Advancing hepatic clearance prediction across in vitro, ex situ, and in vivo systems to facilitate in vitro-to-in vivo extrapolation.

Background: With rapid pharmaceutical development, hepatic clearance has become crucial for assessing drug metabolism. Rapid and effective estimation of hepatic clearance can be achieved through in vitro-to-in vivo extrapolation (IVIVE) without complicated animal experiments. However, this well-established method frequently leads to significant underestimation, limiting its clinical applicability.

Methods: We propose a series of optimizations to address these limitations. Metoprolol was selected as the target drug. In vitro microsomal assays, ex situ isolated perfused rat liver (IPRL) experiments, and in vivo pharmacokinetic studies in rats were performed to systematically investigate the factors that contribute to IVIVE deviation.

Results: To rectify the previously overlooked errors in IVIVE, we incorporated the apparent volume of distribution to refine the estimation of the intrinsic hepatic clearance derived from the Michaelis-Menten equation. Additionally, a more cytosolic-like environment was provided for microsome experiments to better simulate in vivo reactions. By integrating the findings from the IPRL experiments and pharmacokinetic studies, we identified the optimal hepatic clearance model from the perspective of liver drug metabolism-driving concentrations. Ultimately, our results indicate that the IVIVE-predicted hepatic clearance increased from the previously underestimated value of 28.1 to ∼70 mL/min/kg, which is closer to the in vivo value of 73.9 mL/min/kg. Moreover, the HEPES-KOH buffer system exhibits superior performance under these conditions.

Conclusions: We anticipate that the improved IVIVE method will provide a more comprehensive and accurate framework for predicting hepatic clearance, paving the way for its application in clinical drug dosage adjustments and new drug development.