Differences in Intestinal Hydrolytic Activities between Cynomolgus Monkeys and Humans: Evaluation of Substrate Specificities Using Recombinant Carboxylesterase 2 Isozymes

Igawa, Yoshiyuki; Fujiwara, Seiya; Ohura, Kayoko; Hirokawa, Takatsugu; Nishizawa, You; Uehara, Shotaro; Uno, Yasuhiro; Imai, Teruko

doi:10.1021/acs.molpharmaceut.6b00394

Cited by 8 publications

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“…Immunoblotting. Immunoblotting was performed as described previously (Igawa et al, 2016). Briefly, microsomes from cynomolgus monkey liver and small intestine and cytosol fractions of HEK293 cells expressing recombinant CES2 isozymes were added to 7.5% polyacrylamide gel with SDS.…”

Section: Methodsmentioning

confidence: 99%

“…Stable Expression of Recombinant CES2 Isozymes in HEK293 Cells. Recombinant mfCES2Av1 and mfCES2C are stably expressed in HEK293 cells, as described previously (Igawa et al, 2016). Briefly, mfCES2Av1 and mfCES2C cDNA inserted into pTargeT vector (Promega Corporation) were transfected to HEK293 cells (American Type Culture Collection, Rockville, MD; passage 10-12), followed by selection of clones with resistance to G418.…”

Section: Methodsmentioning

confidence: 99%

“…On the other hand, three CES2 genes (mfCES2A, mfCES2B, and mfCES2C) have been reported (Williams et al, 2011;Uno et al, 2014), with expression profiles different from hCE2. It has been reported that the mRNA of mfCES2A is present in most organs (Uno et al, 2014), and the expression of mfCES2A protein has been confirmed in liver and intestine (Hosokawa et al, 1990;Igawa et al, 2016). The amino acid sequence of mfCES2A is 89% homologous with hCE2 (Supplemental Table 1).…”

Section: Introductionmentioning

confidence: 94%

“…In our previous studies, nondenaturing PAGE of cynomolgus intestinal microsomes showed two major bands of proteins with esterase activity in the migration region of the CES2 family of enzymes (Taketani et al, 2007). The protein in the upper band was designated as the imfCES2A (Igawa et al, 2016), therefore it is assumed that the protein on the lower band is mfCES2C.…”

Section: Introductionmentioning

confidence: 99%

“…The amino acid sequence of mfCES2A is 89% homologous with hCE2 (Supplemental Table 1). However, the hydrolytic preferences of mfCES2A recombinant protein for several substrates is different from that of hCE2 (Igawa et al, 2016). In contrast, mfCES2B gene is considered a pseudogene (Uno et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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Identification and Characterization of a New Carboxylesterase 2 Isozyme, mfCES2C, in the Small Intestine of Cynomolgus Monkeys

et al. 2019

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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 STATEMENTIn 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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%