Evaluation of Transport Mechanism of Prodrugs and Parent Drugs Formed by Intracellular Metabolism in Caco-2 Cells with Modified Carboxylesterase Activity: Temocapril as a Model Case

Ohura, Kayoko; Nozawa, Takaaki; Murakami, Kyosuke; Imai, Teruko

doi:10.1002/jps.22628

Cited by 27 publications

(30 citation statements)

References 41 publications

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“…The Papp of BIBR 1087 at 10 mM was 0.89 Â 10 26 cm/s, and the efflux ratio was 1.00. The substantial formation of BIBR 1087 in the donor compartment of the AtoB assay could be explained by esterases expressed on the outer apical membranes of Caco-2 cells (Ohura et al, 2011), as BIBR 1087 will not pass the apical membranes once formed intracellularly as a result of the low Papp and not being a P-gp substrate. This explanation is supported by the fact that BIBR 1087 formation from dabigatran etexilate in human liver microsomes is not completely inhibited by BNPP, which is a selective CES inhibitor, but by paraoxon, which is a strong inhibitor of both CES and (Blech et al, 2008).…”

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

confidence: 99%

“…Dabigatran etexilate is thought to enter the cell as a result of its high Papp (29 Â 10 26 cm/s) ( Table 2) and is then intracellularly hydrolyzed by CES1 to BIBR 1087, which then slowly appears in the receiver compartment. BIBR 1087 was found in both compartments in the AtoB assay in the presence of BNPP, indicating that 200 mM BNPP is not sufficient to completely block esterase activity on the apical membrane of Caco-2 cells (Ohura et al, 2011). Therefore, an experimental condition using […”

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

confidence: 99%

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

confidence: 99%

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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“…The result also showed that the prodrugs are hydrolyzed not only during the permeation across the monolayer by cytosolic esterases, but also on both sides of the monolayer, presumably by secreted esterases. 20,21 Additionally, Caco-2 cells contain more esterases on the apical side than on the basolateral side because the prodrugs were hydrolyzed to a greater extent on the apical side than on the basolateral side. Prodrugs with short promieties such as acetyl 3 or branched chain promoieties such as isobutyryl 9 and 2-ethylbutyryl 10 were hydrolyzed to a lesser extent in these experiments.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis

Dawadi

Kawamura

Rubenstein

et al. 2016

Bioorganic & Medicinal Chemistry

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The nucleoside antibiotic, 5′-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and α-branched alkanoyl groups from two to twelve carbons at the 3′-position of a 2′-fluorinated analogue of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human~rat>>mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2 = 1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5–28 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.

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“…However, a parent drug formed by intestinal metabolism is transported into the lumen as well as the mesenteric vein. In addition, the transport rate through the luminal membrane is threefold to fourfold faster than through the basolateral membrane, resulting in low absorption of prodrug . Therefore, it is desired that the prodrug is resistant to intestinal metabolism, but can be easily converted into the parent drug by a hydrolytic enzyme abundantly expressed in the liver.…”

Section: Introductionmentioning

confidence: 99%

Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein

Ohura

Nakada

Kotani

et al. 2015

Journal of Pharmaceutical Sciences

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The aim of this study was to develop a suitable prodrug for fexofenadine (FXD), a model parent drug, that is resistant to intestinal esterase but converted to FXD by hepatic esterase. Carboxylesterases (CESs), human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2), are the major esterases in human liver and intestine, respectively. These two CESs show quite different substrate specificities, and especially, hCE2 poorly hydrolyzes prodrugs with large acyl groups. FXD contains a carboxyl group and is poorly absorbed because of low membrane permeability and efflux by P-glycoprotein (P-gp). Therefore, two potential FXD prodrugs, ethyl-FXD and 2-hydroxyethyl-FXD, were synthesized by substitution of the carboxyl group in FXD. Both derivatives were resistant to intestinal hydrolysis, indicating their absorption as intact prodrugs. Ethyl-FXD was hydrolyzed by hepatic hCE1, but 2-hydroxyethyl-FXD was not. Both derivatives showed high membrane permeability in human P-gp-negative LLC-PK1 cells. In LLC-GA5-COL300 cells overexpressing human P-gp, ethyl-FXD was transported by P-gp, but its efflux was easily saturated. Whereas 2-hydroxyethyl-FXD showed more efficient P-gp-mediated transport than FXD. Although the structure of 2-hydroxyethyl-FXD only differs from ethyl-FXD by substitution of a hydroxyl group, 2-hydroxyethyl-FXD is unsuitable as a prodrug. However, ethyl-FXD is a good candidate prodrug because of good intestinal absorption and hepatic conversion by hCE1.

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Evaluation of Transport Mechanism of Prodrugs and Parent Drugs Formed by Intracellular Metabolism in Caco-2 Cells with Modified Carboxylesterase Activity: Temocapril as a Model Case

Cited by 27 publications

References 41 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

Synthesis and pharmacological evaluation of nucleoside prodrugs designed to target siderophore biosynthesis in Mycobacterium tuberculosis

Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein

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