Abstract
We previously reported an ontogeny model of hepatic cytochrome P450 (P450) activity that predicts in vivo P450 elimination from in vitro intrinsic clearance. The purpose of this study was to conduct investigations into key assumptions of the P450 ontogeny model using the developing rat model system. We used two developmentally dissimilar enzymes, CYP2E1 and CYP1A2, and male rats (n = 4) at age groups representing critical developmental stages. Total body and liver weights and hepatic microsomal protein contents were measured. Following high-performance liquid chromatography analysis, apparent KM and Vmax estimates were calculated using nonlinear regression analysis for CYP2E1- and CYP1A2-mediated chlorzoxazone 6-hydroxylation and methoxyresorufin O-dealkylation, and Vmax estimates for p-nitrophenol and phenacetin hydroxylations, respectively. Hepatic scaling factors and Vmax values provided estimates for infant scaling factors (ISF). The data show microsomal protein contents increased with postnatal age and reached adult values after postnatal day (PD) 7. Apparent KM values were similar at all developmental stages except at ≤PD7. Developmental increases in probe substrate Vmax values did not correlate with the biphasic increase in immunoquantifiable P450. The activity of two different probe substrates for each P450 covaried as a function of age. A plot of observed ISF values as a function of age reflected the developmental pattern of rat hepatic P450. In summation, these observations diverge from several of the model's assumptions. Further investigations are required to explain these inconsistencies and to investigate whether the developing rat may provide a predictive paradigm for pediatric risk assessment for P450-mediated elimination processes.
Footnotes
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This work was supported by the Natural Sciences and Engineering Research Council of Canada Grant 261219. This study was presented in part at the Seventh International ISSX Meeting, Vancouver, BC, Canada, 2004, and at the Eighth Annual Canadian Society for Pharmaceutical Sciences, Toronto, ON, Canada, 2005, and it was published in part in abstract form in Drug Metab Rev36(Suppl 1):340.
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A small amount of data in this article was recently published in Elbarbry FA, McNamara PJ, and Alcorn J (2007) Ontogeny of hepatic CYP1A2 and CYP2E1 expression in rat. J Biochem Mol Toxicol21:41–50, Fig. 3. Rights to use this data have been granted by John Wiley & Sons, Inc.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017590.
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ABBREVIATIONS: P450, cytochrome P450; CZX, chlorzoxazone; 6-OH CZX, 6-hydroxychlorzoxazone; PNP, p-nitrophenol; 4NC, p-nitrocatechol; HRP, horseradish peroxidase; MROD, methoxyresorufin O-dealkylation; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; PBSt, phosphate-buffered saline/Tween 20; HSF, hepatic scaling factor; ISF, infant scaling factor(s); PD, postnatal day; MP, microsomal protein.
- Received July 10, 2007.
- Accepted September 17, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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