Abstract

To study age-related changes in drug metabolism, we examined the in vitro biotransformation of midazolam (MDZ), a human cytochrome P-450 (CYP) 3A substrate, using liver microsomes from three age groups of male CD-1 mice ranging from 6 weeks to 2 years old. MDZ was metabolized to two major products, α-OH- and 4-OH-MDZ, which were quantified by HPLC. For both metabolites, V_max values were reduced in old livers (P < .05), whileK_m values did not change with age. The net intrinsic clearance (the sum ofV_max/K_m for both pathways) also was reduced in the old animals (P < .05). The capacity of ketoconazole, a CYP3A inhibitor in humans, to inhibit the biotransformation of MDZ and of alprazolam, another human CYP3A substrate, did not differ significantly with age. At 100 μM alprazolam, 0.5μM ketoconazole inhibited metabolite formation by >80%. At 30 μM MDZ, 2.5 μM ketoconazole impaired 4-OH-MDZ formation by 88%, whereas it reduced α-OH-MDZ formation by only 46%. Immunoinhibition studies with polyclonal anti-rat CYP3A1/2 and CYP2C11 antibodies confirmed that 4-OH-MDZ formation was largely CYP3A-dependent, while α-OH-MDZ formation was mediated by CYP3A and -2C isoforms. Western blot analysis revealed decreased microsomal content of CYP3A in old livers. Net intrinsic clearance of MDZ was correlated with total CYP3A content (P < .001). These results demonstrate a reduction in MDZ biotransformation in old male mice, which may be attributable, in part, to decreased CYP3A content in old livers. Changes in expression and activity of CYP2C isoforms also may contribute to age-related changes in MDZ biotransformation, but this requires more investigation.