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

Figures

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  Figure 1.. Pharmacokinetics and pharmacodynamics of prasugrel loading dose in STEMI (ST-segment–elevation myocardial infarction) patients as assessed in the FABOLUS-FASTER trial (Facilitation Through Aggrastat or Cangrelor Bolus and Infusion Over Prasugrel: A Multicenter Randomized Open-Label Trial in Patients With ST-Elevation Myocardial Infarction Referred for Primary Percutaneous Intervention). Plasma concentrations of prasugrel active metabolite (PAM) and prasugrel inactive metabolite (PIM) were determined at predefined time points after the oral administration of 60 mg prasugrel as integral (n=15) or chewed (n=17) tablets in patients with STEMI (A). Significance of differences between chewed and integral tablets at different time points was evaluated by the Mann-Whitney rank-sum test, using Sidak correction to adjust for multiple testing bias. For graphical purposes, data are expressed as mean±SEM. Maximum concentration (Cmax; B) and area under the curve (AUC; C) of PAM and PIM, grouped according to the administration of integral or chewed prasugrel. Individual data and median are shown. The Mann-Whitney U test was applied. Relationships between PAM and PIM AUCs and inhibition of…

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  Figure 2.. Individual prasugrel active metabolite (PAM)/prasugrel inactive metabolite (PIM) ratio towards maximum inhibition of platelet aggregation (4–6 hours after loading dose prasugrel). A, ADP 20 µmol/L as agonist; B, ADP 5 µmol/L as agonist. PAM/PIM ratio was calculated from the concentrations of the 2 compounds measured in samples obtained 2 or 3 hours after the oral loading dose of prasugrel (n=30 at each time point). Spearman correlation was applied. ADP indicates adenosine diphosphate.

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  Figure 3.. Effects of ascending concentrations of prasugrel active metabolite (PAM; 60–500 ng/mL) and prasugrel inactive metabolite (PIM; 60–1000 ng/mL) added in vitro to platelet-rich plasma before platelet aggregation was induced by 5 µmol/L ADP (adenosine diphosphate; n=32). The effects of PAM are shown in A; the effects of PIM are shown in B. Recorded traces are shown as mean±SE of 5 to 12 independent experiments. PAM of 125 to 500 ng/mL significantly inhibited platelet aggregation (P<0.001). Mean difference and 95% CI of differences in platelet aggregation observed in the presence of PIM from platelet aggregation with PAM alone are presented in boxes. Two-way ANOVA was applied to analyze aggregation traces. Effects of high-concentration (500 ng/mL) PIM on ADP-induced expression of P-selectin (C), expression of active fibrinogen receptor (D), and vasodilator-stimulated phosphoprotein (VASP) phosphorylation (E). Experiments (n=9 in each condition) were performed in the presence of acetylsalicylic acid. Student t test for paired data was applied. Individual data are shown. MFI indicates mean fluorescent intensity.

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  Figure 4.. Effects of prasugrel inactive metabolite (PIM; 60–500 ng/mL) added in vitro to platelet-rich plasma 10 minutes before prasugrel active metabolite (PAM; 500, 250, and 125 ng/mL) and effects of PIM (60–500 ng/mL) added to platelet-rich plasma 15 minutes after PAM (500, 250, and 125 ng/mL; n=32). The effects of PIM before PAM 500, 250, and 125 ng/mL are shown in A, B, and C, respectively. The effects of PIM after PAM 500, 250, and 125 ng/mL are shown in D, E, and F, respectively. Platelet aggregation was induced by 5 µmol/L ADP (adenosine diphosphate). Recorded traces are shown as mean±SE of 5 to 9 independent experiments in each set. Two-way ANOVA, followed by Tukey multiple comparison test, was applied to analyze maximum aggregation (last 30 s of the aggregation traces). Mean of differences and 95% confidence interval of differences from platelet aggregation with PAM alone observed in the presence of PIM are presented in boxes. *P<0.05, **P<0.001.

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  Figure 5.. Effects of 500 ng/mL prasugrel inactive metabolite (PIM) added before 500 ng/mL prasugrel active metabolite (PAM) to PRP on adenosine diphosphate (ADP)-induced vasodilator-stimulated phosphoprotein phosphorylation (P-VASP) and P-selectin expression on platelets. A, VASP phosphorylation was abolished by ADP (high PRI), increased by PAM and blunted by preincubation of platelets with PIM before PAM. C, Basal expression of P-selectin increase on cell membrane in 5 µmol/L ADP-stimulated platelets was blunted by PAM. Experiments were performed in the presence of acetylsalicylic acid. In the presence of PIM, a significant reduction in the activities of PAM was observed. Changes (%) in VASP phosphorylation and P-selectin expression in the presence of PIM are shown (B and D). Individual data and mean values from independent experiments, each including all the tested conditions, are shown. Repeated measures ANOVA with Geisser-Greenhouse correction, followed by Tukey multiple comparison test (A and C) and Student t test for paired data (B and D) was applied (n=8–9). MFI indicates mean fluorescent intensity.

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  Figure 6.. Structural in silico analysis of inactive and active metabolites of prasugrel. A, General view of the position of PAM-C973.25 (green), prasugrel inactive metabolite (PIM; magenta) after MD simulations and crystallographic 2-methylthio-adenosine-5′-diphosphate (2MeS-ADP), a close analogue of ADP (yellow): (A) left, side view; right, apical view; (B) close view. PAM indicates prasugrel active metabolite.