A new approach in pediatric drug design: the development of a pediatric pig model. Part II: The maturation of hepatic cytochrome P450 enzymes 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. To determine if the conventional pig is such a potential animal model, the ontogeny of the different eliminating organ processes needs to be unraveled. The liver plays a key role in the biotransformation of drugs due to the presence of the cytochrome P450 enzyme system. 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. The corresponding metabolites, namely 1-hydroxy-midazolam, 4-hydroxy-tolbutamide and 6-hydroxy-chlorzoxazone, were quantified using a validated UHPLC-MS/MS method [2]. Furthermore, the microsomal protein per gram liver (MPPGL) was determined as it is a scaling factor in the extrapolation of the obtained enzyme activities to in vivo [3]. 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: 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. Conclusion: The maturation of porcine CYP450 enzymes shows a growth profile comparable to humans. The increase in activity suggests maturation of the enzymes as well as an increase in the absolute amount of the different CYP450 proteins. 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] De Bock L, Boussery K, Colin P, De Smet J, T'Jollyn H, Van Bocxlaer J. Development and validation of a fast and sensitive UPLC-MS/MS method for the quantification of six probe metabolites for the in vitro determination of cytochrome P450 activity. Talanta. 2012;89:209-16. [3] 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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