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

• Published in: Molecular pharmaceutics 2016-09, Vol.13 (9), p.3176-3186
• Main Authors: Igawa, Yoshiyuki, Fujiwara, Seiya, Ohura, Kayoko, Hirokawa, Takatsugu, Nishizawa, You, Uehara, Shotaro, Uno, Yasuhiro, Imai, Teruko
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Carboxylesterase - chemistry; Carboxylesterase - metabolism; Chromatography, High Pressure Liquid; HEK293 Cells; Humans; Intestine, Small - metabolism; Isoenzymes - chemistry; Isoenzymes - metabolism; Macaca fascicularis; Microsomes - metabolism; Molecular Dynamics Simulation; Substrate Specificity
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.6b00394

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 K m 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 V max 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.