Absolute Quantitation of Drug-Metabolizing Cytochrome P450 Enzymes and Accessory Proteins in Dog Liver Microsomes Using Label-Free Standard-Free Analysis Reveals Interbreed Variability

Abstract

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 mixedbreed 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 breedassociated differences in drug-metabolizing cytochrome P450 expression that may be important for predicting drug disposition variability among a genetically diverse canine population.

Pharmacokinetics and Safety of Mitragynine in Beagle Dogs

“…15S, Supporting Information). Pharmacokinetic studies were performed in female dogs, so the estrus cycle may have an effect on the pharmacokinetics [37], but gender-related differences in the pharmacokinetics of mitragynine are not expected due to the similar expression of CYP3A/CYP3A12 in male and female dogs [38,39]. Mitragynine is metabolized in human liver microsomes predominantly by cytochrome P450 (CYP) 3A4 with minor contribu-tions from CYP2D6 and CYP2C9 [36].…”

Pharmacogenomics of poor drug metabolism in Greyhounds: Cytochrome P450 (CYP) 2B11 genetic variation, breed distribution, and functional characterization

“…Results using these latter probes suggest that CYP3A12 is not deficient in Greyhounds. These results have since been confirmed by us through quantitation of microsomal CYP protein concentrations using proteomic techniques that are more accurate and precise than immunoblotting 15 .…”

“…The HPLC column used was a The relative contributions of individual CYP isoforms to total liver microsome propofol hydroxylation, bupropion hydroxylation, and omeprazole sulfonation activities were estimated by adjustment of specific CYP activities using the average liver microsome abundance of each CYP. Abundance values determined by mass spectrometry in liver microsomes from 59 dogs of differing breeds were 2.8, 82, 11, 7.7, 79, 52, 1.8, 143, 72, 125, and 3.8 pmoles CYP per mg microsomal protein for CYPs 1A1, 1A2, 2A13, 2A25, 2B11, 2C21, 2C41, 2D15, 2E1, 3A12, and 3A26, respectively 15 .…”

Identification and characterization of the enzymes responsible for the metabolism of the non‐steroidal anti‐inflammatory drugs, flunixin meglumine and phenylbutazone, in horses

“…This behavior has been reported previously for both CYP1A1 and CYP3A4 in other species (Inouye, Mizokawa, Saito, Tonomura, & Ohkawa, 2000; Korzekwa et al., 1998). Although equine CYP1A1 is capable of 5‐hydroxylation of FM, it should be noted that in other species, this enzyme is not constitutively expressed (Martinez et al., 2019; Uno et al., 2009), and activity is attributable to induction by other chemicals (Nebert, Dalton, Okey, & Gonazlez, 2004). If the same is found to be true in horses, FM 5‐hydroxylation is most likely mediated primarily by equine CYP3A isoforms with additional contribution from CYP1A1 in horses exposed to CYP1A1 inducers.…”