Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes...

Dogs are commonly used in human and veterinary pharmaceutical development. Physiologically based pharmacokinetic modeling using recombinant cytochrome P450 (CYP) enzymes requires accurate estimates of CYP abundance, particularly in liver. However, such estimates are currently available for only seven CYPs, which were determined in a limited number of livers from one dog breed (beagle). In this study, we used a label-free shotgun proteomics method to quantitate 11 CYPs (including four CYPs not previously measured), cytochrome P450 oxidoreductase, and cytochrome b5 in liver microsomes from 59 dogs representing four different breeds and mixed-breed dogs. Validation included showing correlation with CYP marker activities, immunoquantified protein, as well as CYP1A2 and CYP2C41 null allele genotypes. Abundance values largely agreed with those previously published. Average CYP abundance was highest (>120 pmol/mg protein) for CYP2D15 and CYP3A12; intermediate (40-89 pmol/mg) for CYP1A2, CYP2B11, CYP2E1, and CYP2C21; and lowest (<12 pmol/mg) for CYP2A13, CYP2A25, CYP2C41, CYP3A26, and CYP1A1. The CYP2C41 gene was detected in 12 of 58 (21%) livers. CYP2C41 protein abundance averaged 8.2 pmol/mg in those livers, and was highest (19 pmol/mg) in the only liver with two CYP2C41 gene copies. CYP1A2 protein was not detected in the only liver homozygous for the CYP1A2 stop codon mutation. Large breed-associated differences were observed for CYP2B11 (P < 0.0001; ANOVA) but not for other CYPs. Research hounds and Beagles had the highest CYP2B11 abundance; mixed-breed dogs and Chihuahua were intermediate; whereas greyhounds had the lowest abundance. These results provide the most comprehensive estimates to date of CYP abundance and variability in canine liver. SIGNIFICANCE STATEMENT: This work provides the most comprehensive quantitative analysis to date of the drug-metabolizing cytochrome P450 proteome in dogs that will serve as a valuable reference for physiologically based scaling and modeling used in drug development and research. This study also revealed high interindividual variation and dog breed-associated differences in drug-metabolizing cytochrome P450 expression that may be important for predicting drug disposition variability among a genetically diverse canine population.