Relative contribution of cytochrome P450 3A to midazolam oxidation in cattle liver microsomes.

INTRODUCTION In humans and rodents, members of the cytochrome P450 3A subfamily (CYP3A) are major contributors to midazolam (MDZ) biotransformation into 1-hydroxy-MDZ (1-OHMDZ) and 4-hydroxy-MDZ (4-OHMDZ), and 1-OHMDZ activity is commonly used as a surrogate marker for CYP3A in humans. In veterinary species, it is still crucial to identify isoform- and speciesspecific CYP substrates, to better characterize drug biotransformation and potential for drug–drug-interactions. The aim of this study was to characterize MDZ oxidation in cattle liver microsomes. MATERIALS AND METHODS Pooled microsomes were prepared from the liver of male Piedmontese beef cattle, and the formation of 1-OHMDZ and 4- OHMDZ was evaluated using a slightly modified HPLC-UV method; all the incubations were carried out under linear conditions of metabolite formation, with respect to incubation time and microsomal protein concentration. A confirmatory immunoinhibition study was performed by pre-incubating the pooled liver microsomes with increasing amounts of a polyclonal antibody raised against rat CYP3A1. Finally, MDZ hydroxylation was evaluated in 300 single-donor Piedmontese cattle liver microsomes, and analyzed for correlation with 6b-hydroxylation of testosterone (TST). RESULTS AND CONCLUSIONS Under the adopted chromatographic conditions, 4-OHMDZ, 1-OHMDZ and MDZ were eluted and well separated; the retention times were 13.9, 15.3 and 20.1 min, respectively. Formation of both metabolites conformed to single-enzyme Michaelis-Menten kinetics; Vmax and Km values were 665 pmol/min/mg protein and 6.16 micromolar for 4-OHMDZ, and 64 pmol/min/mg protein and 10.08 micromolar for 1-OHMDZ. The anti-rat CYP3A1 polyclonal antibody inhibited 4-OHMDZ formation up to 94%; however, only a 50% inhibition was noticed for 1-OHMDZ. The rates of formation of 4-OHMDZ and 6b-OHTST in single donor liver microsomes were poorly correlated. In conclusion, cattle liver microsomes are capable of metabolizing MDZ to 1-OHMDZ and 4-OHMDZ. Furthermore, the immunoinhibition results indicate a major contribution of cattle CYP3A to 4-OHMDZ formation, while other CYPs might be involved in drug oxidation to 1-OHMDZ. Finally, the observed poor relationship between 6b-OHTST and 4-OHMDZ deserve further investigation to clarify the specific role played either by individual cattle CYP3A isoforms or other CYPs in MDZ and TST hydroxylation. ACKNOWLEDGEMENTS Project supported by MIUR (2009ZE5HJP).