Abstract
Since the implementation of several legislations to improve pediatric drug research, more pediatric clinical trials are being performed. In order to optimize these pediatric trials, adequate preclinical data are necessary, which are usually obtained by juvenile animal models. The growing piglet has been increasingly suggested as a potential animal model due to a high degree of anatomical and physiological similarities with humans. However, physiological data in pigs on the ontogeny of major organs involved in absorption, distribution, metabolism, and excretion of drugs are largely lacking. The aim of this study was to unravel the ontogeny of porcine hepatic drug metabolizing cytochrome P450 enzyme (CYP450) activities as well as protein abundances. Liver microsomes from 16 conventional pigs (8 males and 8 females) per age group: 2 days, 4 weeks, 8 weeks, and 6–7 months were prepared. Activity measurements were performed with substrates of major human CYP450 enzymes: midazolam (CYP3A), tolbutamide (CYP2C), and chlorzoxazone (CYP2E). Next, the hepatic scaling factor, microsomal protein per gram liver (MPPGL), was determined to correct for enzyme losses during the fractionation process. Finally, protein abundance was determined using proteomics and correlated with enzyme activity. No significant sex differences within each age category were observed in enzyme activity or MPPGL. The biotransformation rate of all three substrates increased with age, comparable with human maturation of CYP450 enzymes. The MPPGL decreased from birth till 8 weeks of age followed by an increase till 6–7 months of age. Significant sex differences in protein abundance were observed for CYP1A2, CYP2A19, CYP3A22, CYP4V2, CYP2C36, CYP2E_1, and CYP2E_2. Midazolam and tolbutamide are considered good substrates to evaluate porcine CYP3A/2C enzymes, respectively. However, chlorzoxazone is not advised to evaluate porcine CYP2E enzyme activity. The increase in biotransformation rate with age can be attributed to an increase in absolute amount of CYP450 proteins. Finally, developmental changes were observed regarding the involvement of specific CYP450 enzymes in the biotransformation of the different substrates.
Highlights
It has been about 20 years since the first pediatric legislations were initiated by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) to stimulate pediatric drug research
Tolbutamide (TB), chlorzoxazone (CZ), chlorpropamide, formic acid, cytochrome c from equine heart, potassium cyanide (KCN), triethylammonium bicarbonate buffer (TEABC), DTT, methyl methanethiosulfonate (MMTS), calcium chloride (CaCl2), dimethyl sulfoxide (DMSO) were purchased from SigmaAldrich
Sadler et al (2016) did not found a direct correlation between cytochrome P450 (CYP450) activity and protein expression. This is in contrast with the results presented here where high correlation coefficients were found between the biotransformation rate and certain CYP450 proteins
Summary
It has been about 20 years since the first pediatric legislations were initiated by the EMA and the FDA to stimulate pediatric drug research. In order to support the safety of these pediatric trials, it is important to improve and optimize pediatric preclinical studies. Pediatric juvenile animal studies can provide essential information regarding the PK, PD and safety of a drug. According to the ICH guideline M3(R2), these studies should only be considered when previous animal data and human safety data are insufficient to support pediatric studies. If a juvenile study is warranted, only one relevant species, preferably rodent, is generally considered adequate. This is in contrast with preclinical studies in adults where both rodent and non-rodent species are mandatory (Food and Drug Administration, HHS, 2010). Still a lot of knowledge gaps remain, such as the maturation of ADME processes both in growing children and piglets
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