In Silico Modeling of Nonspecific Binding to Human Liver Microsomes

Abstract

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...

Defining Desirable Central Nervous System Drug Space through the Alignment of Molecular Properties, in Vitro ADME, and Safety Attributes

“…The ADME assays were performed via reported methods as described previously for MDCK P app (14), MDR-MDCK P-gp (14), and metabolic stability (14,15,22,35).…”

“…Metabolic stability data for the drugs and the candidates, expressed as unbound intrinsic clearance (CL int,u ), was generated using a HT in vitro human liver microsome (HLM) assay and an in silico model of estimated microsomal free fraction (cF u,mic ) according to eq 2 (22). The use of CL int,u has been found to give a more accurate prediction of in vivo clearance than does CL int,app and as such we have used this value for these analyses (23).…”

Utility of MetaSite in Improving Metabolic Stability of the Neutral Indomethacin Amide Derivative and Selective Cyclooxygenase-2 Inhibitor 2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-phenethyl-acetamide

“…b Fraction unbound estimates in rat plasma and rat liver microsomes were obtained using in silico models developed at Pfizer as described previously with human liver microsomes (Gao et al, 2008). Literature free fraction of indomethacin in rat plasma is 0.040 (Dawidowicz et al, 2008), indicating high confidence in the in silico predictions.…”

“…In contrast, indomethacin and the glycine amide 4, which contained free carboxylic acid substituents, were completely resistant to metabolism in liver microsomes from both species. Considering that microsomal unbound fractions of amides 1, 2, and 3 predicted from in silico computational models (Gao et al, 2008) were comparable (Table 1), the free intrinsic clearance values (amide 1: 6646 ml/min/kg; amide 2: 2034 ml/min/kg; and amide 3: 772 ml/min/kg) obtained from scale-up of half-life data (normalized for nonspecific microsomal binding) (Obach, 1999), suggested that the strategy of attenuating metabolism via structural modification of the amide substituent in 1 had worked in our favor. Of much interest in this endeavor were the observations on the similarity in metabolic stability and free microsomal fraction of indomethacin and glycine amide 4 that resulted in comparable intrinsic clearance predictions of Ͻ10 ml/min/kg.…”

“…its neutral amide derivatives (final concentration ϭ 1 M) was assessed by monitoring substrate consumption after incubation with human and rat liver microsomes in the presence of NADPH cofactor for 30 min at 37°C (Table 1). Also shown in Table 1 are the physiochemical parameters (molecular weight and logP) and the unbound fractions of the test compounds in rat plasma and rat liver microsomes that were predicted using the in silico methodology described previously (Gao et al, 2008 , indomethacin-N-phenethyl amide (1) was highly unstable (t 1/2 Յ2 min) in rat and human liver microsomes supplemented with NADPH, whereas amides 2 and 3 were relatively more stable as reflected by the longer t 1/2 values. Unbound fraction estimates in rat microsomes and plasma were comparable for the three neutral amides.…”