME, Birkett DJ (1988) Tolbutamide hydroxylation by human liver microsomes. Kinetic characterisation and relationship to other cytochrome P-450 dependent xenobiotic oxidations. Biochem Pharmacol 37:1137–1144
Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Estabrook RW, Feyereisen R, Gonzales FJ, Coon MJ, Gunsalus JC, Gotoh O, Okuda K, Nebert DW (1993) The P450 superfamily: update on new sequences, gene mapping, acession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol 12:1–51
Omura T, Sato R (1964) The carbon monoxide binding pigment of liver microsomes. J Biol Chem 239:2370–2378
Relling MV, Aoyama T, Gonzalez FJ, Meyer UA (1990) Tolbutamide and mephenytoin hydroxylation by humam cytochrome P 450s in the CYP2C subfamily. J Pharmacol Exp Ther 252:442–447
Rettie AE, Korzekwa KR, Kunze KL, Lawrence RF, Eddy AC, Aoyama T, Gelboin HV, Gonzalez FJ, Trager WF (1992) Hydroxylation of warfarin by human cDNA-expressed cytochrome P-450: A role for P-4502C9 in the etiology of (S)-warfarin-drug interactions. Chem Res Toxicol 5:54–59
Rettie AE, Wienkers LC, Gonzalez FJ, Trager WF, Korzekwa KR (1994) Impaired (S)-warfarin metabolism catalysed by the R144C allelic variant of CYP2C9. Pharmacogenetics 4:39–42
Romkes M, Faletto MB, Blaisdell JA, Raucy JL, Goldstein JA (1991) Cloning and expression of complementary DNAs for multiple members of the human cytochrome P450IIC subfamily. Biochemistry 30:3247–3255
Rutledge DR, Pieper JA, Mirvis DM (1988) Effects of chronic phenobarbital on verapamil disposition in humans. J Pharmacol Exp Ther 246:7–13
Srivastava PK, Yun C-H, Beaune PH, Ged C, Guengerich FP (1991) Separation of human liver microsomal tolbutamide hydroxylase and S-mephenytoin cytochrome P450 enzymes. Mol Pharmacol 40:69–79
Veronese ME, Mackenzie PI, Doecke CJ, McManus ME, Miners JO, Birkett DJ (1991) Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9. Biochem Biophys Res Commun 175:1112–1118
Veronese ME, Doecke CJ, Mackenzie PI, McManus ME, Miners JO, Rees DLP, Gasser R, Meyer UA, Birkett DJ (1993) Site-directed mutation studies of human liver cytochrome P450 isoenzymes in the CYP2C subfamily. Biochemistry 289:533–538
Wilkinson L (1986) SYSTAT: the system for statistics. SYSTAT Inc., Evanston, IL, USA
Wilkinson GR, Guengerich FP, Branch RA (1992) Genetic polymorphism of S-mephenytoin hydroxylation. In: Kalow W (ed) Pharmacogenetics of drug metabolism. Pergamon, New York, pp 657–680
Wrighton SA, Stevens JC, Becker GW, Vanden Branden M (1993) Isolation and characterization of human liver cytochrome P4502C19: correlation between 2C19 and S-mephenytoin 4′-hydroxylation. Arch Biochem Biophys 306:240–245
Yasumori T, Murayama N, Yamazoe Y, Kato R (1990) Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2. Clin Pharmacol Ther 47:313–322
Yasumori T, Yamazoe Y, Kato R (1991) Cytochrome P450 human-2 (P-450IIC9) in mephenytoin hydroxylation polymorphism in human livers: differences in substrate and stereoselectivities among microheterogeneous P-450IIC species expressed in yeasts. J Biochem 109:711–717
Zhao J, Leemann T, Dayer P (1992) In vitro oxidation of oxicam NSAIDS by a human liver cytochrome P450. Life Sci 51:575–581
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Busse, D., Cosme, J., Beaune, P. et al. Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans. Naunyn-Schmiedeberg's Arch Pharmacol 353, 116–121 (1995). https://doi.org/10.1007/BF00168924
Issue Date:
DOI: https://doi.org/10.1007/BF00168924