Dissecting the Activation of Thienopyridines by Cytochromes P450 Using a Pharmacodynamic Assay In Vitro

The thienopyridine antiplatelet drugs, such as ticlopidine, clopidogrel, and prasugrel, require activation by cytochromes P450 in vivo to effectively block platelet aggregation. The study of the metabolic activation of these compounds has been hampered by the lability and reactivity of the ring-opened active metabolite (AM) and by the numerous metabolites that can be formed in such a transformation. We have developed a novel method whereby platelets are incubated with the cytochrome P450 and the thienopyridine of interest for various amounts of time, and the effects on ADP-driven platelet aggregation are directly examined. In this way, the platelet is used as a biosensor for detection of the AM. Using this method, cytochromes P450 capable of converting clopidogrel, prasugrel, and 2-oxo-clopidogrel to metabolites that inhibit ADP-induced platelet aggregation were identified as well as which cytochromes P450 were capable of catalyzing partial reactions (e.g., conversion of 2-oxo-clopidogrel to the AM). These studies show that, in vitro, CYP3A4/5, 2C19, and 2B6 are individually capable of converting clopidogrel and prasugrel to the AM and that the cytochrome P450 preference for these two thienopyridines is very similar.