Microsomal and hepatocyte binding in drug discovery: Method suitability and lipophilicity-based predictions.

Abstract

The accurate determination of the unbound fraction in microsomes (fumic) and hepatocyte incubations (fuhep) is essential for successful predictions of drug clearance and drug-drug interactions. This study systematically compared experimental fumic approaches (rapid equilibrium dialysis (RED), high-throughput dialysis (HTD), and ultracentrifugation) alongside with previously established empirical lipophilicity-based prediction models using a diverse test set of 143 compounds with varying lipophilicity, molecular weight, and ionization. The results suggested broad applicability of ultracentrifugation. RED underperformed for highly bound compounds with fumic ≤ 0.05, whereas HTD demonstrated good performance for the investigated low fumic range of ∼0.01 to 0.15. For a subset of 39 compounds, fuhep was measured using HTD, indicating excellent correlation with measured fumic data. Empirical lipophilicity-based models reliably predicted fumic and fuhep for compounds with low to moderate lipophilicity (logP/D ≤ 3.5) but demonstrated poor performance for highly lipophilic compounds with additional dependence on ionization class and molecular weight. These findings were summarized in a decision tree to guide the selection of empirical and experimental methodologies with minimal requirements for fumic measurements and fully eliminating the need for experimental fuhep data, supporting efficient and robust assessments of incubational binding in drug discovery.

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