Impact of endogenous esterase activity on in vitro p-glycoprotein profiling of dabigatran etexilate in Caco-2 monolayers

• Published in: Drug metabolism and disposition 2014-02, Vol.42 (2), p.250-256
• Main Authors: Ishiguro, Naoki, Kishimoto, Wataru, Volz, Astrid, Ludwig-Schwellinger, Eva, Ebner, Thomas, Schaefer, Olaf
• Format: Article
• Language: English
• Subjects: Antithrombins - metabolism; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; Benzimidazoles - metabolism; Biological Transport; Biotransformation; Caco-2 Cells; Carboxylesterase - antagonists & inhibitors; Carboxylesterase - metabolism; Carboxylic Ester Hydrolases - antagonists & inhibitors; Carboxylic Ester Hydrolases - metabolism; Dabigatran; Enzyme Inhibitors - pharmacology; Humans; Hydrolysis; Intestines - drug effects; Intestines - enzymology; Kinetics; Liver - enzymology; Permeability; Prodrugs - metabolism; Pyridines - metabolism
• ISSN: 0090-9556; 1521-009X
• DOI: 10.1124/dmd.113.053561

Dabigatran etexilate, a double prodrug of dabigatran, is a reversible, competitive, direct thrombin inhibitor that has been approved for use in many countries. A recent guideline from the European Medicines Agency on drug-drug interactions proposed dabigatran etexilate as a sensitive in vivo and in vitro probe substrate for intestinal P-glycoprotein (P-gp) inhibition. We therefore performed a series of in vitro studies to determine the best experimental conditions for evaluation of P-gp involvement on the transport process of dabigatran etexilate across colorectal adenocarcinoma Caco-2 cell monolayers. Experiments using expressed carboxylesterase 1 (CES1) and CES2 bactosomes revealed that dabigatran etexilate was hydrolyzed into BIBR 1087 by CES1 expressed in our Caco-2 cells. The impact of CES1-mediated BIBR 1087 formation during transcellular transport experiments was assessed by comparing several combinations of three experimental approaches: radioactivity detection using [(14)C]dabigatran etexilate as substrate, liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of dabigatran etexilate, and in the presence and absence of a CES inhibitor bis(p-nitrophenyl) phosphate (BNPP). The experimental approach that was based on the use of nonlabeled dabigatran etexilate together with LC-MS/MS quantification and the addition of BNPP was selected as the most favorable condition in which to correctly evaluate the permeability coefficient (Papp) of dabigatran etexilate and its transcellular transport by P-gp. The in vitro Caco-2 study at the selected condition revealed that dabigatran etexilate is a P-gp substrate with an efflux ratio of 13.8 and an intrinsic Papp, which is the Papp under the condition of complete blockage of P-gp by P-gp inhibitor, of 29 × 10(-6) cm/s.