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

Abstract

The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies.CYP2D, CYP2J2, CYP3A4, and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76, and CYP2E1 were not detected in any of the 35 animals examined.On analysis of pooled microsomes (35 animals), CYP3A (3A4 + 3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%), and CYP2D (0.4%). On analysis of individual microsome samples, each P450 content varied 2- to 6-fold between animals, and no sex differences were observed in any P450 content.These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.

Evaluation of 89 Compounds for Identification of Substrates for Cynomolgus Monkey CYP2C76, a New Bupropion/Nifedipine Oxidase

“…In the case of amitriptyline, low BA can be attributed to first-pass intestinal metabolism, resulting in low Fa*Fg (Akabane et al, 2010). Lately, P450 of small intestine was quantified in cynomolgus monkeys by immunoblotting; the content of CYP3A was most abundant (about 80% of immunoquantified total P450 content), similar to humans (Paine et al, 2006;Uehara et al, 2014). Because CYP2C76 was not detected in monkey small intestine (Uehara et al, 2014), CYP2C76 seems to be irrelevant to low Fa*Fg of amitriptyline.…”

“…Lately, P450 of small intestine was quantified in cynomolgus monkeys by immunoblotting; the content of CYP3A was most abundant (about 80% of immunoquantified total P450 content), similar to humans (Paine et al, 2006;Uehara et al, 2014). Because CYP2C76 was not detected in monkey small intestine (Uehara et al, 2014), CYP2C76 seems to be irrelevant to low Fa*Fg of amitriptyline. On the other hand, nifedipine and imipramine were reported to show low hepatic elimination (Takahashi et al, 2009).…”

Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine

“…By comparing the data of the pooled samples to the contents of CYP1A, CYP2C9/19, CYP2D, CYP2J2, CYP3A4, and CYP3A5 which were previously immunoquantified in the same 35 small intestine samples (Uehara et al 2014), CYP4F (CYP4F2/3+CYP4F11+CYP4F12) was the most abundant subfamily (48.1% of total immunoquantified CYP1-4 proteins), followed by CYP3A (CYP3A4+CYP3A5) (41.2%), CYP2J (6.9%), CYP2C(9/19) (2.0%), and CYP2D (0.2%) (Fig. 4B).…”

“…This study was reviewed and approved by Institutional Animal Care and Use Committee at Shin Nippon Biomedical Laboratories, Ltd. Liver and small intestine microsomes were prepared as described previously (Uehara et al 2011; Uehara et al 2014). Concentration of total proteins was determined by the Bradford method using Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions.…”

“…In our previous study, the immunoquantification of cynomolgus CYP1-3 enzymes in 28 livers revealed that CYP3A was the most abundant subfamily as in human liver (Shimada et al 1994), followed by CYP2A, CYP2E1, CYP2B6, CYP2C9/19, CYP2C76, and CYP2D (Uehara et al 2011). Similarly, the immunoquantification of cynomolgus CYP1-3 enzymes in 35 small intestines revealed that CYP3A was the most abundant subfamily as in human small intestine (Paine et al 2006), followed by CYP2J2, CYP2C9/19, CYP1A, and CYP2D (Uehara et al 2014). …”