Identification of Esterases Expressed in Caco-2 Cells and Effects of Their Hydrolyzing Activity in Predicting Human Intestinal Absorption

Imai, Teruko; Imoto, Masumi; Sakamoto, Hisae; Hashimoto, Mitsuru

doi:10.1124/dmd.105.004226

Cited by 100 publications

(94 citation statements)

References 21 publications

(25 reference statements)

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“…As shown in Fig. 3, Caco-2 cells used for dabigatran etexilate evaluation showed higher CES1 expression compared with CES2, comparable to previously reported results (Imai et al, 2005). Therefore, it is reasonable to hypothesize that CES1-catalyzed BIBR 1087 formation occurs during the course of the transcellular transport experiment.…”

Section: Resultssupporting

confidence: 76%

“…Formation of BIBR 1087 from dabigatran etexilate occurred by both nonenzymatic hydrolysis and CES1-catalyzed hydrolysis, whereas formation of BIBR 951 was catalyzed by CES2 only. Caco-2 cells are reported to express CES1 rather than CES2 (Imai et al, 2005), and the level of mRNA expression of CES1 and CES2 in Caco2 cells cultured on transwell filters for 15 to 16 days was investigated. As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Considering its double prodrug nature, a thorough assessment and understanding of in vitro P-gp interaction of dabigatran extexilate are required because the results may trigger extensive clinical DDI assessment. Caco-2 cells express CES1 (Imai et al, 2005), unlike the human small intestine or colon tissue, where CES2 is expressed and dabigatran etexilate requires esterase activity to form pharmacologically active dabigatran. In vitro metabolism studies using CES1/2 bactosomes revealed that the two intermediate prodrugs are formed both by nonenzymatic and CES1-catalyzed hydrolysis (BIBR 1087) and by CES2 only (BIBR 951).…”

Section: Impact Of Esterases On P-gp Profiling Of Dabigatran Etexilatmentioning

confidence: 99%

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Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

et al. 2013

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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 chromatographytandem 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 3 10 26 cm/s.

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Impact Of Esterases On P-gp Profiling Of Dabigatran Etexilatmentioning

confidence: 99%

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Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

et al. 2013

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“…ximelagatran administration . The formation of OH-melagatran and melagatran during passage through Caco-2 cell monolayers can be explained by the presence of hCE-1 in these cells (Imai et al, 2005;Eriksson et al, 2006). In pigs, the intestinal and liver CES isoforms are close to identical, exhibiting 77 and 76% sequence identity with hCE-1, respectively, and 47 and 46% sequence identity with hCE-2 (Musidlowska- Persson and Bornscheuer, 2003).…”

Section: Discussionmentioning

confidence: 99%

Intestinal and Hepatobiliary Transport of Ximelagatran and Its Metabolites in Pigs

Sjödin¹,

Fritsch²,

Eriksson³

et al. 2008

Drug Metab Dispos

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ABSTRACT:The direct thrombin inhibitor melagatran is formed from ximelagatran via two intermediate metabolites, OH-melagatran and ethylmelagatran. The biotransformation of ximelagatran does not involve cytochrome P450 isoenzymes, and it has been suggested that a reported interaction with erythromycin may instead be mediated by transport proteins. A pig model that simultaneously enables bile collection, sampling from three blood vessels and perfusion of a jejunal segment, was used to investigate the biotransformation of ximelagatran and the effect of erythromycin on the intestinal and hepatobiliary transport of ximelagatran and its metabolites. The pigs received enteral ximelagatran (n ‫؍‬ 6), enteral ximelagatran together with erythromycin (n ‫؍‬ 6), i.v. ximelagatran (n ‫؍‬ 4), or i.v. melagatran (n ‫؍‬ 4). The plasma exposure of the intermediates was found to depend on the route of ximelagatran administration. Erythromycin increased the area under the plasma concentration-time curve (AUC) of melagatran by 45% and reduced its biliary clearance from 3.0 ؎ 1.3 to 1.5 ؎ 1.1 ml/min/kg. Extensive biliary exposure of melagatran and ethylmelagatran, mediated by active transport, was evident from the 100-and 1000-fold greater AUC, respectively, in bile than in plasma. Intestinal efflux transporters seemed to be of minor importance for the disposition of ximelagatran and its metabolites considering the high estimated f abs of ximelagatran (80 ؎ 20%) and the negligible amount of the compounds excreted in the perfused intestinal segment. These findings suggest that transporters located at the sinusoidal and/or canalicular membranes of hepatocytes determine the hepatic disposition of ximelagatran and its metabolites, and are likely to mediate the ximelagatran-erythromycin pharmacokinetic interaction.Ximelagatran was the first oral direct thrombin inhibitor to reach the market. As an oral anticoagulant, it had the potential to become an alternative to warfarin and other vitamin K antagonists; however, because of safety concerns involving hepatotoxicity, ximelagatran was withdrawn in 2006. Warfarin currently has an important role in the prevention and treatment of thromboembolic events but is unfortunately also associated with a variable pharmacokinetic and pharmacodynamic response, delayed onset and offset of action, numerous drug-drug interactions, and serious safety issues (Hawkins, 2004;Greenblatt and von Moltke, 2005). The narrow therapeutic index of warfarin and the many sources of variability require monitoring of the anticoagulant effect to maintain blood levels within the therapeutic range. Because of these difficulties and the severe consequences of thrombosis, it is clear that there is a need for new oral anticoagulants. Ximelagatran was developed to improve the low and highly variable bioavailability of the reversible, direct thrombin inhibitor melagatran (Gustafsson et al., 2001). The hydrophilic carboxylic acid and amidine functions of melagatran were protected by introducing an ethylester residue and the less ...

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“…CES1 has been previously biochemically characterized as an oseltamivir-activating enzyme, yet human CES1 polymorphisms do not account for all of the variability in patient prodrug activation 27 . Under the background of this model, we sought to establish if substrate-competitive activity-based profiling could directly annotate oseltamivir-activating enzymes among serine hydrolases present in human Caco-2 cell homogenates, a cell line that expresses multiple carboxylesterases 28 and is widely used for modeling human intestinal absorption 29 . Using this system, FP-PEG-TAMRA completely blocked oseltamivir activation, confirming members of the serine hydrolase enzyme family are solely responsible for ethyl ester hydrolysis ( Figure 1B).…”

Section: Substrate-competitive Profiling Of Oseltamivir-binding Serinmentioning

confidence: 99%

Substrate-competitive activity-based profiling of ester prodrug activating enzymes

Martin

2018

Drug Metabolism and Pharmacokinetics

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Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating pre-clinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporterlinked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a 4-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse, but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substratecompetitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design and preclinical development of ester prodrugs. HHS Public Access

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Identification of Esterases Expressed in Caco-2 Cells and Effects of Their Hydrolyzing Activity in Predicting Human Intestinal Absorption

Cited by 100 publications

References 21 publications

Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

Impact of Endogenous Esterase Activity on In Vitro P-Glycoprotein Profiling of Dabigatran Etexilate in Caco-2 Monolayers

Intestinal and Hepatobiliary Transport of Ximelagatran and Its Metabolites in Pigs

Substrate-competitive activity-based profiling of ester prodrug activating enzymes

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