Development of a Novel System for Estimating Human Intestinal Absorption Using Caco-2 Cells in the Absence of Esterase Activity

Ohura, Kayoko; Sakamoto, Hisae; Ninomiya, Shinichi; Imai, Teruko

doi:10.1124/dmd.109.029413

Cited by 28 publications

(44 citation statements)

References 39 publications

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“…Caco-2 cells express CES; however, the expression profiles of CES isoforms differ from those in human small intestine and colon tissue (Ohura et al, 2010). The involvement of CES1 and CES2 on the formation from dabigatran etexilate to the mono-prodrugs BIBR 1087, BIBR 951, as well as dabigatran was investigated under several experimental conditions (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The effect of CES1-catalyzed BIBR 1087 formation in Caco-2 cells was assessed by conducting transcellular transport experiments with nonlabeled dabigatran etexilate, followed by LC-MS/MS quantification of both dabigatran etexilate and BIBR 1087 in the absence and presence of the CES inhibitor BNPP (Fig. 4) (Ohura et al, 2010). BNPP at a concentration of up to 200 mM did not affect the tightness of the monolayer and P-gp activity, as confirmed by comparing Papp of mannitol, propranolol, and digoxin in the absence and presence of BNPP (data not shown).…”

Section: Resultsmentioning

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: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

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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“…16 As skin homogenate contains all skin components, that is, pieces of cells, fibers, proteoglycan, and so on, it seems that ethyl-FXD binds not only to protein, but also to proteoglycans and to other components with a lipid phase such as cell membranes. Previously we have reported that ethyl-FXD is accumulated into Caco-2 cells during a transport experiment; 10% of ethyl-FXD was transported into the basolateral compartment, 8% remained on the apical side, and 80% accumulated inside the cell after 2 h. 32 We concluded that ethyl-FXD mainly accumulated in the cellular membrane because of its high lipophilicity and basicity. In the skin, ethyl-FXD is able to accumulate in intact whole cells such as keratinocytes, therefore the bound fraction of ethyl-FXD may be large enough for it to be retained in viable skin.…”

Section: Discussionmentioning

confidence: 99%

Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin

Imai

Ariyoshi

Ohura

et al. 2016

Journal of Pharmaceutical Sciences

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The expression of carboxylesterase (CES) and the transdermal movement of an ester prodrug were studied in rat skin. Ethyl-fexofenadine (ethyl-FXD) was used as a model lipophilic prodrug that is slowly hydrolyzed to its parent drug, FXD (MW 502). Among the CES1 and CES2 isozymes, Hydrolase A is predominant in rat skin and this enzyme was involved in 65% of the cutaneous hydrolysis of ethyl-FXD. The similarity of the permeation behavior of ethyl-FXD in full thickness and stripped skin indicated that the stratum corneum was not a barrier to penetration. However, only FXD was observed in receptor fluid, not ethyl-FXD, presumably because of the high degree of binding of ethyl-FXD in viable skin. The rate of hydrolysis of ethyl-FXD was much faster than steady-state flux, such that the influx rate was the rate-limiting process for transdermal permeation. Although Hydrolase A levels gradually increased in skin taken from rats aged from 8 to 90 weeks, variations in the expression levels of the esterase hardly affected the conversion of prodrug. The present data suggest that the slow hydrolysis of the prodrug of an active ingredient in viable skin followed by slow diffusion of active drug may provide a useful approach to topical application.

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“…Masaki et al (2007) also established that nearly complete inhibition of CES is obtained by preperfusion with BNPP (400 M for 40 min). More recently, Ohura et al (2010) reported that BNPP affects neither active transport, e.g., p-glycoprotein, peptide, and organic anion transporters, nor passive diffusion in Caco-2 cell monolayers.…”

Section: Resultsmentioning

confidence: 99%

Prediction of Human Intestinal Absorption of the Prodrug Temocapril by In Situ Single-Pass Perfusion Using Rat Intestine with Modified Hydrolase Activity

Nozawa

Imai²

2011

Drug Metab Dispos

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ABSTRACT:Intestinal absorption of temocapril, a prodrug of temocaprilat, was evaluated in an in situ rat jejunal perfusion model under various conditions of luminal pH and in the presence and absence of carboxylesterase-mediated hydrolysis. Temocapril was more easily taken up by mucosal cells at a luminal pH of 5.4 than at pH 6.4 or 7.4 and was extensively hydrolyzed to temocaprilat in mucosal cells. The hydrolysis was limited by the intrinsic clearance and the influx rate at luminal perfusate pHs of 5.4 and 7.4, respectively. Temocaprilat, derived from temocapril, was transported into both mesenteric vein and jejunal lumen according to pH partition theory. The net absorption of both temocapril and temocaprilat was highest at a luminal perfusate pH of 5.4. When both the luminal and venous fluid were at pH 7.4, temocaprilat was transported approximately 3-fold faster into the lumen than into the vein, due presumably to the greater surface area of the brush border membrane because of the presence of microvilli. Under carboxylesteraseinhibited conditions, the hydrolysis of temocapril was inhibited by only 50%. It is postulated that serine esterases located on the membranes of the epithelial cells were responsible for the residual hydrolysis. We have confirmed that temocapril is most easily absorbed in the proximal intestine after meals, due to prolongation of the gastric emptying time, the lower intraluminal pH caused by secretion of bile acid, and the interaction between serine esterases and the digesta.

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Development of a Novel System for Estimating Human Intestinal Absorption Using Caco-2 Cells in the Absence of Esterase Activity

Cited by 28 publications

References 39 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

Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin

Prediction of Human Intestinal Absorption of the Prodrug Temocapril by In Situ Single-Pass Perfusion Using Rat Intestine with Modified Hydrolase Activity

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