Ohura K

References (22)

Title : Bioconversion and P-gp-mediated transport of depot fluphenazine prodrugs after intramuscular injection - Ohura_2023_J.Pharm.Sci__
Author(s) : Ohura K , Nakada Y , Imai T
Ref : J Pharm Sci , : , 2023
Abstract : Fluphenazine (FPZ) decanoate, an ester-type prodrug formulated as a long-acting injection (LAI), is used in the treatment of schizophrenia. FPZ enanthate was also developed as an LAI formulation, but is no longer in use clinically because of the short elimination half-life of FPZ, the parent drug, after intramuscular injection. In the present study, the hydrolysis of FPZ prodrugs was evaluated in human plasma and liver to clarify the reason for this difference in elimination half-lives. FPZ prodrugs were hydrolyzed in human plasma and liver microsomes. The rate of hydrolysis of FPZ enanthate in human plasma and liver microsomes was 15-fold and 6-fold, respectively, faster than that of FPZ decanoate. Butyrylcholinesterase (BChE) and human serum albumin (HSA) present in human plasma, and two carboxylesterase (CES) isozymes, hCE1 and hCE2, expressed in ubiquitous organs including liver, were mainly responsible for the hydrolysis of FPZ prodrugs. FPZ prodrugs may not be bioconverted in human skeletal muscle at the injection site because of lack of expression of BChE and CESs in muscle. Interestingly, although FPZ was a poor substrate for human P-glycoprotein, FPZ caproate was a good substrate. In conclusion, it is suggested that the shorter elimination half-life of FPZ following administration of FPZ enanthate compared with FPZ decanoate can be attributed to the more rapid hydrolysis of FPZ enanthate by BChE, HSA and CESs.
ESTHER : Ohura_2023_J.Pharm.Sci__
PubMedSearch : Ohura_2023_J.Pharm.Sci__
PubMedID: 37019360

Title : Esterases Involved in the Rapid Bioconversion of Esmolol after Intravenous Injection in Humans - Imai_2022_Biol.Pharm.Bull_45_1544
Author(s) : Imai T , Isozaki M , Ohura K
Ref : Biol Pharm Bull , 45 :1544 , 2022
Abstract : Esmolol is indicated for the acute and temporary control of ventricular rate due to its rapid onset of action and elimination at a rate greater than cardiac output. This rapid elimination is achieved by the hydrolysis of esmolol to esmolol acid. It has previously been reported that esmolol is hydrolyzed in the cytosol of red blood cells (RBCs). In order to elucidate the metabolic tissues and enzymes involved in the rapid elimination of esmolol, a hydrolysis study was performed using different fractions of human blood and liver. Esmolol was slightly hydrolyzed by washed RBCs and plasma proteins while it was extensively hydrolyzed in plasma containing white blood cells and platelets. The negligible hydrolysis of esmolol in RBCs is supported by its poor hydrolysis by esterase D, the sole cytosolic esterase in RBCs. In human liver microsomes, esmolol was rapidly hydrolyzed according to Michaelis-Menten kinetics, and its hepatic clearance, calculated by the well-stirred model, was limited by hepatic blood flow. An inhibition study and a hydrolysis study using individual recombinant esterases showed that human carboxylesterase 1 isozyme (hCE1) is the main metabolic enzyme of esmolol in both white blood cells and human liver. These studies also showed that acyl protein thioesterase 1 (APT1) is involved in the cytosolic hydrolysis of esmolol in the liver. The hydrolysis of esmolol by hCE1 and APT1 also results in its pulmonary metabolism, which might be a reason for its high total clearance (170-285 mL/min/kg bodyweight), 3.5-fold greater than cardiac output (80.0 mL/min/kg bodyweight).
ESTHER : Imai_2022_Biol.Pharm.Bull_45_1544
PubMedSearch : Imai_2022_Biol.Pharm.Bull_45_1544
PubMedID: 36184514

Title : Identification and Characterization of a New Carboxylesterase 2 Isozyme, mfCES2C, in the Small Intestine of Cynomolgus Monkeys - Ohura_2020_Drug.Metab.Dispos_48_146
Author(s) : Ohura K , Igawa Y , Tanaka M , Matsumoto K , Kasahara A , Wada N , Kubota K , Uno Y , Imai T
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 48 :146 , 2020
Abstract : In contrast to a single human carboxylesterase 2 (CES2) isozyme (hCE2), three CES2 genes have been identified in cynomolgus monkeys: mfCES2A, mfCES2B, and mfCES2C . Although mfCES2A protein is expressed in several organs, mfCES2B is a pseudogene and the phenotype of the mfCES2C gene has not yet been clarified in tissues. In previous studies, we detected an unidentified esterase in the region of CES2 mobility upon nondenaturing PAGE analysis of monkey intestinal microsomes, which showed immunoreactivity for anti-mfCES2A antibody. The aim of the present study was to identify this unidentified esterase from monkey small intestine. The esterase was separated on nondenaturing PAGE gel and digested in-gel with trypsin. The amino acid sequences of fragmented peptides were analyzed by tandem mass spectrometry. The unidentified esterase was shown to be identical to mfCES2C (XP_015298642.1, predicted from the genome sequence data). mfCES2C consists of 559 amino acid residues and shows approximately 90% homology with mfCES2A (561 amino acid residues). In contrast to the ubiquitous expression of mfCES2A, mfCES2C is only expressed in the small intestine, kidney, and skin. The hydrolytic properties of recombinant mfCES2C, expressed in HEK293 cells, with respect to p-nitrophenyl derivatives, 4-methylumbelliferyl acetate, and irinotecan were similar to those of recombinant mfCES2A. However, mfCES2C showed a hydrolase activity for O-n-valeryl propranolol higher than mfCES2A. It is concluded that the previously unidentified monkey intestinal CES2 is mfCES2C, which shows different hydrolytic properties to mfCES2A, depending on the substrate. SIGNIFICANCE STATEMENT: In the present research, we determined that mfCES2C, a novel monkey CES2 isozyme, is expressed in the small intestine and kidney of the cynomolgus monkey. Interestingly, mfCES2C showed a relatively wide substrate specificity for ester-containing compounds. These findings may, in early stages of drug development, support the use of in vitro-to-in vivo extrapolation for the intestinal hydrolysis of ester drugs in the cynomolgus monkey.
ESTHER : Ohura_2020_Drug.Metab.Dispos_48_146
PubMedSearch : Ohura_2020_Drug.Metab.Dispos_48_146
PubMedID: 31836607
Gene_locus related to this paper: macfa-a0a2k5uar1

Title : [Evaluation of the Oral Absorption of Ester-type Prodrugs] - Ohura_2020_Yakugaku.Zasshi_140_369
Author(s) : Ohura K
Ref : Yakugaku Zasshi , 140 :369 , 2020
Abstract : The first-pass hydrolysis of oral ester-type prodrugs in the liver and intestine is mediated mainly by hCE1 and hCE2 of the respective predominant carboxylesterase (CES) isozymes. In order to provide high blood concentrations of the parent drugs, it is preferable that prodrugs are absorbed as an intact ester in the intestine, then rapidly converted to active parent drugs by hCE1 in the liver. In the present study, we designed a prodrug of fexofenadine (FXD) as a model parent drug that is resistant to hCE2 but hydrolyzed by hCE1, utilizing the differences in catalytic characteristics of hCE1 and hCE2. In order to precisely predict the intestinal absorption of an FXD prodrug candidate, we developed a novel high-throughput system by modifying Caco-2 cells. Further, we evaluated species differences and aging effects in the intestinal and hepatic hydrolysis of prodrugs to improve the estimation of in vivo first-pass hydrolysis of ester-type prodrugs. Consequently, it was possible to design a hepatotropic prodrug utilizing the differences in tissue distribution and substrate specificity of CESs. In addition, we successfully established three useful in vitro systems for predicting the intestinal absorption of hCE1 substrate using Caco-2 cells. However, some factors involved in estimating the bioavailability of prodrugs in human, such as changes in recognition of drug transporters by esterification, and species differences of the first-pass hydrolysis, should be comprehensively considered in prodrug development.
ESTHER : Ohura_2020_Yakugaku.Zasshi_140_369
PubMedSearch : Ohura_2020_Yakugaku.Zasshi_140_369
PubMedID: 32115554

Title : Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase - Imai_2020_J.Pharm.Sci_109_1417
Author(s) : Imai T , Bahar FG , Ohura K , Toda A
Ref : J Pharm Sci , 109 :1417 , 2020
Abstract : The aim of this experiment was to study the effects of calcium ion on the hydrolysis of cationic and anionic substrate by human butyrylcholinesterase (HuBChE). The hydrolysis of aspirin, an anionic substrate, by HuBChE was markedly increased in the presence of increasing concentrations of calcium ion ( approximately 20 mM), as shown by the increasing kcat ( approximately 18-fold). Butyrylthiocholine (BTC), a cationic substrate, was biphasically hydrolyzed with substrate activation; a second BTC molecule caused a 3-fold increase in kcat. At both lower and higher concentrations of BTC, its hydrolysis by HuBChE was slightly slowed down by the addition of calcium ion. Other cationic substrates, propranolol derivatives with butyryl and valeryl groups, were R-preferentially hydrolyzed by HuBChE; the rate of hydrolysis of these compounds was nearly the same in the absence and presence of calcium ion. These data indicate differential effects of calcium ion on HuBChE activity with anionic and cationic substrates. Furthermore, during the hydrolysis of aspirin in the presence of calcium ions, we demonstrated the existence of 2 additional binding sites for calcium, with Km values of 1.8 and 5.9 mM. These binding sites exhibited much lower affinities than the EF-hand motif, previously identified as a high-affinity calcium-binding site.
ESTHER : Imai_2020_J.Pharm.Sci_109_1417
PubMedSearch : Imai_2020_J.Pharm.Sci_109_1417
PubMedID: 31837977

Title : [Comparative Study of Hydrolase Activity in Skin with Liver and Intestine, and Its Aging Relation of Carboxylesterase Expression in Cynomolgus Monkey and Beagle Dog] - Imai_2019_Yakugaku.Zasshi_139_837
Author(s) : Imai T , Nakada Y , Ohura K
Ref : Yakugaku Zasshi , 139 :837 , 2019
Abstract : The hydrolysis activity and expression level of carboxylesterase (CES) in skin were compared with liver and intestine in the same individual of beagle dog and cynomolgus monkey, and their aging effects were studied. CES1 isozymes were mainly present in skin of both animals. The dermal hydrolysis activity was about 10 and 40% of hepatic activity in beagle dog and cynomolgus monkey, respectively. In beagle dog, the hydrolysis activity and the expression level of CES isozyme in liver and skin were nearly the same between 2- and 11-year-old individuals. On the other hand, the dermal hydrolase activity was lower in young individual than in old, in contrast to slight increase of hepatic and intestinal activity in old cynomolgus monkey. These differences by aging in cynomolgus monkey were related to the expression of CES1 proteins and their mRNA. Furthermore, mRNA level of human CES was investigated using total RNA of two individuals (63 and 85 years old). The two individuals showed approximately 2-fold higher expression of hCE2 than hCE1 in human skin.
ESTHER : Imai_2019_Yakugaku.Zasshi_139_837
PubMedSearch : Imai_2019_Yakugaku.Zasshi_139_837
PubMedID: 31061351

Title : Molecular characterization and polymorphisms of butyrylcholinesterase in cynomolgus macaques - Uno_2018_J.Med.Primatol_47_185
Author(s) : Uno Y , Uehara S , Mahadhi HMD , Ohura K , Hosokawa M , Imai T
Ref : J Med Primatol , 47 :185 , 2018
Abstract : BACKGROUND: Butyrylcholinesterase (BChE), an enzyme essential for drug metabolism, has been investigated as antidotes against organophosphorus nerve agents, and the efficacy and safety have been studied in cynomolgus macaques. BChE polymorphisms partly account for variable BChE activities among individuals in humans, but have not been investigated in cynomolgus macaques. METHODS: Molecular characterization was carried out by analyzing primary sequence, gene, tissue expression, and genetic variants. RESULTS: In cynomolgus and human BChE, phylogenetically closely related, amino acid residues important for enzyme function were conserved, and gene and genomic structure were similar. Cynomolgus BChE mRNA was most abundantly expressed in liver among the 10 tissue types analyzed. Re-sequencing found 26 non-synonymous genetic variants in 121 cynomolgus and 23 rhesus macaques, indicating that macaque BChE is polymorphic, although none of these variants corresponded to the null or defective alleles of human BChE. CONCLUSIONS: These results suggest molecular similarities of cynomolgus and human BChE.
ESTHER : Uno_2018_J.Med.Primatol_47_185
PubMedSearch : Uno_2018_J.Med.Primatol_47_185
PubMedID: 29573432
Gene_locus related to this paper: macfa-BCHE

Title : A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe - Abdel-Daim_2018_Eur.J.Pharm.Sci_114_267
Author(s) : Abdel-Daim A , Ohura K , Imai T
Ref : Eur J Pharm Sci , 114 :267 , 2018
Abstract : In the present study, we established a quantitative western blotting method to measure the expression level of recombinant serine hydrolases based on their catalytic mechanism. Fluorophosphonate (FP)-biotin was selected as a universal probe to quantify their expression levels, since FP moiety irreversibly inhibits serine hydrolases through strong stoichiometric binding to active serine residue. The linearity of detection using FP-biotin was assessed on three serine hydrolases; human carboxylesterase (CES) 1, butyrylcholinesterase and porcine liver esterases (PLE). Similar response signals were obtained from the equimolar concentrations of these enzymes and excellent linearity was observed at the range of 0.4-3.4pmol/lane (r(2)>0.99). Accuracy and precision of the proposed method were proved using PLE with recovery of 97.1-107.2% and relative standard deviation of 5.56%. PLE was selected as a calibration standard because of its high stability and commercial availability. As an application of the developed method, we measured the expression levels of four recombinant CES isozymes from human and cynomolgus macaque in S9 fraction of HEK293 cell homogenates. The expression levels of human CES1 and CES2, and cynomolgus macaque CES1 and CES2 were 2.51+/-0.1, 1.63+/-0.17, 0.79+/-0.09 and 1.37+/-0.13pmol/5mug S9 protein, respectively. Based on these determinations, their hydrolytic activities were accurately assessed. Cynomolgus CESs showed lower hydrolysis activities for p-nitrophenyl esters than human CESs. The hydrolase activities of CES2 isozymes were higher than CES1 in both species. Three to five folds faster hydrolysis for p-nitrophenyl butyrate than p-nitrophenyl acetate was observed in all CES isozymes except of cynomolgus CES1 that showed nearly same hydrolysis for both substrates. The provided method could be widely used for universal quantitative analysis of recombinant serine hydrolases.
ESTHER : Abdel-Daim_2018_Eur.J.Pharm.Sci_114_267
PubMedSearch : Abdel-Daim_2018_Eur.J.Pharm.Sci_114_267
PubMedID: 29289670

Title : Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies - Ishizaki_2018_Biol.Pharm.Bull_41_697
Author(s) : Ishizaki Y , Furihata T , Oyama Y , Ohura K , Imai T , Hosokawa M , Akita H , Chiba K
Ref : Biol Pharm Bull , 41 :697 , 2018
Abstract : Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2(CES1KD) cells). Then, we developed Caco-2(CES1KD) cells that stably express CES2 (CES2/Caco-2(CES1KD) cells) and their control Mock/Caco-2(CES1KD) cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2(CES1KD) cells. Furthermore, we also confirmed that CES2/Caco-2(CES1KD) cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2(CES1KD) cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.
ESTHER : Ishizaki_2018_Biol.Pharm.Bull_41_697
PubMedSearch : Ishizaki_2018_Biol.Pharm.Bull_41_697
PubMedID: 29709907

Title : Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin - Fujii_2017_Eur.J.Pharm.Sci_109_280
Author(s) : Fujii M , Ohara R , Matsumi A , Ohura K , Koizumi N , Imai T , Watanabe Y
Ref : Eur J Pharm Sci , 109 :280 , 2017
Abstract : We studied the effect that three alcohols, ethanol (EA), propanol (PA), and isopropanol (IPA), have on the skin permeation of p-hydroxy benzoic acid methyl ester (HBM), a model ester-type prodrug. HBM was applied to Yucatan micropig skin in a saturated phosphate buffered solution with or without 10% alcohol, and HBM and related materials in receptor fluid and skin were determined with HPLC. In the absence of alcohol, p-hydroxy benzoic acid (HBA), a metabolite of HBM, permeated the skin the most. The three alcohols enhanced the penetration of HBM at almost the same extent. The addition of 10% EA or PA to the HBM solution led to trans-esterification into the ethyl ester or propyl ester of HBA, and these esters permeated skin as well as HBA and HBM did. In contrast, the addition of 10% IPA promoted very little trans-esterification. Both hydrolysis and trans-esterification in the skin S9 fraction were inhibited by BNPP, an inhibitor of carboxylesterase (CES). Western blot and native PAGE showed the abundant expression of CES in micropig skin. Both hydrolysis and trans-esterification was simultaneously catalyzed by CES during skin permeation. Our data indicate that the alcohol used in dermal drug preparations should be selected not only for its ability to enhance the solubility and permeation of the drug, but also for the effect on metabolism of the drug in the skin.
ESTHER : Fujii_2017_Eur.J.Pharm.Sci_109_280
PubMedSearch : Fujii_2017_Eur.J.Pharm.Sci_109_280
PubMedID: 28821439

Title : Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin - Imai_2016_J.Pharm.Sci_105_714
Author(s) : Imai T , Ariyoshi S , Ohura K , Sawada T , Nakada Y
Ref : J Pharm Sci , 105 :714 , 2016
Abstract : 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.
ESTHER : Imai_2016_J.Pharm.Sci_105_714
PubMedSearch : Imai_2016_J.Pharm.Sci_105_714
PubMedID: 26444870

Title : Differences in Intestinal Hydrolytic Activities between Cynomolgus Monkeys and Humans: Evaluation of Substrate Specificities Using Recombinant Carboxylesterase 2 Isozymes - Igawa_2016_Mol.Pharm_13_3176
Author(s) : Igawa Y , Fujiwara S , Ohura K , Hirokawa T , Nishizawa Y , Uehara S , Uno Y , Imai T
Ref : Mol Pharm , 13 :3176 , 2016
Abstract : Cynomolgus monkeys, used as an animal model to predict human pharmacokinetics, occasionally show different oral absorption patterns to humans due to differences in their intestinal metabolism. In this study, we investigated the differences between intestinal hydrolytic activities in cynomolgus monkeys and humans, in particular the catalyzing activities of their carboxylesterase 2 (CES2) isozymes. For this purpose we used both human and monkey microsomes and recombinant enzymes derived from a cell culture system. Monkey intestinal microsomes showed lower hydrolytic activity than human microsomes for several substrates. Interestingly, in contrast to human intestinal hydrolysis, which is not enantioselective, monkey intestine showed preferential R-form hydrolysis of propranolol derivatives. Recombinant CES2 isozymes from both species, mfCES2v3 from monkeys and human hCE2, showed similar metabolic properties to their intestinal microsomes when expressed in HEK293 cells. Recombinant hCE2 and mfCES2v3 showed similar Km values for both enantiomers of all propranolol derivatives tested. However, recombinant mfCES2v3 showed extreme R-enantioselective hydrolysis, and both hCE2 and mfCES2v3 showed lower activity for O-3-methyl-n-butyryl propranolol than for O-n-valeryl and O-2-methyl-n-butyryl propranolol. This lower hydrolytic activity was characterized by lower Vmax values. Docking simulations of the protein-ligand complex demonstrated that the enantioselectivity of mfCES2v3 for propranolol derivatives was possibly caused by the orientation of its active site being deformed by an amino acid change of Leu107 to Gln107 and the insertion of Met309, compared with hCE2. In addition, molecular dynamics simulation indicated the possibility that the interatomic distance between the catalytic triad and the substrate was elongated by a 3-positioned methyl in the propranolol derivatives. Overall, these findings will help us to understand the differences in intestinal hydrolytic activities between cynomolgus monkeys and humans.
ESTHER : Igawa_2016_Mol.Pharm_13_3176
PubMedSearch : Igawa_2016_Mol.Pharm_13_3176
PubMedID: 27454346

Title : Establishment and Characterization of a Novel Caco-2 Subclone with a Similar Low Expression Level of Human Carboxylesterase 1 to Human Small Intestine - Ohura_2016_Drug.Metab.Dispos_44_1890
Author(s) : Ohura K , Nishiyama H , Saco S , Kurokawa K , Imai T
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 44 :1890 , 2016
Abstract : Caco-2 cells predominantly express human carboxylesterase 1 (hCE1), unlike the human intestine that predominantly expresses human carboxylesterase 2 (hCE2). Transport experiments using Caco-2 cell monolayers often lead to misestimation of the intestinal absorption of prodrugs because of this difference, as prodrugs designed to increase the bioavailability of parent drugs are made to be resistant to hCE2 in the intestine, so that they can be hydrolyzed by hCE1 in the liver. In the present study, we tried to establish a new Caco-2 subclone, with a similar pattern of carboxylase expression to human intestine, to enable a more accurate estimation of the intestinal absorption of prodrugs. Although no subclone could be identified with high expression levels of only hCE2, two subclones, #45 and #78, with extremely low expression levels of hCE1 were subcloned from parental Caco-2 cells by the limiting dilution technique. Unfortunately, subclone #45 did not form enterocyte-like cell monolayers due to low expression of claudins and beta-actin. However, subclone #78 formed polarized cell monolayers over 4 weeks and showed similar paracellular and transcellular transport properties to parental Caco-2 cell monolayers. In addition, the intestinal transport of oseltamivir, a hCE1 substrate, could be evaluated in subclone #78 cell monolayers, including P-glycoprotein-mediated efflux under nonhydrolysis conditions, unlike parental Caco-2 cells. Consequently, it is proposed that subclone #78 may provide a more effective system in which to evaluate the intestinal absorption of prodrugs that are intended to be hydrolyzed by hCE1.
ESTHER : Ohura_2016_Drug.Metab.Dispos_44_1890
PubMedSearch : Ohura_2016_Drug.Metab.Dispos_44_1890
PubMedID: 27638507
Gene_locus related to this paper: human-CES1

Title : Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein - Ohura_2015_J.Pharm.Sci_104_3076
Author(s) : Ohura K , Nakada Y , Kotani S , Imai T
Ref : J Pharm Sci , 104 :3076 , 2015
Abstract : 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. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3076-3083, 2015.
ESTHER : Ohura_2015_J.Pharm.Sci_104_3076
PubMedSearch : Ohura_2015_J.Pharm.Sci_104_3076
PubMedID: 25953731

Title : 3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells - Matsusaki_2015_Biochem.Biophys.Res.Commun_457_363
Author(s) : Matsusaki M , Hikimoto D , Nishiguchi A , Kadowaki K , Ohura K , Imai T , Akashi M
Ref : Biochemical & Biophysical Research Communications , 457 :363 , 2015
Abstract : Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications.
ESTHER : Matsusaki_2015_Biochem.Biophys.Res.Commun_457_363
PubMedSearch : Matsusaki_2015_Biochem.Biophys.Res.Commun_457_363
PubMedID: 25576862

Title : Distinct patterns of aging effects on the expression and activity of carboxylesterases in rat liver and intestine - Ohura_2014_Drug.Metab.Dispos_42_264
Author(s) : Ohura K , Tasaka K , Hashimoto M , Imai T
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 42 :264 , 2014
Abstract : The age-associated alteration in expression levels of carboxylesterases (CESs) can affect both intestinal and hepatic first-pass metabolism after oral administration of xenobiotic esters such as prodrugs. In this study, the age-related expression of CES isozymes and hydrolase activities were simultaneously investigated in liver, jejunum, and ileum from 8-, 46-, and 90-week-old rats. Rat liver expresses three major CES1 isozymes, Hydrolase A, Hydrolase B, and Hydrolase C, as well as one minor CES1 (Egasyn) and three minor CES2 isozymes (RL4, AY034877, and D50580). The mRNA and protein levels of major hepatic CES1 isozymes were decreased in an age-dependent manner, while those of minor CESs were maintained in all age groups. The hepatic hydrolase activity for temocapril was decreased in an age-dependent manner, accompanied by downregulation of Hydrolase B/C mRNA, while age-independent hydrolysis of propranolol derivatives was observed in rat liver, due to the contribution of Egasyn. Rat small intestine expresses one major CES2 (RL4) and four minor CESs (Hydrolase B, Hydrolase C, Egasyn, and AY034877). Interestingly, the expression of RL4 was age-dependently increased in both jejunum and ileum, while minor isozymes showed a constant expression across a wide age range. The up-regulation of RL4 expression with aging led to an increase of intestinal hydrolase activities for temocapril and propranolol derivatives. Consequently, age-dependent changes in the expression of CES isozymes affect the hydrolysis of xenobiotics in both rat liver and small intestine.
ESTHER : Ohura_2014_Drug.Metab.Dispos_42_264
PubMedSearch : Ohura_2014_Drug.Metab.Dispos_42_264
PubMedID: 24271336
Gene_locus related to this paper: human-CES1

Title : Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV - Ustun-Aytekin_2014_Cytotech_66_159
Author(s) : Ustun-Aytekin O , Gurhan ID , Ohura K , Imai T , Ongen G
Ref : Cytotechnology , 66 :159 , 2014
Abstract : Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL(-1)) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L(-1) h(-1)).
ESTHER : Ustun-Aytekin_2014_Cytotech_66_159
PubMedSearch : Ustun-Aytekin_2014_Cytotech_66_159
PubMedID: 23715645

Title : Species difference of esterase expression and hydrolase activity in plasma - Bahar_2012_J.Pharm.Sci_101_3979
Author(s) : Bahar FG , Ohura K , Ogihara T , Imai T
Ref : J Pharm Sci , 101 :3979 , 2012
Abstract : Differences in esterase expression among human, rhesus monkey, cynomolgus monkey, dog, minipig, rabbit, rat, and mouse plasma were identified using native polyacrylamide gel electrophoresis. Paraoxonase (PON) and butyrylcholinesterase (BChE) were ubiquitous in all species, but were highly expressed in primates and dogs, whereas carboxylesterase (CES) was only abundant in rabbits, mice, and rats. Several unknown esterases were observed in minipig and mouse plasma. These differences in plasma esterases and their expression levels result in species differences with respect to hydrolase activity. These differences were characterized using several different substrates. In contrast to the high hydrolase activity found for p-nitrophenylacetate (PNPA), a substrate of several hydrolase enzymes, irinotecan, a carbamate compound, was resistant to all plasma esterases. Oseltamivir, temocapril, and propranolol (PL) derivatives were rapidly hydrolyzed in mouse and rat plasma by their highly active CES enzyme, but rabbit plasma CES hydrolyzed only the PL derivatives. Interestingly, PL derivatives were highly hydrolyzed by monkey plasma BChE, whereas BChE from human, dog, and minipig plasma showed negligible activity. In conclusion, the esterase expression and hydrolyzing pattern of dog plasma were found to be closest to that of human plasma. These differences should be considered when selecting model animals for preclinical studies.
ESTHER : Bahar_2012_J.Pharm.Sci_101_3979
PubMedSearch : Bahar_2012_J.Pharm.Sci_101_3979
PubMedID: 22833171

Title : 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_2011_J.Pharm.Sci_100_3985
Author(s) : Ohura K , Nozawa T , Murakami K , Imai T
Ref : J Pharm Sci , 100 :3985 , 2011
Abstract : The intestinal absorption mechanism of temocapril, an ester-type prodrug of temocaprilat, was evaluated using Caco-2 cell monolayers with or without active carboxylesterase (CES)-mediated hydrolysis. The inhibition of CES-mediated hydrolysis was achieved by pretreatment of the monolayers with bis-p-nitrophenyl phosphate (BNPP), which inhibited 94% of the total hydrolysis of temocapril in the Caco-2 cells. The remaining 6% hydrolysis was due to the presence of serine esterases, other than CES, on the cell membranes. Transport experiments under CES-inhibited conditions showed temocapril not to be a substrate for peptide transporter 1 (PEPT1) or organic anion transporting polypeptides (OATPs), but to be an inhibitor of PEPT1; P-glycoprotein (P-gp) and breast-cancer-resistant protein (BCRP) were responsible for the efflux of temocapril, which was mainly absorbed by passive diffusion at low apical pH. In Caco-2 cell monolayers with CES-mediated hydrolysis intact, temocaprilat derived from temocapril, was 2.5-fold more rapidly transported into the apical compartment than into the basolateral compartment due to the presence of microvilli on the apical membrane. In contrast, temocaprilat at low intracellular concentrations, was preferentially transported across the basolateral membrane under CES-inhibited conditions.
ESTHER : Ohura_2011_J.Pharm.Sci_100_3985
PubMedSearch : Ohura_2011_J.Pharm.Sci_100_3985
PubMedID: 21618543

Title : Development of a novel system for estimating human intestinal absorption using Caco-2 cells in the absence of esterase activity - Ohura_2010_Drug.Metab.Dispos_38_323
Author(s) : Ohura K , Sakamoto H , Ninomiya S , Imai T
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 38 :323 , 2010
Abstract : Both mRNA and protein levels of the carboxylesterase (CES) isozymes, hCE1 and hCE2, in Caco-2 cells increase in a time-dependent manner, but hCE1 levels are always higher than those of hCE2. In human small intestine, however, the picture is reversed, with hCE2 being the predominant isozyme. Drugs hydrolyzed by hCE1 but not by hCE2 can be hydrolyzed in Caco-2 cells, but they are barely hydrolyzed in human small intestine. The results in Caco-2 cells can be misleading as a predictor of what will happen in human small intestine. In the present study, we proposed a novel method for predicting the absorption of prodrugs in the absence of CES-mediated hydrolysis in Caco-2 cells. The specific inhibition against CES was achieved using bis-p-nitrophenyl phosphate (BNPP). The optimal concentration of BNPP was determined at 200 microM by measuring the transport and hydrolysis of O-butyryl-propranolol (butyryl-PL) as a probe. BNPP concentrations of more than 200 microM inhibited 86% of hydrolysis of butyryl-PL, resulting in an increase in its apparent permeability. Treatment with 200 microM BNPP did not affect paracellular transport, passive diffusion, or carrier-mediated transport. Furthermore, the proposed evaluation system was tested for ethyl fexofenadine (ethyl-FXD), which is a superior substrate for hCE1 but a poor one for hCE2. CES-mediated hydrolysis of ethyl-FXD was 94% inhibited by 200 microM BNPP, and ethyl-FXD was passively transported as an intact prodrug. From the above observations, the novel evaluation system is effective for the prediction of human intestinal absorption of ester-type prodrugs.
ESTHER : Ohura_2010_Drug.Metab.Dispos_38_323
PubMedSearch : Ohura_2010_Drug.Metab.Dispos_38_323
PubMedID: 19923255

Title : The role of intestinal carboxylesterase in the oral absorption of prodrugs - Imai_2010_Curr.Drug.Metab_11_793
Author(s) : Imai T , Ohura K
Ref : Curr Drug Metab , 11 :793 , 2010
Abstract : The bioavailability of therapeutic agents can be improved by using prodrugs which have better passive diffusion than the active agents. Intestinal hydrolysis is an important reaction in the bioconversion of prodrugs, and may be the rate-limiting factor in their absorption. Carboxylesterase (CES) is ubiquitous in most organs and is located in the endoplasmic reticulum. Single-pass perfusion experiments in rat intestine have shown that CES is the main enzyme involved in intestinal first-pass hydrolysis. In man, intestinal CESs belong to the CES2 gene family and their activity is nearly constant along the jejunum and ileum. The predominant human intestinal CES, hCE2, preferentially hydrolyzes prodrugs in which the alcohol group of a pharmacologically active molecule has been modified by the addition of a small acyl group. In preclinical animal models, CES2 isozymes are also the major intestinal enzymes although they have different substrate specificities to human CES2, while CES1 isozymes and other unidentified enzymes are also present. It is therefore difficult to predict human intestinal absorption from animal experiments. Caco-2 cells mainly express the human CES1 isozyme, hCE1, which shows quite different substrate specificity from hCE2, making Caco-2 cells unsuitable for prediction of human intestinal absorption of prodrugs. However, we have developed a novel experimental method for predicting the human intestinal absorption of prodrugs using Caco-2 cells in which CES-mediated hydrolysis has been inhibited. The expression of hCE2 shows inter-individual variation and is regulated by several mechanisms, such as gene polymorphism and epigenetic processes. There are no reports suggesting that severe toxicity is associated with prodrugs due to genetic polymorphism of the CES2 gene.
ESTHER : Imai_2010_Curr.Drug.Metab_11_793
PubMedSearch : Imai_2010_Curr.Drug.Metab_11_793
PubMedID: 21189138

Title : Carboxylesterase in the liver and small intestine of experimental animals and human - Taketani_2007_Life.Sci_81_924
Author(s) : Taketani M , Shii M , Ohura K , Ninomiya S , Imai T
Ref : Life Sciences , 81 :924 , 2007
Abstract : Native polyacrylamide gel electrophoresis showed carboxylesterase (CES) to be the most abundant hydrolase in the liver and small intestine of humans, monkeys, dogs, rabbits and rats. The liver contains both CES1 and CES2 enzymes in all these species. The small intestine contains only enzymes from the CES2 family in humans and rats, while in rabbits and monkeys, enzymes from both CES1 and CES2 families are present. Interestingly, no hydrolase activity at all was found in dog small intestine. Flurbiprofen derivatives were R-preferentially hydrolyzed in the liver microsomes of all species, but hardly hydrolyzed in the small intestine microsomes of any species except rabbit. Propranolol derivatives were hydrolyzed in the small intestine and liver microsomes of all species except dog small intestine. Monkeys and rabbits showed R-preferential and non-enantio-selective hydrolysis, respectively, for propranolol derivatives in both organs. Human and rat liver showed R- and S-preferential hydrolysis, respectively, in spite of non-enantio-selective hydrolysis in their small intestines. The proximal-to-distal gradient of CES activity in human small intestine (1.1-1.5) was less steep than that of CYP 3A4 and 2C9 activity (three-fold difference). These findings indicate that human small intestine and liver show extensive hydrolase activity attributed to CES, which is different from that in species commonly used as experimental animals.
ESTHER : Taketani_2007_Life.Sci_81_924
PubMedSearch : Taketani_2007_Life.Sci_81_924
PubMedID: 17764701