Genetic polymorphisms and the impact of a higher clopidogrel dose regimen on active metabolite exposure and antiplatelet response in healthy subjects.

A double-blind crossover study was conducted in four CYP2C19 genotype-defined metabolizer groups to assess whether increase in clopidogrel dosing can overcome reduced pharmacodynamic response in CYP2C19 poor metabolizers (PMs). Ten healthy subjects in each of four metabolizer groups were randomized to a clopidogrel regimen of a 300-mg loading dose (LD) and a 75-mg/day maintenance dose (MD) for 4 days followed by 600-mg LD and 150 mg/day MD, or vice versa. The exposure levels of clopidogrel's active metabolite H4 (clopi-H4) in PMs were 71% lower on the 75-mg/day regimen and 64% lower on the 150-mg/day regimen than the corresponding exposure levels in extensive metabolizers (EMs). In PMs, the maximal platelet aggregation (MPA) induced by adenosine diphosphate (ADP) 5 µmol/l was 10.5% lower on the 75-mg/day regimen and 7.9% lower on the 150-mg/day regimen than the corresponding values in EMs. PMs who were on the clopidogrel regimen of 600-mg LD/150 mg/day MD showed clopi-H4 exposure and MPA levels similar to those in EMs who were on the regimen of 300-mg LD/75 mg/day MD. In a pooled analysis evaluating CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP3A5, CYP2D6, ABCB1, and P2RY12 polymorphisms (N = 396 healthy subjects), only CYP2C19 had a significant impact on antiplatelet response. In healthy CYP2C19 PMs, a clopidogrel regimen of 600-mg LD/150 mg/day MD largely overcomes diminished clopi-H4 exposure and antiplatelet response, as assessed by MPA levels.

Differential effects of omeprazole and pantoprazole on the pharmacodynamics and pharmacokinetics of clopidogrel in healthy subjects: randomized, placebo-controlled, crossover comparison studies.

Four randomized, placebo-controlled, crossover studies were conducted among 282 healthy subjects to investigate whether an interaction exists between clopidogrel (300-mg loading dose/75-mg/day maintenance dose) and the proton-pump inhibitor (PPI) omeprazole (80 mg) when they are administered simultaneously (study 1); whether the interaction, if any, can be mitigated by administering clopidogrel and omeprazole 12 h apart (study 2) or by increasing clopidogrel to 600-mg loading/150-mg/day maintenance dosing (study 3); and whether the interaction applies equally to the PPI pantoprazole (80 mg) (study 4). Relative to levels after administration of clopidogrel alone in studies 1,2,3, and 4, coadministration of PPI decreased the AUC(0-24) of the clopidogrel active metabolite H4 by 40, 47, 41, and 14% (P ≤ 0.002), respectively; increased maximal platelet aggregation (MPA) induced by 5 micromol/l adenosine diphosphate (ADP) by 8.0, 5.6, 8.1, and 4.3% (P ≤ 0.014), respectively; and increased the vasodilator-stimulated phosphoprotein phosphorylation-platelet reactivity index (VASP-PRI) by 20.7, 27.1, 19.0 (P < 0.0001), and 3.9% (P = 0.3319), respectively. The results suggest that a metabolic drug-drug interaction exists between clopidogrel and omeprazole but not between clopidogrel and pantoprazole.

Comparison of the stability of some major cytochrome P450 and conjugation reactions in rat, dog and human hepatocyte monolayers.

The stability of four major cytochrome P450 isoenzymes (CYPIA, CYP2B, CYP2E1 and CYP3A) and of two phase II conjugation enzymes (glucuronyl- and sulfotransferases) was investigated in primary cultures of rat, dog and human hepatocytes in the same conditions. 7-ethoxyresorufin deethylation (EROD), 7-methoxycoumarin demethylation (MCOD), chlorzoxazone (CLOX) 6-hydroxylation, 1'- and 4-hydroxylation of midazolam (MDZ), and p-nitrophenol glucuronidation and sulfation, were used respectively. The EROD activity was stable over 72 hours in rat and dog and only 48 hours in human hepatocytes. The MCOD activity was also stable in rat but decreased in dog by 30% within 72 hours The CLOX hydroxylase activity was most stable in human whereas in rat and dog it fell down to 30% within 72 and 24 hours, respectively. The MDZ hydroxylase activity showed the same unstability profile in the three species investigated. Both conjugation reactions were either stable or showed an increase by up to 60-70% in all three species over 72 hours. The enzymes tested showed different stabilities in rat, dog and human hepatocytes over 72 hours, thus demonstrating the limitations of hepatocyte monolayers as models for metabolic investigations and emphasising the need for validation/characterization studies before routine use.

Estimation of flavin-containing monooxygenase activity in intact hepatocyte monolayers of rat, hamster, rabbit, dog and human by using N-oxidation of benzydamine.

The flavin-containing monooxygenase (FMO)-dependent N-oxidation of benzydamine has been assessed as a method for monitoring the activity of FMOs in monolayer cultures of hepatocytes from rat, dog, rabbit, hamster and human. The advantage of this substrate is that benzydamine N-oxide formation can be measured directly in extracts of cellular incubations without an intensive work-up procedure. Benzydamine and its N-oxide are readily separated by HPLC with fluorometric detection. This assay proved sensitive enough to monitor FMOs activity in intact monolayer of cultured hepatocytes. The formation of benzydamine N-oxide was inhibited when hepatocytes were coincubated with methimazole (another FMO substrate) in a dose-dependent manner, whereas N-octylamine (an inhibitor of cytochrome P450) had no inhibitory effect. In contrast to cytochrome P450, FMO activity assessed by benzydamine N-oxidation was relatively stable for all species studied during 72-h cultures.