Drug transporter - enzyme crossroads: Does OATP2B1 shape Cyp3a-driven midazolam metabolism in rats?

OATP2B1, encoded by SLCO2B1, is an uptake transporter in liver. Humanized SLCO2B1^+/+ rats showed increased hepatic Cyp3a1 expression compared to rSlco2b1^-/- rats. To assess the impact of humanization on Cyp3a activity, we conducted a phenotyping study applying midazolam in SLCO2B1^+/+ and rSlco2b1^-/- rats. Analysis of the serum levels and pharmacokinetic parameters for midazolam, 1'-hydroxymidazolam and 4-hydroxymidazolam revealed no significant differences between humanized and rSlco2b1^-/- rats, indicating minimal impact of genotype on systemic exposure. In contrast, ex vivo experiments with liver microsomes from untreated male SLCO2B1^+/+ and rSlco2b1^-/- rats, showed slightly higher Cyp3a activity in the knockout group. To resolve this apparent discrepancy, we characterized midazolam metabolism using recombinant enzyme systems, where we found Cyp3a2 to exhibit a higher metabolic capacity for midazolam when comparing formation of both metabolites. Determination of transcript levels of Cyp3a isoforms in rat livers revealed consistently higher mRNA expression of Cyp3a2 irrespective of genotype. These findings suggest that Cyp3a2 is the predominant isoform responsible for midazolam metabolism in rats. The modestly elevated microsomal activity in rSlco2b1^-/- rats is therefore most likely attributable to the subtle variation in Cyp3a2 abundance. In conclusion, our findings indicate that even if OATP2B1 affects hepatic abundance of Cyp3a1 this does not translate into relevant changes in pharmacokinetics of midazolam most likely due to the dominance of Cyp3a2 in its metabolism.