Synthesis and evaluation of indomethacin prodrugs with a diester structure that are metabolically activated by human carboxylesterases.

1. Carboxylesterase (CES) has been studied extensively, mostly with substrates in the monoester structures. We investigated the relationship between indomethacin diester prodrugs and metabolic activation by microsomes and recombinant human CES.2. Eight indomethacin diester prodrugs were synthesised in two steps. They were used as substrates and hydrolysis rates were calculated.3. As a result, the major hydrolysis enzyme was CES. The hydrolysis rate of recombinant CES2A1 was comparable to that of recombinant CES1A1.4. In this study, by changing the structure of the prodrug to a diester structure, it was found that CES2 activity was equivalent to CES1 activity.5. It should be noted that the use of diester prodrugs in prodrug discovery, where organ-specific hydrolysis reactions are expected, may not yield the expected results.

Functional roles and localization of hydrolases in the Japanese mitten crab Eriocheir japonica.

The Japanese mitten crab Eriocheir japonica inhabits rivers throughout Japan and is being cultivated for food. To conduct aquaculture efficiently, it is crucial to comprehend the physiological functions of the target organisms. However, there is a lack of fundamental information on Japanese mitten crabs. In this study, hydrolases were extracted from the midgut glands of Japanese mitten crabs and their metabolic activities were analyzed. An enzyme with hydrolytic activity was discovered within the cytosol of the midgut gland. Western blot analysis also revealed that the Japanese mitten crab contains a hydrolase with cross-reactivity to human carboxylesterase 1 (hCES1) antibodies. The substrate specificity of the S9 fraction of the midgut gland was investigated and, interestingly, it was revealed that it reacts well with indomethacin phenyl ester and fluorescein diacetate, which are substrates of hCES2, not substrates of hCES1. Furthermore, this enzyme was observed to metabolize the ester derivative of astaxanthin, which is a red pigment inherent to the Japanese mitten crab. These findings underscore the significance the midgut gland in the Japanese mitten crab as an important organ for metabolizing both endogenous and exogenous ester-type compounds.

Effects of steric hindrance and electron density of ester prodrugs on controlling the metabolic activation by human carboxylesterase.

Carboxylesterase (CES) plays an important role in the hydrolysis metabolism of ester-type drugs and prodrugs. In this study, we investigated the change in the hydrolysis rate of hCE1 by focusing on the steric hindrance of the ester structure and the electron density. For 26 kinds of synthesized indomethacin prodrugs, the hydrolytic rate was measured in the presence of human liver microsomes (HLM), human small intestine microsomes (HIM), hCE1 and hCE2. The synthesized prodrugs were classified into three types: an alkyl ester type that is specifically metabolized by hCE1, a phenyl ester type that is more easily metabolized by hCE1 than by hCE2, and a carbonate ester type that is easily metabolized by both hCE1 and hCE2. The hydrolytic rate of 1-methylpentyl (hexan-2-yl) ester was 10-times lower than that of 4-methylpentyl ester in hCE1 solution. hCE2 was susceptible to electron density of the substrate, and there was a difference in the hydrolysis rate of up to 3.5-times between p-bromophenyl ester and p-acetylphenyl ester. By changing the steric hindrance and electron density of the alkoxy group, the factors that change the hydrolysis rate by CES were elucidated.

Design, synthesis and biological evaluation of water-soluble phenytoin prodrugs considering the substrate recognition ability of human carboxylesterase 1.

Human carboxylesterase 1 (hCES1) is a hydrolase that is mainly expressed in the liver and lung and plays the most important role in the metabolic activation of ester-type prodrugs. In this study, design, synthesis and evaluation of water-soluble phenytoin prodrugs were performed with consideration of the substrate recognition ability of hCES1. The phenytoin prodrugs were synthesized in two steps without column chromatography. It was confirmed that all prodrugs are efficiently converted to phenytoin in a human liver microsome (HLM) solution (up to 54.6 nmol/mg protein/min). Although some of the prodrugs were degraded in strongly basic solution, the solubility of all prodrugs was greater than that of phenytoin in buffer solutions at pH 7.4 and 8.3. Among the synthesized phenytoin prodrugs, the 3,3-dimethylglutarate prodrug was superior in terms of solubility and stability, and it showed solubility of 10 mg/mL or more (phenytoin: <0.1 mg/mL) in a solution of pH 8.3. It was also found that the 3,3-dimethylglutarate prodrug was selectively activated by hCES1 but not hCES2 or arylacetamidodeacetylase.

Structure-activity relationship of atorvastatin derivatives for metabolic activation by hydrolases.

1. We investigated the structure-activity relationship of 31 kinds of synthesized atorvastatin esters, thioesters, amides and lactone, selected as prodrug models, for metabolic activation by microsomes and hydrolases.2. The susceptibility to human carboxylesterase 1 (hCES1) was influenced not only by the size of the acyl group and alkoxy group but also by the degree of steric crowding around the alkoxy group.3. The susceptibility to human carboxylesterase 2 (hCES2) increased with a decrease in electron density around the alkoxy group of the substrate.4. Lactone was specifically hydrolyzed by paraoxonase 3 (PON3).5. These findings should be useful in prodrug design for controlling metabolic activation.

Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters.

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the Vmax value of the (R) form was 10 times larger than that of the (S) form.

Indole-3-propionic acid has chemical chaperone activity and suppresses endoplasmic reticulum stress-induced neuronal cell death.

Insoluble aggregated proteins are often associated with neurodegenerative diseases. Previously, we investigated chemical chaperones that prevent the aggregation of denatured proteins. Among these, 4-phenyl butyric acid (4-PBA) has well-documented chemical chaperone activity, but is required at doses that have multiple effects on cells, warranting further optimization of treatment regimens. In this study, we demonstrate chemical chaperone activities of the novel compound indole-3-propionic acid (IPA). Although it has already been reported that IPA prevents ß-amyloid aggregation, herein we show that this compound suppresses aggregation of denatured proteins. Our experiments with a cell culture model of Parkinson's disease are the first to show that IPA prevents endoplasmic reticulum (ER) stress and thereby protects against neuronal cell death. We suggest that IPA has potential for the treatment of neurodegenerative diseases and other diseases for which ER stress has been implicated.

Synthesis and evaluation of haloperidol ester prodrugs metabolically activated by human carboxylesterase.

Two types of haloperidol prodrugs in which a chemical modification was carried out on the hydroxyl group or carbonyl group were synthesized, and their metabolic activation abilities were evaluated in a human liver microsome (HLM) solution, a human small intestine microsome (HIM) solution and solutions of human recombinant carboxylesterases (hCESs). The metabolic activation rates of alcohol ester prodrugs in HLM solution were similar to those in hCES2 solution, and haloperidol pentanoate and haloperidol hexanoate showed high metabolic activation rates in the synthesized alcohol ester prodrugs. In addition, haloperidol acetate and haloperidol 2-methylbutanoate were hydrolyzed as slowly as haloperidol decanoate. The results suggested that haloperidol prodrugs with a small chain or a branched chain are useful as prodrugs for sustained release. The metabolic activation rate of the enol ester prodrug in HLM solution was similar to that in hCES1 solution, and the enol ester prodrug was found to behave differently from alcohol ester prodrugs, which were metabolically activated by hCES2.

Analysis of carboxylesterase 2 transcript variants in cynomolgus macaque liver.

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. 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. 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. 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. It is concluded that CES2A is the major cynomolgus hepatic CES2, and new transcript variant, CES2Av2, has similar functions to CES2Av1.

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.

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-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD 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-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD 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-2CES1KD 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.

Molecular characterization and polymorphisms of butyrylcholinesterase in cynomolgus macaques.

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.

Chemical synthesis of an indomethacin ester prodrug and its metabolic activation by human carboxylesterase 1.

It is necessary to consider the affinity of prodrugs for metabolic enzymes for efficient activation of the prodrugs in the body. Although many prodrugs have been synthesized with consideration of these chemical properties, there has been little study on the design of a structure with consideration of biological properties such as substrate recognition ability of metabolic enzymes. In this report, chemical synthesis and evaluation of indomethacin prodrugs metabolically activated by human carboxylesterase 1 (hCES1) are described. The synthesized prodrugs were subjected to hydrolysis reactions in solutions of human liver microsomes (HLM), human intestine microsomes (HIM) and hCES1, and the hydrolytic parameters were investigated to evaluate the hydrolytic rates of these prodrugs and to elucidate the substrate recognition ability of hCES1. It was found that the hydrolytic rates greatly change depending on the steric hindrance and stereochemistry of the ester in HLM, HIM and hCES1 solutions. Furthermore, in a hydrolysis reaction catalyzed by hCES1, the Vmax value of n-butyl thioester with chemically high reactivity was significantly lower than that of n-butyl ester.

Virtual Clinical Studies to Examine the Probability Distribution of the AUC at Target Tissues Using Physiologically-Based Pharmacokinetic Modeling: Application to Analyses of the Effect of Genetic Polymorphism of Enzymes and Transporters on Irinotecan Induced Side Effects.

PURPOSE: To establish a physiologically-based pharmacokinetic (PBPK) model for analyzing the factors associated with side effects of irinotecan by using a computer-based virtual clinical study (VCS) because many controversial associations between various genetic polymorphisms and side effects of irinotecan have been reported. METHODS: To optimize biochemical parameters of irinotecan and its metabolites in the PBPK modeling, a Cluster Newton method was introduced. In the VCS, virtual patients were generated considering the inter-individual variability and genetic polymorphisms of enzymes and transporters. RESULTS: Approximately 30 sets of parameters of the PBPK model gave good reproduction of the pharmacokinetics of irinotecan and its metabolites. Of these, 19 sets gave relatively good description of the effect of UGT1A1 *28 and SLCO1B1 c.521T>C polymorphism on the SN-38 plasma concentration, neutropenia, and diarrhea observed in clinical studies reported mainly by Teft et al. (Br J Cancer. 112(5):857-65, 20). VCS also indicated that the frequency of significant association of biliary index with diarrhea was higher than that of UGT1A1 *28 polymorphism. CONCLUSION: The VCS confirmed the importance of genetic polymorphisms of UGT1A1 *28 and SLCO1B1 c.521T>C in the irinotecan induced side effects. The VCS also indicated that biliary index is a better biomarker of diarrhea than UGT1A1 *28 polymorphism.

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.

Synthesis and evaluation of atorvastatin esters as prodrugs metabolically activated by human carboxylesterases.

We synthesized 11 kinds of prodrug with an esterified carboxylic acid moiety of atorvastatin in moderate to high yields. We discovered that they underwent metabolic activation specifically by the human carboxylesterase 1 (CES1) isozyme. The results suggested that these ester compounds of atorvastatin have the potential to act as prodrugs in vivo.

Analysis of a child who developed abnormal neuropsychiatric symptoms after administration of oseltamivir: a case report.

BACKGROUND: Neuropsychiatric side effects of oseltamivir occur occasionally, especially in infants and young patients, but nothing is known about possible contributory factors. CASE PRESENTATION: We report a case of a 15-year-old Japanese female with influenza infection who developed abnormal psychiatric symptoms after administration of standard doses of oseltamivir. She had no history of neurological illness, had never previously taken oseltamivir, and had not developed psychiatric reactions during previous influenza infection. Her delirium-like symptoms, including insomnia, visual hallucinations, and a long-term memory deficit, disappeared after cessation of oseltamivir and administration of benzodiazepine. Detailed assessment was performed, including neurological examination (electroencephalogram, brain magnetic resonance imaging, single photon emission computed tomography with 99mTc-ethyl cysteinate dimer and with (123)I-iomazenil, cerebrospinal fluid analysis and glutamate receptor autoantibodies), drug level determination and simulation, and genetic assessment (OAT1, OAT3, CES1, Neu2). CONCLUSIONS: Abnormal slowing in the electroencephalogram, which is characteristic of influenza-associated encephalopathy, was not observed in repeated recordings. The serum level determination of active metabolite Ro 64-0802 determined at 154 h after final dosing of oseltamivir was higher than the expected value, suggesting delayed elimination of Ro 64-0802. Thus, abnormal exposure to Ro 64-0802 might have contributed, at least in part, to the development of neuropsychiatric symptoms in this patient. The score on Naranjo's adverse drug reaction probability scale was 6. Mutation of c.122G > A (R41Q) in the sialidase Neu2 gene, increased CSF glutamate receptor autoantibodies, and limbic GABAergic dysfunction indicated by SPECT with (123)I-iomazenil were found as possible contributory factors to the CNS side effects.

A new methodology for functionalization at the 3-position of indoles by a combination of boron Lewis acid with nitriles.

We discovered that a reagent comprising a combination of PhBCl2 and nitriles was useful for syntheses of both 3-acylindoles and 1-(1H-indol-3-yl)alkylamine from indoles. The reaction proceeded selectively at the 3-position of indoles providing 3-acylindoles in moderate to high yields on treatment with the above reagent. Furthermore, the reaction provided the corresponding amine products in moderate to high yields after the intermediate imine was reduced by NaBH3CN. These reactions proceeded under mild conditions and are applicable to the formation of indoles functionalized at the 3-position.

Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques.

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.

Systematic identification and characterization of carboxylesterases in cynomolgus macaques.

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.