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Ohura K, Nakada Y, Imai T Ref: J Pharm Sci, :, 2023 : PubMed ESTHER: Ohura_2023_J.Pharm.Sci__ PubMedSearch: Ohura 2023 J.Pharm.Sci PubMedID: 37019360 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. Title: Esterases Involved in the Rapid Bioconversion of Esmolol after Intravenous Injection in Humans Imai T, Isozaki M, Ohura K Ref: Biol Pharm Bull, 45:1544, 2022 : PubMed ESTHER: Imai_2022_Biol.Pharm.Bull_45_1544 PubMedSearch: Imai 2022 Biol.Pharm.Bull 45 1544 PubMedID: 36184514 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). Title: Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase Imai T, Bahar FG, Ohura K, Toda A Ref: J Pharm Sci, 109:1417, 2020 : PubMed ESTHER: Imai_2020_J.Pharm.Sci_109_1417 PubMedSearch: Imai 2020 J.Pharm.Sci 109 1417 PubMedID: 31837977 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. Title: Identification and Characterization of a New Carboxylesterase 2 Isozyme, mfCES2C, in the Small Intestine of Cynomolgus Monkeys 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 : PubMed 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 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. 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 T, Nakada Y, Ohura K Ref: Yakugaku Zasshi, 139:837, 2019 : PubMed ESTHER: Imai_2019_Yakugaku.Zasshi_139_837 PubMedSearch: Imai 2019 Yakugaku.Zasshi 139 837 PubMedID: 31061351 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. Title: A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe Abdel-Daim A, Ohura K, Imai T Ref: Eur J Pharm Sci, 114:267, 2018 : PubMed ESTHER: Abdel-Daim_2018_Eur.J.Pharm.Sci_114_267 PubMedSearch: Abdel-Daim 2018 Eur.J.Pharm.Sci 114 267 PubMedID: 29289670 Inhibitor(s) related to this paper: FP-Biotin 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. 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 Y, Furihata T, Oyama Y, Ohura K, Imai T, Hosokawa M, Akita H, Chiba K Ref: Biol Pharm Bull, 41:697, 2018 : PubMed ESTHER: Ishizaki_2018_Biol.Pharm.Bull_41_697 PubMedSearch: Ishizaki 2018 Biol.Pharm.Bull 41 697 PubMedID: 29709907 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. Title: Development of simultaneous quantification method of loteprednol etabonate (LE) and its acidic metabolites, and analysis of LE metabolism in rat Samir A, Kage A, Kayoko O, Imai T Ref: Xenobiotica, :1, 2018 : PubMed ESTHER: Samir_2018_Xenobiotica__1 PubMedSearch: Samir 2018 Xenobiotica 1 PubMedID: 29781759 Abstract 1. Loteprednol etabonate (LE) is a soft corticosteroid with two labile ester bonds at 17alpha- and 17beta-positions. Its corticosteroidal activity disappears upon hydrolysis of either ester bond. Hydrolysis of both ester bonds produces the inactive metabolite, Delta(1)-cortienic acid (Delta(1)-CA). 2. The simple high performance liquid chromatography method using acetic acid gradient was developed for the simultaneous determination of LE and its acidic metabolites. 3. LE was hydrolyzed in rat plasma with a half-life of 9 minutes. However, LE hydrolysis was undetectable in rat liver and intestine. LE hydrolysis in rat plasma was completely inhibited by paraoxon and bis (p-nitrophenyl) phosphate, thus identifying carboxylesterase as the LE hydrolase. Rat plasma carboxylesterase had a Km of 6.7 muM for LE. 4. In contrast to the disappearance rate of LE in rat plasma, the formation rate of 17alpha-monoester and Delta(1)-CA was markedly low, and a main hydrolysate of LE was not detected in rat plasma. 5. The metabolism of LE proceeded via different pathways in human and rat plasma. LE was slowly hydrolyzed by paraoxonase in human plasma to 17alpha-monoester with a half-life of 12 hours, but by carboxylesterase in rat plasma to yield undetectable products, presumed to include the unstable 17beta-monoester. Title: Prescription of Antidementia Drugs and Antipsychotics for Elderly Patients in Japan: A Descriptive Study Using Pharmacy Prescription Data Tsukada T, Sato I, Matsuoka N, Imai T, Doi Y, Arai M, Fujii Y, Matsunaga T, Kawakami K Ref: J Geriatr Psychiatry Neurol, :891988718785775, 2018 : PubMed ESTHER: Tsukada_2018_J.Geriatr.Psychiatry.Neurol__891988718785775 PubMedSearch: Tsukada 2018 J.Geriatr.Psychiatry.Neurol 891988718785775 PubMedID: 30016896 Abstract BACKGROUND: Antipsychotics are commonly used for managing behavioral and psychological symptoms of dementia among elderly patients with dementia receiving antidementia drugs (ADDs). However, the use of antipsychotics among these patients has not been investigated since 3 ADDs were approved in 2011 in Japan. METHOD: We conducted a descriptive study using pharmacy prescription data and identified patients aged >/=65 years who were newly prescribed donepezil, memantine, rivastigmine, and galantamine between January 1, 2012, and September 30, 2014. We determined the proportion of antipsychotic prescription and the factors affecting antipsychotic prescription using multivariable Cox proportional hazard models. RESULT: Of 13 876 patients, 1705 were memantine users, and the proportion of antipsychotic prescription among them was the highest (11.1%). Adjusted hazard ratios for donepezil, rivastigmine, and galantamine were 0.66, 0.56, and 0.66, respectively, relative to that for memantine. CONCLUSION: Compared to other ADD users, new memantine users were most likely to be prescribed antipsychotics. Title: Analysis of carboxylesterase 2 transcript variants in cynomolgus macaque liver Uno Y, Igawa Y, Tanaka M, Kayoko O, Hosokawa M, Imai T Ref: Xenobiotica, :1, 2018 : PubMed ESTHER: Uno_2018_Xenobiotica__1 PubMedSearch: Uno 2018 Xenobiotica 1 PubMedID: 29384423 Abstract 1. Carboxylesterase (CES) is important for the detoxification of a wide range of drugs and xenobiotics. In this study, the hepatic level of CES2 mRNA was examined in cynomolgus macaques used widely in preclinical studies for drug metabolism. 2. Three CES2 mRNAs were present in cynomolgus macaque liver. The mRNA level was highest for cynomolgus CES2A (formerly CES2v3), much lower for cynomolgus CES2B (formerly CES2v1), and extremely low for cynomolgus CES2C (formerly CES2v2). Most various transcript variants produced from cynomolgus CES2B gene did not contain a complete coding region. Thus, CES2A is the major CES2 enzyme in cynomolgus liver. 3. A new transcript variant of CES2A, CES2Av2, was identified. CES2Av2 contained exon 3 region different from wild-type (CES2Av1). In cynomolgus macaques expressing only CES2Av2 transcript, CES2A contained the sequence of CES2B in exon 3 and vicinity, probably due to gene conversion. 4. On genotyping, this CES2Av2 allele was prevalent in Indochinese cynomolgus macaques, but not in Indonesian cynomolgus or rhesus macaques. CES2Av2 recombinant protein showed similar activity to CES2Av1 protein for several substrates. 5. It is concluded that CES2A is the major cynomolgus hepatic CES2, and new transcript variant, CES2Av2, has similar functions to CES2Av1. Title: Molecular characterization and polymorphisms of butyrylcholinesterase in cynomolgus macaques Uno Y, Uehara S, Mahadhi HMD, Ohura K, Hosokawa M, Imai T Ref: J Med Primatol, 47:185, 2018 : PubMed 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 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. Title: Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin Fujii M, Ohara R, Matsumi A, Ohura K, Koizumi N, Imai T, Watanabe Y Ref: Eur J Pharm Sci, 109:280, 2017 : PubMed ESTHER: Fujii_2017_Eur.J.Pharm.Sci_109_280 PubMedSearch: Fujii 2017 Eur.J.Pharm.Sci 109 280 PubMedID: 28821439 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. Title: Differences in Intestinal Hydrolytic Activities between Cynomolgus Monkeys and Humans: Evaluation of Substrate Specificities Using Recombinant Carboxylesterase 2 Isozymes Igawa Y, Fujiwara S, Ohura K, Hirokawa T, Nishizawa Y, Uehara S, Uno Y, Imai T Ref: Mol Pharm, 13:3176, 2016 : PubMed ESTHER: Igawa_2016_Mol.Pharm_13_3176 PubMedSearch: Igawa 2016 Mol.Pharm 13 3176 PubMedID: 27454346 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. Title: Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin Imai T, Ariyoshi S, Ohura K, Sawada T, Nakada Y Ref: J Pharm Sci, 105:714, 2016 : PubMed ESTHER: Imai_2016_J.Pharm.Sci_105_714 PubMedSearch: Imai 2016 J.Pharm.Sci 105 714 PubMedID: 26444870 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. 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 K, Nishiyama H, Saco S, Kurokawa K, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 44:1890, 2016 : PubMed 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 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. Title: 3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells Matsusaki M, Hikimoto D, Nishiguchi A, Kadowaki K, Ohura K, Imai T, Akashi M Ref: Biochemical & Biophysical Research Communications, 457:363, 2015 : PubMed ESTHER: Matsusaki_2015_Biochem.Biophys.Res.Commun_457_363 PubMedSearch: Matsusaki 2015 Biochem.Biophys.Res.Commun 457 363 PubMedID: 25576862 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. Title: Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein Ohura K, Nakada Y, Kotani S, Imai T Ref: J Pharm Sci, 104:3076, 2015 : PubMed ESTHER: Ohura_2015_J.Pharm.Sci_104_3076 PubMedSearch: Ohura 2015 J.Pharm.Sci 104 3076 PubMedID: 25953731 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. Title: Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques Uno Y, Hosokawa M, Imai T Ref: J Vet Med Sci, 77:721, 2015 : PubMed ESTHER: Uno_2015_J.Vet.Med.Sci_77_721 PubMedSearch: Uno 2015 J.Vet.Med.Sci 77 721 PubMedID: 25715734 Abstract Arylacetamide deacetylase (AADAC), a microsomal serine esterase, hydrolyzes drugs, such as flutamide, phenacetin and rifampicin. Because AADAC has not been fully investigated at molecular levels in cynomolgus macaques, the non-human primate species widely used in drug metabolism studies, cynomolgus AADAC cDNA was isolated and characterized. The deduced amino acid sequence, highly homologous (92%) to human AADAC, was more closely clustered with human AADAC than the dog, rat or mouse ortholog in a phylogenetic tree. AADAC was flanked by AADACL2 and SUCNR1 in the cynomolgus and human genomes. Moreover, relatively abundant expression of AADAC mRNA was found in liver and jejunum, the drug-metabolizing organs, in cynomolgus macaques, similar to humans. The results suggest molecular similarities of AADAC between cynomolgus macaques and humans. Title: Distinct patterns of aging effects on the expression and activity of carboxylesterases in rat liver and intestine Ohura K, Tasaka K, Hashimoto M, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 42:264, 2014 : PubMed 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 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. Title: Quality of life in purely ocular myasthenia in Japan Suzuki S, Murai H, Imai T, Nagane Y, Masuda M, Tsuda E, Konno S, Oji S, Nakane S and Utsugisawa K <2 more author(s)> Suzuki S, Murai H, Imai T, Nagane Y, Masuda M, Tsuda E, Konno S, Oji S, Nakane S, Motomura M, Suzuki N, Utsugisawa K (- 2) Ref: BMC Neurol, 14:142, 2014 : PubMed ESTHER: Suzuki_2014_BMC.Neurol_14_142 PubMedSearch: Suzuki 2014 BMC.Neurol 14 142 PubMedID: 24996227 Abstract BACKGROUND: Since there has been no conclusive evidence regarding the treatment of ocular myasthenia, treatment guidelines were recently issued by the European Federation of Neurological Societies/European Neurological Society (EFNS/ENS). However, the therapeutic outcomes concerning the quality-of-life (QOL) of patients with ocular myasthenia are not yet fully understood. Title: Systematic identification and characterization of carboxylesterases in cynomolgus macaques Uno Y, Uehara S, Hosokawa M, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 42:2002, 2014 : PubMed ESTHER: Uno_2014_Drug.Metab.Dispos_42_2002 PubMedSearch: Uno 2014 Drug.Metab.Dispos 42 2002 PubMedID: 25256558 Gene_locus related to this paper: macfa-CES1v2, macfa-CES1v1, macfa-CES5, macfa-CES2v3 Abstract Carboxylesterase (CES) is important for detoxification of a wide range of drugs and xenobiotics and catalyzes cholesterol and fatty acid metabolism. Cynomolgus macaques are widely used in drug metabolism studies; however, cynomolgus CES has not been fully investigated at molecular levels, partly due to the lack of gene information. In this study, we isolated and characterized cDNAs for CES homologous to human CES1, CES2, and CES5A in cynomolgus macaques. By genome analysis, in the cynomolgus macaque genome, three gene sequences were found for CES1(v1-3) and CES2(v1-3), whereas one gene sequence was found for CES5A. Cynomolgus CES1, CES2, and CES5A genes were located in the genomic regions corresponding to the human genes. We successfully identified CES1v1, CES1v2, CES2v1, CES2v3, and CES5A cDNAs from cynomolgus liver. Sequence analysis showed that amino acid sequences of each CES were highly homologous to that of the human homolog. All five CESs had sequences characteristic for CES enzymes, including the catalytic triad and oxyanion hole loop. By quantitative polymerase chain reaction, the most abundant expression of CES mRNAs among the 10 tissue types analyzed was observed in liver (CES1v1 and CES2v3 mRNAs), jejunum (CES2v1 mRNAs), and kidney (CES1v2 and CES5A mRNA), the organs important for drug metabolism and excretion. The results indicated that cynomolgus macaques express at least five CES genes, which potentially encode intact CES proteins. Title: Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV Ustun-Aytekin O, Gurhan ID, Ohura K, Imai T, Ongen G Ref: Cytotechnology, 66:159, 2014 : PubMed ESTHER: Ustun-Aytekin_2014_Cytotech_66_159 PubMedSearch: Ustun-Aytekin 2014 Cytotech 66 159 PubMedID: 23715645 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)). Title: Aspirin hydrolysis in human and experimental animal plasma and the effect of metal cations on hydrolase activities Bahar FG, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 41:1450, 2013 : PubMed ESTHER: Bahar_2013_Drug.Metab.Dispos_41_1450 PubMedSearch: Bahar 2013 Drug.Metab.Dispos 41 1450 PubMedID: 23649702 Abstract The hydrolyzing properties of plasma esterases for aspirin were investigated in human plasma and plasma from experimental animals. The observed rates of aspirin hydrolysis were in the following order: rabbit > human > monkey > rat > mouse > dog > minipig. In human, monkey, and dog plasma, aspirin was hydrolyzed by their major hydrolases, paraoxonase (PON), butyrylcholinesterase (BChE), and albumin. In rabbit, mouse, and rat plasma, carboxylesterase (CES) was determined to be the enzyme responsible for aspirin hydrolysis, and in mouse and rat plasma, especially the latter, hydrolase activity was increased by the addition of ethopropazine, a specific inhibitor of BChE. Interestingly, divalent cations affected the plasma activity by enhancing or inhibiting the hydrolase activity of plasma BChE. The addition of 2 mM calcium increased the hydrolysis of aspirin in human, monkey, and dog plasma by 2.7-, 1.9-, and 2.3-fold, respectively. Magnesium showed a similar but lesser effect. Increasing concentrations of calcium and magnesium resulted in a two-phase stimulatory effect on aspirin hydrolysis in human plasma. In contrast, the addition of zinc had an inhibitory effect on plasma BChE activity. It is postulated that calcium and magnesium bind to BChE and thereby change the conformation of the enzyme to a more appropriate position for aspirin hydrolysis. Title: Involvement of Carboxylesterase in Hydrolysis of Propranolol Prodrug during Permeation across Rat Skin Imai T, Takase Y, Iwase H, Hashimoto M Ref: Pharmaceutics, 5:371, 2013 : PubMed ESTHER: Imai_2013_Pharmaceutics_5_371 PubMedSearch: Imai 2013 Pharmaceutics 5 371 PubMedID: 24300511 Abstract The use of a prodrug, a conjugate of an active drug with a lipophilic substituent, is a good way of increasing the cutaneous absorption of a drug. However, the activity of dermal hydrolases has rarely been investigated in humans, or experimental animals. In the present study, we focused on the identification of rat dermal esterases and the hydrolysis of a prodrug during permeation across rat skin. We found that carboxylesterase (CES), especially the rat CES1 isozyme, Hydrolase A, is expressed in rat skin and that the hydrolysis of p-nitrophenyl acyl derivatives and caproyl-propranolol (PL) was 20-fold lower in the 9000g supernatant fraction of skin homogenate than in liver microsomes. A permeation study of caproyl-PL was performed in rat full-thickness and stripped skin using a flow-through diffusion cell. Caproyl-PL was easily partitioned into the stratum corneum and retained, not only in the stratum corneum, but also in viable epidermis and dermis. Caproyl-PL could barely be detected in the receptor fluid after application to either full-thickness or stripped skin. PL, derived from caproyl-PL, was, however, detected in receptor fluid after extensive hydrolysis of caproyl-PL in viable skin. Permeation of PL was markedly decreased under CES inhibition, indicating that the net flux of caproyl-PL is dependent on its conversion rate to PL. Title: Species difference of esterase expression and hydrolase activity in plasma Bahar FG, Ohura K, Ogihara T, Imai T Ref: J Pharm Sci, 101:3979, 2012 : PubMed ESTHER: Bahar_2012_J.Pharm.Sci_101_3979 PubMedSearch: Bahar 2012 J.Pharm.Sci 101 3979 PubMedID: 22833171 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. Title: Prediction of human intestinal absorption of the prodrug temocapril by in situ single-pass perfusion using rat intestine with modified hydrolase activity Nozawa T, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 39:1263, 2011 : PubMed ESTHER: Nozawa_2011_Drug.Metab.Dispos_39_1263 PubMedSearch: Nozawa 2011 Drug.Metab.Dispos 39 1263 PubMedID: 21474683 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 carboxylesterase-inhibited 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. 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 K, Nozawa T, Murakami K, Imai T Ref: J Pharm Sci, 100:3985, 2011 : PubMed ESTHER: Ohura_2011_J.Pharm.Sci_100_3985 PubMedSearch: Ohura 2011 J.Pharm.Sci 100 3985 PubMedID: 21618543 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. Title: Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins Holmes RS, Wright MW, Laulederkind SJ, Cox LA, Hosokawa M, Imai T, Ishibashi S, Lehner R, Miyazaki M and Maltais LJ <9 more author(s)> Holmes RS, Wright MW, Laulederkind SJ, Cox LA, Hosokawa M, Imai T, Ishibashi S, Lehner R, Miyazaki M, Perkins EJ, Potter PM, Redinbo MR, Robert J, Satoh T, Yamashita T, Yan B, Yokoi T, Zechner R, Maltais LJ (- 9) Ref: Mamm Genome, 21:427, 2010 : PubMed ESTHER: Holmes_2010_Mamm.Genome_21_427 PubMedSearch: Holmes 2010 Mamm.Genome 21 427 PubMedID: 20931200 Abstract Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species. Title: Prodrug approach using carboxylesterases activity: catalytic properties and gene regulation of carboxylesterase in mammalian tissue Imai T, Hosokawa M Ref: Journal of Pesticide Science, 35:229, 2010 : PubMed ESTHER: Imai_2010_J.Pestic.Sci_35_229 PubMedSearch: Imai 2010 J.Pestic.Sci 35 229 Abstract A prodrug is a pharmacologically inactive derivative of an active parent drug, and is bioconverted to the active drug in vivo. Through chemical modification of a drug to a prodrug, we are able to deliver drugs to the target site, to optimize therapy and minimize toxicity. A major pathway for the bioconversion of prodrugs to the active parent drugs is via carboxylesterase (CES) activity. Among human CES isozymes, hCE1 and hCE2 predominantly participate in the hydrolysis of prodrugs in the liver and small intestine, respectively, although the substrate specificity is quite different between two isozymes; therefore, we can rationally design prodrugs based on the enzyme characteristics. However, since the expression levels of CES vary among individuals, there is a range of pharmacological responses following prodrug administration. Species differences are caused by tissue-dependent hydrolase activity mediated by CES, which makes it difficult to predict effectiveness in humans from a preclinical study using animals. Accordingly, understanding the regulation of CES expression and species difference of CES catalytic properties will be helpful in the design of prodrugs with increased specificity and enhanced physicochemical and biological properties. Title: The role of intestinal carboxylesterase in the oral absorption of prodrugs Imai T, Ohura K Ref: Curr Drug Metab, 11:793, 2010 : PubMed ESTHER: Imai_2010_Curr.Drug.Metab_11_793 PubMedSearch: Imai 2010 Curr.Drug.Metab 11 793 PubMedID: 21189138 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. Title: Development of a novel system for estimating human intestinal absorption using Caco-2 cells in the absence of esterase activity Ohura K, Sakamoto H, Ninomiya S, Imai T Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 38:323, 2010 : PubMed ESTHER: Ohura_2010_Drug.Metab.Dispos_38_323 PubMedSearch: Ohura 2010 Drug.Metab.Dispos 38 323 PubMedID: 19923255 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. Title: [Carboxylesterase: from drug metabolism to drug discovery] Imai T Ref: Nihon Yakurigaku Zasshi, 134:281, 2009 : PubMed ESTHER: Imai_2009_Nihon.Yakurigaku.Zasshi_134_281 PubMedSearch: Imai 2009 Nihon.Yakurigaku.Zasshi 134 281 PubMedID: 19915289 Title: [Hydrolysis by carboxylesterase and disposition of prodrug with ester moiety] Imai T Ref: Yakugaku Zasshi, 127:611, 2007 : PubMed ESTHER: Imai_2007_Yakugaku.Zasshi_127_611 PubMedSearch: Imai 2007 Yakugaku.Zasshi 127 611 PubMedID: 17409690 Abstract Prodrug is a useful approach for improving the bioavailability of therapeutic agents through increased passive transport. Carboxylesterases (CESs, EC.3.1.1.1.) that show ubiquitous expression profiles play an important role in the biotransformation of ester-containing prodrugs into their therapeutically active forms in the body. High levels of CESs are found in the liver, small intestine and lungs where prodrugs are firstly hydrolyzed before entering the systemic circulation. Rat intestine single-pass perfusion experiments have shown that CES is involved in the intestinal first-pass hydrolysis. Extensive pulmonary first-pass hydrolysis has been observed in accordance to the substrate specificity of CES1 isozyme. Hydrolysis in the human liver and lungs is mainly catalyzed by hCE1 (a human CES1 family isozyme), whereas that in the small intestine is predominantly mediated by hCE2 (a human CES2 family isozyme). hCE2 preferentially hydrolyzes substrates with a small acyl moiety such as CPT-11, due to conformational steric hindrance in its active site. In contrast, hCE1 is able to hydrolyze a variety of substrates due to spacious and flexible substrate binding region in its active site. In addition, hCE1 has been found to catalyze transesterification. Caco-2 cells mainly expresses CES1 isozyme but not CES2 isozyme. Because of the differences in substrate specificity between CES1 and CES2 enzymes, Caco-2 cell monolayer is not suitable for predicting intestinal absorption of prodrugs. These findings indicate that identification of substrate specificity of CES isozymes and development of an in vitro experimental method are essential to support rational design of prodrug. Title: Thyroglobulin gene mutations producing defective intracellular transport of thyroglobulin are associated with increased thyroidal type 2 iodothyronine deiodinase activity Kanou Y, Hishinuma A, Tsunekawa K, Seki K, Mizuno Y, Fujisawa H, Imai T, Miura Y, Nagasaka T and Murata Y <3 more author(s)> Kanou Y, Hishinuma A, Tsunekawa K, Seki K, Mizuno Y, Fujisawa H, Imai T, Miura Y, Nagasaka T, Yamada C, Ieiri T, Murakami M, Murata Y (- 3) Ref: J Clinical Endocrinology Metab, 92:1451, 2007 : PubMed ESTHER: Kanou_2007_J.Clin.Endocrinol.Metab_92_1451 PubMedSearch: Kanou 2007 J.Clin.Endocrinol.Metab 92 1451 PubMedID: 17244789 Abstract CONTEXT: Most patients with defective synthesis and/or secretion of thyroglobulin (Tg) present relatively high serum free T3 (FT3) concentrations with disproportionately low free T4 (FT4) resulting in a high FT3/FT4 ratio. The mechanism of this change in FT3/FT4 ratio remains unknown. OBJECTIVE: We hypothesize that increased type 2 iodothyronine deiodinase (D2) activity in the thyroid gland may explain the higher FT3/FT4 ratio that is frequently observed in patients with abnormal Tg synthesis. DESIGN: We recently identified a compound heterozygous patient (patient A) with a Tg G2356R mutation and one previously described (C1245R) that is known to cause a defect in intracellular transport of Tg. In the current study, after determining the abnormality caused by G2356R, we measured D2 activity as well as its mRNA level in the thyroid gland. We also measured the thyroidal D2 activity in three patients with Tg transport defect and in normal thyroid tissue. RESULTS: Morphological and biochemical analysis of the thyroid gland from patient A, complemented by a pulse-chase experiment, revealed that G2356R produces a defect in intracellular Tg transport. D2 activity but not type 1 deiodinase in thyroid glands of patients with abnormal Tg transport was significantly higher than in normal thyroid glands, whereas D2 mRNA level in patient A was comparable with that in normal thyroid glands. Furthermore, there was a positive correlation between D2 activity and FT3/FT4 ratios. CONCLUSION: Increased thyroidal D2 activity in the thyroid gland is responsible for the higher FT3/FT4 ratios in patients with defective intracellular Tg transport.. Title: Carboxylesterase in the liver and small intestine of experimental animals and human Taketani M, Shii M, Ohura K, Ninomiya S, Imai T Ref: Life Sciences, 81:924, 2007 : PubMed ESTHER: Taketani_2007_Life.Sci_81_924 PubMedSearch: Taketani 2007 Life.Sci 81 924 PubMedID: 17764701 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. Title: Involvement of up-regulation of hepatic breast cancer resistance protein in decreased plasma concentration of 7-ethyl-10-hydroxycamptothecin (SN-38) by coadministration of S-1 in rats Yokoo K, Hamada A, Watanabe H, Matsuzaki T, Imai T, Fujimoto H, Masa K, Saito H Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 35:1511, 2007 : PubMed ESTHER: Yokoo_2007_Drug.Metab.Dispos_35_1511 PubMedSearch: Yokoo 2007 Drug.Metab.Dispos 35 1511 PubMedID: 17537871 Inhibitor(s) related to this paper: SN-38 Abstract The safety and efficacy of combination therapy with 7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin (CPT-11, irinotecan) and S-1 composed of tegafur, a prodrug of 5-fluorouracil, gimeracil, and potassium oxonate, have been confirmed in patients with colorectal cancer. Previously, we showed that p.o. coadministration of S-1 decreased the plasma concentration of both CPT-11 and its metabolites in a patient with advanced colorectal cancer. The aim of this study was to clarify the mechanism of drug interaction using both in vivo and in vitro methods. Rats were administered S-1 p.o. (10 mg/kg) once a day for 7 consecutive days. On day 7, CPT-11 (10 mg/kg) was administered by i.v. injection. Coadministration of S-1 affected the pharmacokinetic behavior of CPT-11 and its metabolites, with a decrease in the C(max) and area under the plasma concentration curve (AUC) of the active metabolite 7-ethyl-10-hydroxycampothecin (SN-38) lactone form. Furthermore, the rate of biliary excretion of the SN-38 carboxylate form increased on coadministration of S-1. In the liver, the level of breast cancer resistance protein (BCRP), but not P-glycoprotein and multidrug resistance-associated protein 2, was elevated after administration of S-1. Enzymatic conversion of CPT-11 to SN-38 by carboxylesterase (CES) was unaffected by the liver microsomes of rats treated with S-1. In addition, components of S-1 did not inhibit the hydrolysis of p-nitrophenylacetate, a substrate of CES, in the S9 fraction of HepG2 cells. Therefore, the mechanism of drug interaction between CPT-11 and S-1 appears to involve up-regulation of BCRP in the liver, resulting in an increased rate of biliary excretion of SN-38 accompanied by a decrease in the C(max) and AUC of SN-38. Title: Human carboxylesterase isozymes: catalytic properties and rational drug design Imai T Ref: Drug Metab Pharmacokinet, 21:173, 2006 : PubMed ESTHER: Imai_2006_Drug.Metab.Pharmacokinet_21_173 PubMedSearch: Imai 2006 Drug.Metab.Pharmacokinet 21 173 PubMedID: 16858120 Gene_locus related to this paper: human-CES1 Abstract Human carboxylesterase 1 (hCE-1, CES1A1, HU1) and carboxylesterase 2 (hCE-2, hiCE, HU3) are a serine esterase involved in both drug metabolism and activation. Although both hCE-1 and hCE-2 are present in several organs, the hydrolase activity of liver and small intestine is predominantly attributed to hCE-1 and hCE-2, respectively. The substrate specificity of hCE-1 and hCE-2 is significantly different. hCE-1 mainly hydrolyzes a substrate with a small alcohol group and large acyl group, but its wide active pocket sometimes allows it to act on structurally distinct compounds of either large or small alcohol moiety. In contrast, hCE-2 recognizes a substrate with a large alcohol group and small acyl group, and its substrate specificity may be restricted by a capability of acyl-hCE-2 conjugate formation due to the presence of conformational interference in the active pocket. Furthermore, hCE-1 shows high transesterification activity, especially with hydrophobic alcohol, but negligible for hCE-2. Transesterification may be a reason for the substrate specificity of hCE-1 that hardly hydrolyzes a substrate with hydrophobic alcohol group, because transesterification can progress at the same time when a compound is hydrolyzed by hCE-1. From the standpoint of drug absorption, the intestinal hydrolysis by CES during drug absorption is evaluated in rat intestine and Caco2-cell line. The rat in situ single-pass perfusion shows markedly extensive hydrolysis in the intestinal mucosa. Since the hydrolyzed products are present at higher concentration in the epithelial cells rather than blood vessels and intestinal lumen, hydrolysates are transported by a specific efflux transporter and passive diffusion according to pH-partition. The expression pattern of CES in Caco-2 cell monolayer, a useful in vitro model for rapid screening of human intestinal drug absorption, is completely different from that in human small intestine but very similar to human liver that expresses a much higher level of hCE-1 and lower level of hCE-2. Therefore, the prediction of human intestinal absorption using Caco-2 cell monolayers should be carefully monitored in the case of ester and amide-containing drugs such as prodrugs. Further experimentation for an understanding of detailed substrate specificity for CES and development of in vitro evaluation systems for absorption of prodrug and its hydrolysates will help us to design the ideal prodrug. Title: Substrate specificity of carboxylesterase isozymes and their contribution to hydrolase activity in human liver and small intestine Imai T, Taketani M, Shii M, Hosokawa M, Chiba K Ref: Drug Metabolism & Disposition: The Biological Fate of Chemicals, 34:1734, 2006 : PubMed ESTHER: Imai_2006_Drug.Metab.Dispos_34_1734 PubMedSearch: Imai 2006 Drug.Metab.Dispos 34 1734 PubMedID: 16837570 Gene_locus related to this paper: human-CES1 Abstract Hydrolase activity from human liver and small intestine microsomes was compared with that of recombinant human carboxylesterases, hCE-1 and hCE-2. Although both hCE-1 and hCE-2 are present in human liver, the dominant component was found to be hCE-1, whereas the hydrolase activity of the human small intestine was found to be predominantly hCE-2. hCE-2 has a limited ability to hydrolyze large acyl compound substrates. Interestingly, propranolol derivatives, good substrates for hCE-2, were easily hydrolyzed by substitution of the methyl group on the 2-position of the acyl moiety, but were barely hydrolyzed when the methyl group was substituted on the 3-position. These findings suggest that hCE-2 does not easily form acylated intermediates because of conformational interference in its active site. In contrast, hCE-1 could hydrolyze a variety of substrates. The hydrolytic activity of hCE-2 increased with increasing alcohol chain length in benzoic acid derivative substrates, whereas hCE-1 preferentially catalyzed the hydrolysis of substrates with short alcohol chains. Kinetic data showed that the determining factor for the rate of hydrolysis of p-aminobenzoic acid esters was V(max) for hCE-1 and K(m) for hCE-2. Furthermore, the addition of hydrophobic alcohols to the reaction mixture with p-aminobenzoic acid propyl ester induced high and low levels of transesterification by hCE-1 and hCE-2, respectively. When considering the substrate specificities of hCE-1, it is necessary to consider the transesterification ability of hCE-1, in addition to the binding structure of the substrate in the active site of the enzyme. Title: Assessment of induction of cytochrome P450 by NO-1886 (ibrolipim), a lipoprotein lipase-promoting agent, in primary cultures of human hepatocytes and in female rat liver Morioka Y, Nishimura M, Imai T, Suzuki S, Harada M, Satoh T, Naito S Ref: Drug Metab Pharmacokinet, 21:19, 2006 : PubMed ESTHER: Morioka_2006_Drug.Metab.Pharmacokinet_21_19 PubMedSearch: Morioka 2006 Drug.Metab.Pharmacokinet 21 19 PubMedID: 16547390 Abstract The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA. A higher concentration (50 microM) of NO-1886 induced an increase in CYP2C8 mRNA and a decrease in CYP2C19 mRNA, and these changes continued after additional culture for 24 hr in fresh medium without NO-1886. The expression level of CYP3A4 mRNA after exposure to NO-1886 for 24 hr at 50 microM was about twice that in controls. Following additional culture for 24 hr in fresh medium without NO-1886, the expression of CYP3A4 mRNA was comparable to that in controls. On the other hand, the expression levels of CYP2C9 and CYP3A5 mRNA showed small and variable changes in each donor even at a high concentration (50 microM) of NO-1886. Furthermore, the pharmacokinetics of NO-1886 during repeated oral administration for 14 days was studied in female rats. The pharmacokinetic parameters of NO-1886 were nearly the same on days 1, 7, and 14 of repeated administration. The hepatic microsomal content of CYP isoforms was not affected by repeated administration for 7 days at a dose of 1 to 30 mg/kg in female rats, although the total CYP content was increased at a dose of 30 mg/kg. The expression levels of CYP1A2, CYP2B2, CYP2C12, and CYP2E1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2, 10, or 50 microM. The expression levels of CYP3A1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2 or 10 microM, but were increased, with large individual variation, by exposure at 50 microM. The mRNA expression levels in rat hepatocytes exposed to concentrations comparable to free plasma levels did not change significantly, which was consistent with the equivalence in the in vivo plasma concentrations observed on days 1 and 14 of repeated administration. These results suggest that repeated administration of NO-1886 at clinical doses does not significantly affect the expression levels of CYP isoforms in human liver, although the mRNA levels of the CYP isoforms involved in the metabolism of NO-1886 were increased by exposure to higher concentrations of NO-1886 in human hepatocytes in vitro. Title: Effects of NO-1886 (Ibrolipim), a lipoprotein lipase-promoting agent, on gene induction of cytochrome P450s, carboxylesterases, and sulfotransferases in primary cultures of human hepatocytes Nishimura M, Imai T, Morioka Y, Kuribayashi S, Kamataki T, Naito S Ref: Drug Metab Pharmacokinet, 19:422, 2004 : PubMed ESTHER: Nishimura_2004_Drug.Metab.Pharmacokinet_19_422 PubMedSearch: Nishimura 2004 Drug.Metab.Pharmacokinet 19 422 PubMedID: 15681896 Abstract In the present study, the effects on expression of cytochrome P450 (CYP1A1, CYP1A2, CYP3A4 and CYP3A5), carboxylesterase (CES1 and CES2) and sulfotransferase (CHST1, CHST3, CHST4, CST, SULT2A1 and TPST2) mRNA in primary cultures of cryopreserved human hepatocytes were evaluated after exposure to NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) for 48 hr at 2, 10, and 50 microM. Analysis was performed by RT-PCR in the presence of TaqMan probe. CYP1A1 and CYP1A2 mRNA levels after exposure to 50 microM omeprazole (positive control for CYP1As) were increased by 162 (p<0.001) and 37 times (p<0.001), respectively, compared with untreated controls. However, these mRNA levels were increased by 2 times or less after exposure to NO-1886. CYP3A4 and CYP3A5 mRNA levels after exposure to 50 microM rifampicin (positive control for CYP3As) were significantly increased by 5.8 (p<0.01) and 2.0 times (p<0.01), respectively, compared with untreated controls. The CYP3A4 mRNA level after exposure to 10 microM NO-1886 was increased by 1.3 times (p<0.05). Further, the CYP3A4 mRNA level after exposure to 50 microM NO-1886 was significantly increased by 3.6 times (p<0.001). However, the CYP3A5 mRNA level after exposure to 50 microM NO-1886 was not significantly increased. CES1 and CES2 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 1.4 (p<0.05) and 2.6 times (p<0.01), respectively, compared with untreated controls. CHST1, CST and SULT2A1 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 3.8 (p<0.001), 1.8 (p<0.01) and 4.4 times (p<0.01), respectively. CHST3, CHST4 and TPST2 mRNA levels after exposure to 50 microM NO-1886 were not significantly increased. This in vitro technique using primary cultured human hepatocytes is expected to be very useful for the preclinical evaluation of the induction of drug-metabolizing enzymes in humans. | |||||||
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