title: Impact of endogenous esterase activity on in vitro p-glycoprotein profiling of dabigatran etexilate in Caco-2 monolayers.
abstract: 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.
authors.affiliation: Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan (N.I., W.K., O.S.); and Drug Metabolism and Pharmacokinetics, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany (A.V., E.L., T.E.).
authors.forename: Naoki
authors.initials: N
authors.lastname: Ishiguro
authors.forename: Wataru
authors.initials: W
authors.lastname: Kishimoto
authors.forename: Astrid
authors.initials: A
authors.lastname: Volz
authors.forename: Eva
authors.initials: E
authors.lastname: Ludwig-Schwellinger
authors.forename: Thomas
authors.initials: T
authors.lastname: Ebner
authors.forename: Olaf
authors.initials: O
authors.lastname: Schaefer
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000991:Antithrombins / Q000378:metabolism*; D001562:Benzimidazoles / Q000378:metabolism*; D001692:Biological Transport; D001711:Biotransformation; D018938:Caco-2 Cells; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000069604:Dabigatran; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007422:Intestines / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010539:Permeability; D011355:Prodrugs / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism*
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000991:Antithrombins; D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D011355:Prodrugs; D011725:Pyridines; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; C517204:CES2 protein, human; D043182:Carboxylesterase; D000069604:Dabigatran