Characterization of Porcine Hepatic and Intestinal Drug Metabolizing CYP450: Comparison with Human Orthologues from A Quantitative, Activity and Selectivity Perspective

Abstract

Over the past two decades, the pig has gained attention as a potential model for human drug metabolism. Cytochrome P450 enzymes (CYP450), a superfamily of biotransformation enzymes, are pivotal in drug metabolism. Porcine CYP450 has been demonstrated to convert typical substrates of human CYP450. Nevertheless, knowledge and insight into porcine CYP450 quantity and substrate selectivity is scant, especially regarding intestinal CYP450. The current study aimed to map the quantities of hepatic and intestinal CYP450 in the conventional pig by using a proteomic approach. Moreover, the selectivity of the six most common used probe substrates (phenacetin, coumarin, midazolam, tolbutamide, dextromethorphan, and chlorzoxazone) for drug metabolizing enzyme subfamilies (CYP1A, CYP2A, CYP3A, CYP2C, CYP2D and CYP2E respectively), was investigated. Hepatic relative quantities were 4% (CYP1A), 31% (CYP2A), 14% (CYP3A), 10% (CYP2C), 28% (CYP2D) and 13% (CYP2E), whereas for the intestine only duodenal CYP450 could be determined with 88% for CYP3A and 12% for CYP2C. Furthermore, the results indicate that coumarin (CYP2A), midazolam (CYP3A), tolbutamide (CYP2C), and dextromethorphan (CYP2D) are as selective for porcine as for human CYP450. However, phenacetin (CYP1A2) and chlorzoxazone (CYP2E1) are less selective for the specific enzyme, despite similarities in selectivity towards the different enzymes involved compared to humans.

Figure 1

Relative amounts of hepatic (a) and duodenal (b) CYP450 proteins in conventional pigs and humans (c,d respectively) Total detected hepatic CYP450 was 365.3 ± 27.41 pmol/mg protein (n = 16; age 12 weeks, 8 males and 8 females), total detected duodenal CYP450 was 3.44 ± 2.42 pmol/mg protein (n = 3, pool of 16 pigs; 8 males and 8 females). Data for human CYP450 pie charts are derived from^,,,.

Figure 2

Eadie-Hofstee plots of CYP450 activity for tolbutamide, chlorzoxazone, coumarin, phenacetin, midazolam, and dextromethorphan, in porcine liver (n = 16, 8 males, 8 females, 12 weeks of age).

Figure 3

Eadie-Hofstee plots of CYP450 activity for tolbutamide, chlorzoxazone, and dextromethorphan in porcine duodenum (DD), and of phenacetin, midazolam in duodenum, jejunum (JJ) and ileum (IL).

Figure 4

General decision tree for enzyme involvement. No distinction is made between enzymes belonging to the same subfamily. For a specific flow scheme for each substrate, the reader is referred to the Supplementary Data.