Itraconazole Metabolite Biotransformation by Hepatic Cytochrome P450 Enzymes in the Dog

Abstract: Background – Itraconazole (ITZ) is a commonly used azole antifungal in dogs but has an unacceptable rate of treatment failure and adverse reactions. Understanding species-specific metabolism of ITZ may be important in predicting clinical outcomes. ITZ undergoes serial metabolism by the hepatic cytochrome P450 (CYP) enzymes to hydroxy-ITZ, keto-ITZ, and N-desalkyl-ITZ. The first step is mediated by CYP2D15 and CYP3A12, but the specific isoforms responsible for the latter reactions are unknown.

Hypothesis/Objectives – Identify the CYP isoforms that mediate the hydroxy-ITZ to keto-ITZ and keto-ITZ to N-desalkyl-ITZ reactions in dog liver.

Animals – None.

Methods – Reaction conditions were optimized by incubating substrates (hydroxy-ITZ or keto-ITZ) with dog liver microsomes and products (keto-ITZ or N-desalkyl-ITZ) were quantified by LC-MS. Substrates were incubated with recombinant canine CYP enzymes (CYP1A1, CYP1A2, CYP2B11, CYP2C21, CYP2C41, CYP2D15, CYP3A12, CYP3A26). Reaction velocities were compared between CYPs before and after correction for relative abundance of isoforms within dog liver microsomes. For CYPs with the highest corrected reaction velocities, activities for the respective reactions were confirmed using isoform-specific inhibitors.

Results – CYP2D15 and CYP3A12 showed the highest uncorrected and corrected reaction velocities for both hydroxy-ITZ to keto-ITZ and keto-ITZ to N-desalkyl-ITZ reactions. Quinidine and erythromycin selectively inhibited both reactions in recombinant CYP2D15 and CYP3A12, respectively.

Conclusions and clinical importance – CYP2D15 and CYP3A12 both appear to contribute to the second and third steps of ITZ metabolism in dog liver. Genetic or acquired factors affecting expression or function of these isoforms could impact clinical outcomes of this drug.