Prasugrel Intermediate Metabolite Modulates Platelet Inhibition by Negatively Interfering With an Active Metabolite: An Ex Vivo, In Vitro, and In Silico Study

Background: Prasugrel is converted into prasugrel active metabolite (PAM; R-138727) through the cytochrome P450-mediated conversion of an intermediate metabolite (PIM; R-95913). It is unknown whether PIM exerts any biological function. The FABOLUS-FASTER trial showed that chewed prasugrel does not improve bioactivity, in spite of accelerated PAM kinetics. Methods: PIM and PAM pharmacokinetics were assessed by mass spectrometry in blood samples collected from ST-segment-elevation myocardial infarction patients randomized to chewed (n=17) or integral (n=15) 60 mg prasugrel. The ex vivo and in vitro effects of PAM and PIM were assessed on ADP-induced platelet activation. The binding sites of PIM and PAM were investigated by molecular dynamics simulation. Results: Chewed prasugrel was associated with higher PIM levels compared with integral prasugrel: PIM median area under the curve (25-75 p): 73 (41.5-92.0) versus 33 (0.0-50.0) ng·h/mL (P<0.05). PIM plasma concentrations negatively correlated with inhibition of ADP-induced platelet aggregation, which strongly correlated to the PAM/PIM ratio (ρ=0.782; P<0.001; n=30) than PAM, suggesting an antagonistic role of PIM on PAM-induced P2Y12 inhibition. Subsequent in vitro tests confirmed the dose-dependent, reversible antagonistic effect of PIM on PAM inhibition of aggregation (maximum effect, -49.5% [95% CI, -54.4% to -44.6%]; P<0.001), confirmed by P-selectin expression and vasodilator-stimulated phosphoprotein phosphorylation as readouts at the signaling level. At molecular dynamics simulations of the drug-receptor systems, PIM accommodates through noncovalent reversible binding in the same PAM-binding site, distinct from that of 2-methylthio-adenosine-5'-diphosphate. Conclusions: PIM negatively interferes with PAM, thereby reducing its inhibitory activity, likely competing at the P2Y12 receptor-binding site. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02978040. URL: https://www.clinicaltrialsregister.eu; Unique identifier: EudraCT 2017-001065-24.