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
Joske Millecam(1), Elke Gasthuys(1), Mathias Devreese(1), Dieter Deforce(2), Lies De Bock(3), Jan Van Bocxlaer(3), Siska Croubels(1)
1Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium ; 2Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium ; 3Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium
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.
Conclusions: 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.
