Carboxylesterase Isozymes Research Articles

Bioconversion and P-gp-Mediated Transport of Depot Fluphenazine Prodrugs after Intramuscular Injection

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.

Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3mL/min. Calibration curves were linear within the concentration range of 100-20,000ng/mL for CCAM and 1-200ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1mg/mL, incubation time was 60min for clopidogrel and 30min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

The role of carboxylesterase enzymes in capecitabine therapy

Introduction: Capecitabine ( CAP ) is an oral antineoplastic pro-drug, whose initial step of activation is carboxylesterase (CES) dependent. The main conversion of CAP to 5-DFCR occurs in the liver by CES1 and a minor part - in the gastrointestinal tract (GIT) by CES2. Usually, the enteral pro-drug activation is associated with the appearance of fluoropyrimidine GIT toxicity, which may be dose-limiting and life-threatening for the patient. Thus, it is important to clear out the factors that could influence on the activity of both CES isozymes. Aim: The aim of the present study was to present the mechanism of hydrolysis, sources of variability and modulation possibilities of CESs, that could affect the treatment with CAP . Materials and Methods: A systematic review of the scientific databases in PubMed, Science Direct and Google Scholar was conducted. Results: The literature data showed up to 89% inter-individual variability in the plasma content of CAP and its metabolites. It was also established that factors, such as genetic polymorphisms, age, gender, and diseases, are responsible for these variabilities. Enzyme inhibitors and inductors, on the other hand, are among the factors that could be controlled and used as reliable modulators for CAP therapy. In fact, some authors found that the inhibition of CAP hydrolysis at CES2 level could reduce the common GIT toxicity and improve the bioavailability of the pro-drug. Conclusion : In accordance with the individual patient, the CES activity modulation approach could be used for the enhancement of the CAP therapeutic index. However, further detailed in vivo researches are needed to achieve categorical and applicable results.

An Activity-Based Fluorogenic Probe Enables Cellular and in Vivo Profiling of Carboxylesterase Isozymes.

Carboxylesterases (CEs) exist as multiple types of isomers in humans, and two major types are CE1 and CE2. They are widely distributed in human tissues and well-known for their important roles in drug metabolism and pathology of various diseases. Thus, the detection of CEs in living systems could provide efficient proof in disease diagnostics, as well as important information regarding chemotherapeutic effects of antitumor drugs and prognosis. To develop a specific probe to discriminate CEs from other hydrolases, especially cholinesterases, is quite challenging due to their structural similarities and substrate specificity. To date, almost all of the fluorescent probes developed for CEs have been constructed with an acetyl group as the recognition unit. Herein we proposed a new design strategy of probe-cavity matching, which led to the identification of a new fluorogenic substrate (termed as HBT-CE) with high specificity toward both CE isomers and improved sensitivity, considering the higher binding affinity and catalysis efficiency. The promising capability of HBT-CE was further demonstrated for endogenous CEs imaging in living cells, zebrafish, and nude mice. In addition, HBT-CE was successfully applied in kinetically monitoring drug-induced CE regulation in cancer cells. All of these findings suggest that HBT-CE is a valuable tool for tracking and imaging endogenous CEs in complex biological systems.

Evaluation of the Oral Absorption of Ester-type Prodrugs

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.

Abstract 989: The relationship between TP53 gene status and carboxylesterase 2 expression in human gastric cancer

Abstract Carboxylesterases are serine hydrolases that are involved in the metabolisms of various endogenous and exogenous compounds. They are also required for activation of many anti-cancer prodrugs. For example, irinotecan (CPT-11), an anti-cancer prodrug that has been approved for the treatment of many types of solid tumors including gastric cancer, is converted by the carboxylesterase CES2 to its active metabolite 7-ethyl-10-hydroxycamptothesin (SN-38), a very potent topoisomerase I inhibitor. Among carboxylesterase isozymes, CES2 is most highly expressed in the gastrointestinal tract. Thus, the expression of CES2 may play an important role in local (i.e., intratumoral) activation of anti-cancer prodrugs such as irinotecan in the gut. Recent studies with cultured cancer cell lines have shown that CES2 expression is regulated by the tumor suppressor protein p53. However, whether CES2 expression is affected by the presence of p53 mutation in clinical cancer samples still remains unclear. In this study, we focused on the regulatory mechanism of CES2 expression in gastric cancer. First, we examined the relationship between TP53 gene status and CES2 expression using gastric cancer cell lines. Several gastric cancer cell lines expressing wild-type p53 (AGS, NUGC4, MKN74, and HSC58) were treated with nutlin-3a, a drug that inhibits the interaction between p53 and the E3 ubiquitin ligase MDM2 and thereby directly activates p53 signaling without genotoxic side effects. The expression of p21, a downstream target of p53, was increased following nutlin-3a treatment in two p53 wild-type cell lines NUGC4 and HSC58. The expression of CES2 was also upregulated by nutlin-3a in three p53 wild-type cell lines AGS, NUGC4, and HSC58. As expected, the expression levels of p21 and CES2 were not largely affected by nutlin-3a in gastric cancer cell lines with TP53 mutations. These results indicate that CES2 expression is positively regulated by the p53 pathway in most gastric cancer cells. We also investigated the relationship between TP53 gene status and CES2 expression in human gastric cancer samples. Our results may provide useful information for predicting the efficacy of anti-cancer prodrugs that are activated by CES2 in gastric cancer. Note: This abstract was not presented at the meeting. Citation Format: Yoshinori Kohira, Hyeon-Cheol Lee, Momoko Ishimine, Hajime Orita, Toshiyuki Kobayashi, Koichi Sato, Takehiko Yokomizo, Tetsu Fukunaga. The relationship between TP53 gene status and carboxylesterase 2 expression in human gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 989.

Differential expression of carboxylesterases in larva and adult of Culex quinquefasciatus Say (Diptera: Culicidae) from sub-Himalayan West Bengal, India

AbstractCulex quinquefasciatusSay, a widely distributed mosquito in tropical and subtropical areas, is the most important vector of the filarial parasiteWuchereria bancrofti, the causative agent of lymphatic filariasis. In India, filariasis is endemic in 17 States and six Union Territories, putting about 553 million people at risk of infection. Vector control, which aims to prevent pathogen transmission through interventions targeting adult mosquito vectors, is a significant component of control of the disease. Chemical-based control represents a chief strategy in the management of mosquito vectors; however, continuous application of insecticides has led to the development of resistance in many mosquito vectors around the world. The current study aims to observe the variability of expression of carboxylesterase isozymes that play a role in detoxifying insecticides into non-toxic compounds, in different life stages ofCulexmosquitoes, to understand levels of insecticide susceptibility that may be used in integrated mosquito management for efficient vector control.C. quinquefasciatus werecollected from different localities of sub-Himalayan West Bengal, India, and adult and larval bioassays were performed against one organophosphate insecticide (chlorpyrifos) and two synthetic pyrethroid insecticides (deltamethrin and lambda-cyhalothrin). The activity of α- and β-carboxylesterases (quantitative assay) were measured in larvae and adults ofC. quinquefasciatususing a microplate assay, and measured qualitative expression by native polyacrylamide gel electrophoresis. The study shows a differential activity of α- and β-carboxylesterases both in quantitative and qualitative assays. The quantitative assay reveals that larvae exhibit a 12.2-fold higher level activity of α-carboxylesterase and about 5.0-fold higher level of activity of β-carboxylesterase than adults. Some carboxylesterase isozymes, i.e., α-Est I-IV, α-Est VII and α-Est XI-XV were exclusively expressed in larvae, whereas α-Est V-VI and α-Est IX were expressed only in adults. In larvae, all β-Est I-IX were expressed, while in adults only β-Est IV-V was expressed. The results of adult and larval insecticide bioassay are also as per the above findings showing an LC90value of 0.017 ppm, 0.097 ppm and 0.072 ppm in the larva, and LC90value of 0.0015 ppm, 0.721 ppm, 0.364 ppm in adults against chlorpyrifos, deltamethrin and lambda-cyhalothrin, respectively.

Presence and inter-individual variability of carboxylesterases (CES1 and CES2) in human lung

Lungs are pharmacologically active organs and the pulmonary drug metabolism is of interest for inhaled drugs design. Carboxylesterases (CESs) are enzymes catalyzing the hydrolysis of many structurally different ester, amide and carbamate chemicals, including prodrugs. For the first time, the presence, kinetics, inhibition and inter-individual variations of the major liver CES isozymes (CES1 and CES2) were investigated in cytosol and microsomes of human lungs from 20 individuals using 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD) as substrates the rates of hydrolysis (Vmax) for pNPA and 4-MUA, unlike FD, were double in microsomes than in cytosol. In these cellular fractions, the Vmax of pNPA, as CES1 marker, were much greater (30–50-fold) than those of FD, as a specific CES2 marker. Conversely, the Km values were comparable suggesting the involvement of the same enzymes. Inhibition studies revealed that the FD hydrolysis was inhibited by bis-p-nitrophenylphosphate, phenylmethanesulfonyl fluoride, and loperamide (specific for CES2), whereas the pNPA and 4-MUA hydrolysis inhibition was limited. Inhibitors selective for other esterases missed having any effect on above-mentioned activities. In cytosol and microsomes of 20 lung samples, inter-individual variations were found for the hydrolysis of pNPA (2.5–5-fold), FD or 4-MUA (8–15-fold). Similar variations were also observed in CES1 and CES2 gene expression, although determined in a small number (n = 9) of lung samples. The identification of CES1 and CES2 and their variability in human lungs are important for drug metabolism and design of prodrugs which need to be activated in this organ.

A novel quantification method for serine hydrolases in cellular expression system using fluorophosphonate-biotin probe

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 (r2>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/5μg 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.

Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes

Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

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

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.

Distinct Patterns of Aging Effects on the Expression and Activity of Carboxylesterases in Rat Liver and Intestine

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.

Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues

1. Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure–metabolic activity relationship was examined.2. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains.3. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben.4. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity.5. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes.6. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens.7. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively.

Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity. Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts. Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P ≤ 0.001). CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression.

Contribution of Carboxylesterase in Hamster to the Intestinal First-Pass Loss and Low Bioavailability of Ethyl Piperate, an Effective Lipid-Lowering Drug Candidate

Ethyl piperate is an effective lipid-lowering drug candidate synthesized from piperine. However, its pharmacokinetic characteristics and oral absorption process remain unclear. A liquid chromatography-tandem mass spectrometry method was applied to determine the oral bioavailability of ethyl piperate. Simulated gastrointestinal pH conditions and intestinal washings were prepared to investigate their contributions to the loss of ethyl piperate. Hydrolysis by carboxylesterase (CES) was evaluated in vitro using microsomes and S9 fractions. In situ intestinal single-pass perfusion experiments were performed to estimate the role of CES in ethyl piperate absorption. The bioavailability of ethyl piperate was extremely low (0.47%) in hamster independent of gastrointestinal environmental effects. Ethyl piperate was a typical substrate of CES with kinetic parameters K(m) and V(max) of 7.56 ± 1.491 μM and 0.16 ± 0.008 nmol · min(-1) · mg protein(-1), respectively. CES was responsible for 85.8% of the intestinal hydrolysis of ethyl piperate. Specific inhibition of CES with bis-p-nitrophenyl phosphate (BNPP), decreased degradation clearance to 36% of control with no significant change in absorption clearance. This contrasted with the results of Caco-2 monolayer experiments, which showed a dramatic increase in the apparent permeability coefficient after BNPP treatment. mRNA levels for the CES isozyme, CES2A3, were similar among the three regions of hamster intestine and 60% less than those in liver; CES1B1 mRNA levels were even lower in the intestine and showed a proximal-to-distal decrease. In conclusion, CES markedly contributes to intestinal first-pass hydrolysis of ethyl piperate that is sufficient, but not necessary, to cause the observed extremely low bioavailability.

Carboxylesterases: structure, function and polymorphism in mammals

This review covers current developments in molecular-based studies of the structure and function of carboxylesterases. To allay the confusion of the classic classification of carboxylesterase isozymes, we propose a novel nomenclature and classification of mammalian carboxylesterases on the basis of molecular properties. Mechanisms of the regulation of the gene expression of carboxylesterases by xenobiotics, and the involvement of carboxylesterase in drug metabolism are also described. The novel biomarker for organophosphate pesticide exposure developed here is much more useful and reliable than cholinesterase inhibition.

Prodrug approach using carboxylesterases activity: catalytic properties and gene regulation of carboxylesterase in mammalian tissue

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.

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

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.

Inhibition of cholinesterases and carboxylesterases of two invertebrate species, Biomphalaria glabrata and Lumbriculus variegatus, by the carbamate pesticide carbaryl

In this study, the effects of sublethal concentrations of the carbamate carbaryl on the cholinesterase (ChE) and carboxylesterase (CES) activities present in the oligochaete Lumbriculus variegatus and in the pigmented Biomphalaria glabrata gastropod were investigated. The results showed that ChE activity from both species was inhibited by in vivo and in vitro exposure to carbaryl, with EC 50 and IC 50 values approximately 20 times lower for the oligochaete than for the gastropod. On the other hand, the recovery process in uncontaminated media was more efficient in oligochaetes than in snails. Thus, in only 2 h the oligochaetes showed no inhibition with respect to control values whereas the snails did not reach control values even after 48 h of being in pesticide-free water. CES activity was investigated in whole body soft tissue homogenates using three different substrates: p-nitrophenyl butyrate, 1-naphthyl acetate (NA) and 2-NA. In addition, the presence of multiple CES isozymes in L. variegatus and B. glabrata extracts, with activity towards 1- and 2-NA, was confirmed by native polyacrylamide electrophoresis. In both species, the activities measured using the naphthyl substrates were higher than the activity towards p-nitrophenyl butyrate. In addition, B. glabrata showed a higher CES activity than L. variegatus independently of the substrate used. In L. variegatus, in vivo CES activity towards the different substrates was less sensitive to carbaryl inhibition than ChE activity. In contrast, in B. glabrata, CES activity towards p-nitrophenyl butyrate was inhibited at lower insecticide concentrations than ChE. The results of this study contribute to the knowledge of the sensitivity of non-target freshwater invertebrate Type B-esterases towards pesticides.