Toward improved clearance predictions and distribution profiles employing the isolated perfused rat liver model: Experimental optimization.

To reduce the drug attrition due to failure in clinical trials, an early accurate hepatic clearance (CL_H) prediction for small molecule drugs is critical. However, the routinely used in vitro to in vivo extrapolation (IVIVE) methods to predict human CL_H have led to significant underpredictions for many drug discovery programs. The ex vivo isolated perfused rat liver (IPRL) can be employed as a model to gain a mechanistic understanding of the root cause(s) of the frequent suboptimal performance of IVIVE methods. Although the IPRL perfusate composition is a critical factor to obtain reliable IPRL data to bridge the in vitro to in vivo clearance gap, no studies have experimentally explored the effect of different perfusate compositions on drug disposition profiles. Initially, in this study, acetaminophen and atazanavir, showing low or high protein binding, respectively, were tested in the IPRL system with 2 reperfusion media. One medium contained 3% bovine serum albumin, whereas the other was 10% rat blood to determine optimal experimental conditions, analogous to best practices in literature. This research provides new insights into the effect of IPRL perfusate composition on accurate CL_H predictions and liver disposition profiles. Specifically, this study demonstrates for the first time that 3% bovine serum albumin and 0.01% bovine α-1-acid glycoprotein are essential in an IPRL experiment to mimic biorelevant conditions and to achieve in vivo-relevant unbound clearance values, alongside 10% blood to maintain liver viability and functionality. The unique mechanistic insights derived from these robust IPRL data will aid in the development of improved IVIVE methods for CL_H. SIGNIFICANCE STATEMENT: This study explored the effect of perfusate composition on compound disposition profiles in an isolated perfused rat liver system. Reliable disposition profiles and improved clearance predictions were obtained, employing biorelevant plasma protein concentrations alongside 10% blood to obtain in vivo-relevant unbound clearance values and to preserve liver viability and functionality. Robust conditions are critical in isolated perfused rat liver experiments to gain mechanistic insights into the in vitro to in vivo clearance gap, enabling optimization of in vitro to in vivo extrapolation methods.