Abstract

Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted to the active thrombin inhibitor, dabigatran (DAB), by serine esterases. The aims of the present study were to detail the in vitro kinetics and pathway of DABE hydrolysis by human carboxylesterase enzymes, and the effect of alcohol on these transformations. The kinetics of DABE hydrolysis by two recombinant carboxylesterase enzymes (CES1 and CES2), and in human liver S9 were performed. The effects of alcohol (a known CES1 inhibitor) on the formation of DABE metabolites in carboxylesterase enzymes and human liver S9 were also examined. Because chemical standards were not commercially available for them, the two intermediate metabolites in the study samples were quantified by our recently developed standard-free quantitative-estimation approach. The inhibitory effect of bis(4-nitrophenyl)phosphate on the carboxylesterase-mediated metabolism of DABE, and alcohol on the hydrolysis of a classic carboxylesterase substrate (cocaine) were studied to validate our in vitro model. The ethyl ester of DABE was hydrolyzed exclusively by CES1 (Km 51 ± 12 μM, Vmax 794 ± 78 pmol/min/mg protein) and the carbamate ester of DABE was exclusively hydrolyzed by CES2 (Km 1.2 ± 0.2 μM, Vmax 70.3 ± 2.6 pmol/min/mg protein). In human liver S9 incubations containing both CES1 and CES2, DAB was formed in significant amounts, while only negligible quantities of DAB were formed in the individual incubations with CES1 or CES2. The results suggest sequential hydrolysis of DABE by intestinal CES2 and hepatic CES1 as the major pathway for the formation of the active metabolite (DAB) from DABE. Alcohol showed no inhibition of hydrolysis.