The Antiaggregating Activity of Clopidogrel Is due to a Metabolic Activation by the Hepatic Cytochrome P450-1A

Summary

Clopidogrel and ticlopidine are two well known selective anti-ADP agents which are inactive in vitro and must be administered in vivo to fully exhibit their antiaggregating and antithrombotic effects. Since previous studies have clearly demonstrated that the activation steps take place in the liver, we examined the effect of specific induction or inhibition of cytochrome P450 subfamilies on the antiaggregating activity of clopidogrel. SKF 525-A, a global cytochrome P450 inhibitor, dramatically decreased the antiaggregating effect of clopidogrel, therefore indicating that cytochrome P450 enzymes are involved in the hepatic activation of clopidogrel. The efficacy of clopidogrel was increased in animals pretreated with 3-methylcholanthrene and (3-naphthoflavone, indicating that the cytochrome P450-1A subfamily pathway was mainly involved in the activating metabolism of clopidogrel. The use of specific antibodies directed against the various cytochrome P450 subfamilies ascertained this observation.

• References

• 1 Panak E, Maffrand JP, Picard-Fraire C, Vallee E, Blanchard J, Roncucci R. Ticlopidine, a promise for the prevention and treatment of thrombosis and its complications. Haemostasis 1983; 13: 1-52
• 2 ThÉbault JJ, Blatrix CE, Blanchard J, Panak E. Effects of Ticlopidine, a new platelet aggregation inhibitor in man. Clin Pharmacol Ther 1975; 18: 485-490
• 3 DelebassÉe D, Bernat A, Frehel D, Defreyn G, Maffrand JP. Antiaggregating and antithrombotic effects in the rat of two thienopyridine enantiomers structurally related to ticlopidine. Xth International congress on thrombosis; Athens: 1988. Abst 197
• 4 Savi P, Herbert JM, Pflieger AM, Dol F, DelebassÉe D, Combalbert J, Defreyn G, Maffrande JP. Importance of hepatic metabolism in the antiaggregating activity of thienopyridine clopidogrel. Biochem Pharmacol 1992; 44: 527-532
• 5 Nebert DW, Nelson DR, Coon MJ, Estabrook RW, Feyereisen R, Fujii-Kuriyama Y, Gonzalez FJ, Gengerich FP, Gunsalus IC, Johnson EF, Loper JC, Sato R, Waterman MR, Waxman DJ. The P450 superfamily: update on new sequences, gene mapping, a recommended nomenclature. DNA Cell Biol 1991; 10: 1-14
• 6 Porter TD, Coon MJ. Cytochrome P450: Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem 1991; 266: 13469-13472
• 7 Combalbert J, Fabre I, Fabre G, Dalet I, Derancourt J, Cano JP, Maurel P. Metabolism of cyclosporin A; IV. Purification and identification of rifampicin-inductible human liver cytochrome P450 (Cyclosporine A oxidase) as a product of P45QIIIA gene subfamily. Drug Metab Djspos 1989; 17: 197-207
• 8 Guengerich FP. Oxidatiop of 17α-ethynylestradiol by human liver cytochrome P450. Mol Pharmacol 1988; 33: 500-508
• 9 Geneve J, Larrey D, Lettreton P, Descatoire V, Tinel M, Amouyal G, Pessayre D. Metabolic activation of antidepressant amineptine: Cytochrome P450 mediated in vitro covalent binding. Biochem Pharmacol 1987; 36: 323-329
• 10 Pessayre D, Wandscheer JC, Descatoire V, Dolder A, Degott C, Benhamou JP. Cu mulative effects of repeated doses of compounds transformed into reactive metabolites. Biochem Pharmacol 1980; 29: 1041-1047
• 11 Kronbach T, Mathys D, Umeno M, Gonzalez FJ, Meyer UA. Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol 1989; 36: 89-96
• 12 Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ. Characterization of rat and human liver cytochrome P450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 1986; 261: 5051-5060
• 13 Lopez-Garcia MP, Dansette P, Valadon P, Amar C, Beaune P, Guengerich FB, Man-suye D. Human liver cytochrome P450 expressed in yeast as tools for reactive activation formation studies. Oxidative activation of tienilic acid by cytochrome P450 2C9 and 2C10. Eur J Biochem 1993; 213: 223-232
• 14 Born GV. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature (Lond) 1962; 194: 927-929
• 15 Pollard HB, Menard R, Brandt HA, Pazoles CJ, Creutz CE, Ramu A. Application of Bradford’s protein assay to adrenal gland subcellular fractions. Anal Biochem 1978; 86: 761-763
• 16 Omura T, Sato R. Carbon monoxide binding pigment of liver microsomes. J Biol Chem 1974; 239: 2370-2378
• 17 Burke MD, Mayer RT. Differential effects of phenobarbitone and 3-methyl-cholan-threne induction on the hepatic microsomal metabolism and cytochrome P450 binding of phenoxazone and homologous series of its n-alkyl ethers (alkoly resorufins). Chem Biol Interact 1983; 45: 243-247
• 18 Murray S, BoobisE AR. An assay for paracetamol, produced by O-deethylation of phenacetine in vitro, using gas chromatography/electron capture negative ion chemical ionization mass spectrometry. Biomed Environ Mass Spec 1986; 13: 91-93
• 19 Guengerich FP, Dannan GA, Wright ST, Martin MV, Kaminsky LS. Purification and characterization of liver microsomal cytochrome P450. Electrophoretic, spectral, catalytic and immunological properties and inducibility of eight isozymes isolated from rats treated with phenobarbital and β-naphthoflavone. Biochemistry 1982; 21: 6019-6030
• 20 Nash T. The colorimetric estimation of formaldehyde by means of Manzech reaction. Biochem J 1953; 55: 416-421
• 21 Mieyal JJ, Blumers JL. Acceleration of the autooxidation of human oxyhemoglobin by aniline and its relation to hemoglobin-catalysed aniline hydroxylation. J Biol Chem 1976; 252: 3442-3446
• 22 Liu G, Gelboin HV, Myers MJ. Role of cytochrome P450IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies. Arch Biochem Biophys 1991; 284: 400-406
• 23 Sesardic D, Edwards RJ, Davies DS, Thomas PE, Levin W, Boobis AR. High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat. Biochem Pharmacol 1990; 39: 489-498
• 24 Ornate Y, Friedman FK. Cytochrome P450-benzphetamine interactions in the endoplasmic reticulum: studies using monoclonal antibody to P450b. Biochemistry 1992; 31: 8862-8867
• 25 Waltz FG, Vlasuk GP, Omiecinski CJ, Bresnick E, Thomas PE, Ryan DE, Levin W. Multiple immunoidentical forms of phenobarbital-induced rat liver cytochrome P450 are encoded by different mRNAs. J Biol Chem 1982; 257: 4023-4026
• 26 Srivastava G, Bawden MJ, Anderson A, May BK. Drug induction of P450IIB1/IIB2 and 5-aminolevulinate synthase mRNAs in rat tissues. Biochem Biophys Acta 1989; 1007: 192-195
• 27 Leighton JK, DeBrunner-Vossbrinck BA, Kemper B. Isolation and sequence analysis of three cloned cDNAs for rabbit liver proteins that are related to rabbit cytochrome P450 (form 2), the major phenobarbital-inducible form. Biochemistry 1984; 23: 204-210
• 28 Raucy J, Fernandes P, Black M, Yang SL, Koop DR. Identification of human liver cytochrome P450 exhibiting catalytic and immunochemical similarities to cytochrome P450-3a of rabbit liver. Biochem Pharmacol 1987; 36: 2921-2926
• 29 Ingelman-Sundberg M, Johansson I, Hansson A. Catalytic properties of the liver microsomal hydrolase system in reconstituted phospholipids vesicles. Acta Biol Med Germ 1979; 38: 379-382
• 30 Daujat M, Pichard L, Dalet C, Larroque C, Bonfils C, Pompon D, Li D, Guzelian PS, Maurel P. Expression of five forms of microsomal cytochrome P450 in primary cultures of rabbit hepatocytes treated with various classes of inducers. Biochem Pharmacol 1987; 36: 3597-3606
• 31 Distlerath LM, Guengerich FP. Enzymology of human liver cytochrome P450. In: Mammalian cytochromes P450, volume I CRC Press Inc; eds 1987
• 32 Maffrand JP, Defreyn G, Bernat A, Delebassee D, Tissinier AM. Reviewed pharmacology of ticlopidine. Angiologie 1988; 77: 6-13
• 33 Herbert JM, Frehel D, Vallee E, Kieffer G, Gouy D, Berger Y, Defreyn G, Maffrand JP. Clopidogrel, a novel antiplatelet and antithrombotic agent. Cardiovasc Drug Rev, in press