ABSTRACT:Estimation of unbound fraction of substrate in microsomal incubation media is important in accurately predicting hepatic intrinsic clearance and drug-drug interactions. In this study, the unbound fraction of 1223 drug-like molecules in human liver microsomal incubation media has been determined using equilibrium dialysis. These compounds, which include 27 marketed drug molecules, cover a much broader range of physiochemical properties such as hydrophobicity, molecular weight, ionization state, and degree of binding than those examined in previous work. In developing the in silico model, we have used two-dimensional molecular descriptors including cLogP, Kier connectivity, shape, and E-state indices, a subset of MOE descriptors, and a set of absorption, disposition, metabolism, and excretion structural keys used for our in-house absorption, disposition, metabolism, excretion, and toxicity modeling. Hydrophobicity is the most important molecular property contributing to the nonspecific binding of substrate to microsomes. The prediction accuracy of the model is validated using a subset of 100 compounds, and 92% of the variance is accounted for by the model with a root mean square error (RMSE) of 0.10. For the training set of compounds, 99% of variance is accounted for by the model with a RMSE of 0.02. The performance of the developed model has been further tested using the 27 marketed drug molecules with a RMSE of 0.10 between the observed and the predicted unbound fraction values.In drug discovery, the ultimate goal of a medicinal chemist is to rationally design compounds that are safe and efficacious at a marketable dose regimen. Such chemical design efforts frequently rely upon intrinsic clearance (CL int ) and P450 inhibitory potency (IC 50 ) estimates derived from human liver microsomal incubations. With relatively few assumptions, these estimates can be expected to have proportional implications for oral dose requirements and therapeutic windows against pharmacokinetic drug-drug interactions. For example, the classic well stirred model of hepatic clearance can be used to illustrate the directly proportional relationship between CL int and oral dose by eq. 1 (Wagner et al., 1965). Other oral dose determinants include the unbound steady-state average concentration necessary for the desired pharmacologic response (C ss, u ), dose interval (), and fraction of dose absorbed (fa):In addition, by assuming that inhibition of P450 occurs via a competitive mechanism, eq. 2 is a valid and commonly used approach to calculate the therapeutic window against pharmacokinetic drug-drug interactions:Therapeutic window ϭ C ss, u IC 50It has now been well established in the scientific literature that nonspecific binding of compounds into microsomal phospholipids will confound the experimental estimation of CL int and IC 50 by decreasing the fraction of unbound drug (fu mic ) to metabolizing enzymes within the incubation (Kalvass et al., 2001;Margolis and Obach, 2003;Obach, 1997Obach, , 1999Tran et al., 2002). In such ca...