Mass spectrometry-based quantification of CYP enzymes to establish in vitro/in vivo scaling factors for intestinal and hepatic metabolism in beagle dog.

ABSTRACT

PURPOSE: Physiologically based models, when verified in pre-clinical species, optimally predict human pharmacokinetics. However, modeling of intestinal metabolism has been a gap. To establish in vitro/in vivo scaling factors for metabolism, the expression and activity of CYP enzymes were characterized in the intestine and liver of beagle dog. METHODS: Microsomal protein abundance in dog tissues was determined using testosterone-6ß-hydroxylation and 7-hydroxycoumarin-glucuronidation as markers for microsomal protein recovery. Expressions of 7 CYP enzymes were estimated based on quantification of proteotypic tryptic peptides using multiple reaction monitoring mass spectrometry. CYP3A12 and CYP2B11 activity was evaluated using selective marker reactions. RESULTS: The geometric mean of total microsomal protein was 51 mg/g in liver and 13 mg/cm in intestine, without significant differences between intestinal segments. CYP3A12, followed by CYP2B11, were the most abundant CYP enzymes in intestine. Abundance and activity were higher in liver than intestine and declined from small intestine to colon. CONCLUSIONS: CYP expression in dog liver and intestine was characterized, providing a basis for in vitro/in vivo scaling of intestinal and hepatic metabolism.