Abstract 272: In vitro Platelet Aggregation as a Biosensor for Clopidogrel Active Metabolite Formation
The thienopyridine anti-platelet drugs, such as clopidogrel, require metabolic activation in vivo to effectively block platelet aggregation. The study of the activation of these compounds has been hampered by the lability and reactivity of the ring-opened active metabolite. Many studies have relied solely on the disappearance of the parent drug as an indicator of bioactivation by a particular cytochrome P450 (CYP). We have developed an alternative method for studying the formation of the active metabolite. Conditions were optimized whereby washed human platelets can be incubated in the presence of an individual, recombinant CYP and clopidogrel. At various time points during this incubation, an aliquot is removed and platelet aggregation is measured using 2-(Methylthio)adenosine 5′-diphosphate (2MeSADP) or adenosine 5′-diphosphate (ADP) as the agonist. Inhibition of platelet aggregation, compared to the control lacking active enzyme, suggests the formation of the thienopyridine active metabolite and its reaction with the platelet P2Y12 receptor. The inhibition measured showed both a dependence on the concentration of clopidogrel and the concentration of the CYP. No inhibition of platelet aggregation was observed in this system when thrombin was used as the agonist. Of the CYP’s tested, CYP3A4, CYP2B6 and CYP2C19 showed increasing inhibition of platelet aggregation with time in this system while CYP1A2, CYP2D6 and CYP2C9 did not. The CYP3A4 reaction was inhibited by ketoconazole and by antibodies against CYP3A4. Increasing the concentration of CYP3A4 and CYP2B6 from 20 nM to 80 nM allowed for complete inhibition of platelet aggregation to be achieved over the 20 minute time course of the experiment. This level of inhibition was not achieved with CYP2C19 suggesting that CYP3A4 and CYP2B6 generate the thienopyridine active metabolite more efficiently than CYP2C19. While it is clear from these and other studies that CYP3A4 plays an important role in clopidogrel metabolism, it is now also clear that other cytochrome P450’s can convert clopidogrel to its active metabolite and could play a role in its conversion in vivo.