Regional Distribution of Drug-metabolizing Enzyme Activities in the Liver and Small Intestine of Cynomolgus Monkeys

Abstract

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Immunochemical Detection of Cytochrome P450 Enzymes in Liver Microsomes of 27 Cynomolgus Monkeys

“…After centrifugation at 1500g for 10 min, the supernatant or extract was analyzed by reverse-phase high-performance liquid chromatography with a fluorescence or UV detector. Metabolic assays using diclofenac and testosterone as substrates were carried out as described previously (Nakanishi et al, 2011). To estimate a correlation between drugmetabolizing enzyme activities and P450 amounts, linear regression analysis was performed using Origin7.5J software (OriginLab Corp., Northampton, MA).…”

CYP2C19 polymorphisms account for inter-individual variability of drug metabolism in cynomolgus macaques

“…From these samples, genomic DNA was prepared using the Puregene DNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Microsome samples were prepared from the livers of 7 Indochinese cynomolgus macaques as described previously [11]. The study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd.…”

Immunochemical detection of cytochrome P450 enzymes in small intestine microsomes of male and female untreated juvenile cynomolgus monkeys

“…CYP3A content in small intestine was lower in cynomolgus monkey (21 pmol/mg protein) (Table 1) than in human (74 pmol/mg protein) (Paine et al 2006). A previous study reported that testosterone 6β-hydroxylation in small intestine (jejunum) is comparable in cynomolgus monkeys and humans (Nakanishi et al 2011). This could be attributable to the higher metabolic activity of CYP3A enzymes in cynomolgus monkeys compared to that in human (Iwasaki et al 2010).…”

“…Catalytic activities mediated by cynomolgus CYP2C9/19, such as tolbutamide methylhydroxylation, diclofenac 4′-hydroxylation, and S -mephenytoin 4′-hydroxylation, are much lower in small intestine than in liver (Nakanishi et al 2011). These results suggest that small intestine would make a small contribution to first-pass metabolism of CYP2C9/19-metabolized drugs.…”

“…This study was reviewed and approved by the Institutional Animal Care and Use Committee at Shin Nippon Biomedical Laboratories, Ltd. (Kainan, Japan). Intestinal microsomes were prepared as described previously (Nakanishi et al 2011). Microsomal protein concentrations were measured by the Bradford method using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories) with bovine serum albumin as the standard.…”