Improving In Vitro-In Vivo Extrapolation (IVIVE) of Clearance Using Rat Liver Microsomes for Highly Plasma Protein Bound Molecules .

It is common practice in drug discovery and development to predict in vivo hepatic clearance from in vitro incubations with liver microsomes or hepatocytes using the well-stirred model (WSM). When applying the WSM to a set of about 3000 Novartis research compounds, 73% of neutral and basic compounds (extended clearance classification system ECCS class 2) were well-predicted within 3-fold. In contrast, only 44% (ECCS class 1A) or 34% (ECCS class 1B) of acids were predicted within 3-fold. To explore the hypothesis whether the higher degree of plasma protein binding for acids contributes to the in vitro in vivo correlation (IVIVC) disconnect, 68 proprietary compounds were incubated with rat liver microsomes (RLM) in the presence and absence of 5% plasma. A minor impact of plasma on clearance IVIVC was found for moderately bound compounds (fu p {greater than or equal to} 1%). However, addition of plasma significantly improved the IVIVC for highly bound compounds (fu p < 1%) as indicated by an increase of the average fold error (AFE) from 0.10 to 0.36. Correlating fu p with the unbound intrinsic clearance (CL int,u ) ratio in the presence or absence of plasma allowed the establishment of an empirical, non-linear correction equation that depends on fu p Taken together, estimation of the metabolic clearance of highly bound compounds was enhanced by the addition of plasma to microsomal incubations. For standard incubations in buffer only, application of an empirical correction provided improved clearance predictions. Significance Statement Application of the well-stirred liver model for clearance IVIVE in rat generally underpredicts the clearance of acids. The strong protein binding of acids is suspected to be one responsible factor for the underprediction. CL int,u determinations using rat liver microsomes incubations supplemented with 5% plasma resulted in an improved IVIVC. An empirical equation was derived from fu p and the ratio of CL int,u in the presence and absence of 5% plasma which can be applied to correct CL int,u -values from incubations in buffer.