title: Differences in Intestinal Hydrolytic Activities between Cynomolgus Monkeys and Humans: Evaluation of Substrate Specificities Using Recombinant Carboxylesterase 2 Isozymes.
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.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University , 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.|Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd. , 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Igawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University , 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University , 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Ohura
authors.affiliation: Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology , 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Hirokawa
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University , 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
authors.forename: You
authors.initials: Y
authors.lastname: Nishizawa
authors.affiliation: Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd. , 16-1 Minami Akasaka, Kainan, Wakayama 642-0017, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Uehara
authors.affiliation: Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd. , 16-1 Minami Akasaka, Kainan, Wakayama 642-0017, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Uno
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kumamoto University , 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan.
authors.forename: Teruko
authors.initials: T
authors.lastname: Imai
mesh_terms: D000818:Animals; D015153:Blotting, Western; D043182:Carboxylesterase / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D007421:Intestine, Small / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008252:Macaca fascicularis; D008861:Microsomes / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; C517204:CES2 protein, human; D043182:Carboxylesterase
keywords: carboxylesterase; cynomolgus monkey; docking simulation; enantioselective hydrolysis; small intestine; species differences