Improving In Vitro–In Vivo Extrapolation 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 approximately 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 in the presence and absence of 5% plasma. A minor impact of plasma on clearance IVIVC was found for moderately bound compounds (fraction unbound in plasma [fu_p] ≥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 from 0.10 to 0.36. Correlating fu_p with the scaled unbound intrinsic clearance ratio in the presence or absence of plasma allowed the establishment of an empirical, nonlinear 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 in vitro–in vivo extrapolation (IVIVE) in rat generally underpredicts the clearance of acids and the strong protein binding of acids is suspected to be one responsible factor. Unbound intrinsic in vitro clearance (CL_int,u) determinations using rat liver microsomes supplemented with 5% plasma resulted in an improved IVIVE. An empirical equation was derived that can be applied to correct CL_int,u-values in dependance of fraction unbound in plasma (fu_p) and measured CL_int in buffer.