The ontogeny of hepatic cytochrome P450 enzymes in conventional pigs using enzyme activity and proteomics

Objectives: Development of appropriate animal models taking growth and maturation into account is pivotal for pediatric preclinical pharmacokinetic and pharmacodynamic (PK/PD) research. Literature reports have demonstrated a high homology between human and porcine CYP450 enzymes in adults, suggesting the pig as a suited animal model for PK/PD and safety studies [1]. However data regarding the ontogeny of porcine hepatic CYP enzymes are lacking. Methods: The in vitro CYP450 enzyme activity of the following probe substrates was measured in microsomes: midazolam, tolbutamide and chlorzoxazone. The microsomes were prepared of each time 16 pigs (8 ♂ and 8 ♀, Hybrid sow x Piétrain boar) aging 2 days, 4 weeks, 8 weeks and 6-7 months. Furthermore, the microsomal protein per gram liver (MPPGL) was determined [2]. In addition to these in vitro activity experiments, the CYP isoenzymes in the same microsomes were determined by high definition data directed analysis (HD-DDA) mass spectrometry. The data analysis was performed using Progenesis QI. Results and conclusion: The microsomal activity of the three substrates increased with age. Significant sex differences were observed at 8 weeks of age for the three substrates and at 6 months of age for chlorzoxazone. The activity per gram liver, as calculated with the MPPGL, also showed a maturation profile. The increase in microsomal activity is reflected in an increase in CYP450 proteins in the microsomes. A total of 17 CYP isoenzymes was identified from which 10 had 2 or more unique peptides. These results show similar trends with human CYP ontogeny. Acknowledgements This study was supported by the "Agency for Innovation by Science and Technology in Flanders (IWT)" (IWT 141427). References [1] Puccinelli E, Gervasi PG, Longo V. Xenobiotic metabolizing cytochrome P450 in pig, a promising animal model. Curr Drug Metab. 2011;12(6):507-25. [2] Guengerich FP, Martin MV, Sohl CD, Cheng Q. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase. Nature protocols. 2009;4(9):1245-51.

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