Drug Design and Success of Prospective Mouse In Vitro–In Vivo Extrapolation (IVIVE) for Predictions of Plasma Clearance (CLp) from Hepatocyte Intrinsic Clearance (CLint)

Hepatocyte intrinsic clearance (CL_int) and methods of in vitro-in vivo extrapolation (IVIVE) are often used to predict plasma clearance (CL_p) in drug discovery. While the prediction success of this approach is dependent on the chemotype, specific molecular properties and drug design features that govern these outcomes are poorly understood. To address this challenge, we investigated the success of prospective mouse CL_p IVIVE across 2142 chemically diverse compounds. Dilution scaling, which assumes that the free fraction in hepatocyte incubations (f_u,inc) is governed by binding to the 10% of serum in the incubation medium, was used as our default CL_p IVIVE approach. Results show that predictions of CL_p are better for smaller (molecular weight (MW) < 500 Da), less polar (total polar surface area (TPSA) < 100 Ų, hydrogen bond donor (HBD) ≤1, hydrogen bond acceptor (HBA) ≤ 6), lipophilic (log D > 3), and neutral compounds, with low HBD count playing the key role. If compounds are classified according to their chemical space, predictions were good for compounds resembling central nervous system (CNS) drugs [average absolute fold error (AAFE) of 2.05, average fold error (AFE) of 0.90], moderate for classical druglike compounds (according to Lipinski, Veber, and Ghose guidelines; AAFE of 2.55; AFE of 0.68), and poor for nonclassical "beyond the rule of 5" compounds (AAFE of 3.31; AFE of 0.41). From the perspective of measured druglike properties, predictions of CL_p were better for compounds with moderate-to-high hepatocyte CL_int (>10 μL/min/10⁶ cells), high passive cellular permeability (P_app > 100 nm/s), and moderate observed CL_p (5-50 mL/min/kg). Influences of plasma protein binding (f_u,p) and P-glycoprotein (Pgp) apical efflux ratio (AP-ER) were less pronounced. If the extended clearance classification system (ECCS) is applied, predictions were good for class 2 (P_app > 50 nm/s; neutral or basic; AAFE of 2.35; AFE of 0.70) and acceptable for class 1A compounds (AAFE of 2.98; AFE of 0.70). Classes 1B, 3 A/B, and 4 showed poor outcomes (AAFE > 3.80; AFE < 0.60). Functional groups trending toward weaker CL_p IVIVE were esters, carbamates, sulfonamides, carboxylic acids, ketones, primary and secondary amines, primary alcohols, oxetanes, and compounds liable to aldehyde oxidase metabolism, likely due to multifactorial reasons. Multivariate analysis showed that multiple properties are relevant, combining together to define the overall success of CL_p IVIVE. Our results indicate that the current practice of prospective CL_p IVIVE is suitable only for CNS-like compounds and well-behaved classical druglike space (e.g., high permeability or ECCS class 2) without challenging functional groups. Unfortunately, based on existing mouse data, prospective CL_p IVIVE for complex and nonclassical chemotypes is poor and hardly better than random guessing. This is likely due to complexities such as extrahepatic metabolism and transporter-mediated disposition which are poorly captured by this methodology. With small-molecule drug discovery increasingly evolving toward nonclassical and complex chemotypes, existing CL_p IVIVE methodology will require improvement. While empirical correction factors may bridge the gap in the near future, improved and new in vitro assays, data integration models, and machine learning (ML) methods are increasingly needed to address this challenge and reduce the number of nonclinical pharmacokinetic (PK) studies.