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.

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.

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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.

ABSTRACT:The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester-and amide-containing drugs such as prodrugs.

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Three typical absorption enhancers, i.e., sodium caprate (Cap-Na), sodium deoxycholate (Deo-Na), and dipotassium glycyrrhizinate (Grz-K), were compared in terms of their permeability-enhancing effects on hydrophilic and hydrophobic model compounds in Caco-2 cell monolayers. The transepithelial electrical resistance (TEER) of the monolayers was reduced concentration-dependently by treatment with Cap-Na and Deo-Na, while treatment with Grz-K increased the TEER. Two patterns of TEER reduction were observed: one pattern indicated that Cap-Na had a rapid reducing effect, and another indicated that Deo-Na had a delayed reducing effect. These reductions in the TEER were accompanied by the increased transepithelial transport of two hydrophilic model compounds, sodium fluorescein (Flu-Na; MW = 376, log P = -1.52) and fluorescein isothiocyanate-dextran 4000 (FD-4; MW = 4400, log P = -2.0), and one hydrophobic model compound, rhodamine 123 hydrate (Rh123; MW = 381, log P = 1.13). The transport-enhancing effects of Cap-Na and Deo-Na on these model compounds decreased in the following order: FD-4 > Rh123 > Flu-Na, while Grz-K was found to have no effect on the transport of any of these model compounds. Confocal laser scanning microscopy (CLSM) of Caco-2 cell monolayers revealed that Cap-Na and Deo-Na enhanced the transepithelial transport of the hydrophilic model compounds via the paracellular route and that of the hydrophobic model compound via both paracellular and transcellular routes. Semiquantitative visual information obtained from CLSM images reflected the results of the transport experiment.