Abstract

Dabigatran etexilate (DABE), a double ester prodrug of dabigatran (DAB), is a probe substrate of intestinal P-glycoprotein (P-gp) commonly used in clinical drug-drug interaction (DDI) studies. When compared to its therapeutic dose at 150 mg, microdose DABE (375 µg) showed approximately 2-fold higher in DDI magnitudes with CYP3A/P-gp inhibitors. In this study, we conducted several in vitro metabolism studies to demonstrate that DABE at a theoretical gut concentration after microdosing significantly underwent NADPH-dependent oxidation (~40-50%) in parallel to carboxylesterase-mediated hydrolysis in human intestinal microsomes. Furthermore, NADPH-dependent metabolism of its intermediate monoester BIBR0951 was also observed in both human intestinal and liver microsomes, accounting for 100% and 50% of total metabolism, respectively. Metabolite profiling using LC-MS/MS confirmed the presence of several novel oxidative metabolites of DABE and of BIBR0951 in the NADPH-fortified incubations. CYP3A was identified as the major enzyme catalyzing the oxidation of both compounds. The metabolism of DABE and BIBR0951 was well described by Michaelis-Menten kinetics, with K_m ranging 1-3 µM, significantly below the expected concentrations following the therapeutic dose of DABE. Overall, the present results suggested that CYP3A played a significant role in the presystemic metabolism of DABE and BIBR0951 following microdose DABE administration, thus attributing partly to the apparent overestimation in the DDI magnitude observed with the CYP3A/P-gp inhibitors. Therefore, DABE at the microdose, unlike the therapeutic dose, would likely be a less predictive tool and should be considered as a clinical dual substrate for P-gp and CYP3A when assessing potential P-gp-mediated impacts by dual CYP3A/P-gp inhibitors.

Significance Statement This is the first study demonstrating a potentially significant role of CYP-mediated metabolism of the prodrug DABE following a microdose, but not a therapeutic dose. This additional pathway coupled with its susceptibility to P-gp may make DABE a clinical dual substrate for both P-gp and CYP3A at a microdose. The study also highlights the need for better characterization of the pharmacokinetics and metabolism of a clinical DDI probe substrate over the intended study dose range for proper result interpretations.