The Influence of Nonspecific Microsomal Binding on Apparent Intrinsic Clearance, and Its Prediction from Physicochemical Properties

Abstract

ABSTRACT:The apparent intrinsic clearance of 13 drugs has been determined using rat liver microsomes at three different concentrations of microsomal protein. The kinetics was studied using the in vitro half-life method. The nonspecific binding of these drugs to the microsomes was also studied under the same conditions, except for cofactor removal, using equilibrium dialysis. The intrinsic clearances are shown to be dependent on the microsomal concentration, but are approximately constant when corrected for the extent of nonspecific binding to the microsomes. The large difference between observed intrinsic clearance and unbound intrinsic clearance that exists for some compounds, particularly lipophilic bases, is highlighted. A simple model has been developed for understanding the binding of compounds to microsomes and is demonstrated to accurately predict the extent of microsomal binding at one concentration of microsomes from measurement at another. The binding of a further 25 drugs to rat liver microsomes at a microsomal concentration of 1 mg/ml was also studied, along with measurements of lipophilicity using octanol-water partition coefficients. It is shown that the extent of microsomal binding is correlated with lipophilicity, but that basic compounds show a different behavior to acidic and neutral compounds. Microsomal binding is shown to be best predicted using a model where logP is used for basic compounds, and logD 7.4 is used for acidic and neutral compounds. This model has been developed further so that the extent of binding to microsomes of any given concentration can be estimated purely from a knowledge of lipophilicity and ionization.In recent years, kinetic measurements using liver microsomes have been increasingly used as a measure of the rate of metabolic drug oxidation in drug discovery. The results from these in vitro assays are then frequently processed, through a variety of methods, to produce predictions of the in vivo metabolic clearance of compounds in humans (Iwatsubu et al., 1997;Obach et al., 1997;Lave et al., 1999). Methods that are often used for scaling such in vitro data to in vivo predictions are the well stirred model and the parallel tube model, and these models contain plasma free fraction terms that account for the effect of plasma binding on the clearance of compounds (Pang and Rowland, 1977). However, it is frequently found that inclusion of the plasma binding correction can lead to underprediction of the in vivo clearance, particularly for nonacidic compounds, and better performance can often be obtained by ignoring the correction altogether (Obach 1997(Obach , 1999Obach et al., 1997). It has been recognized that nonspecific microsomal binding in the in vitro metabolic assays can significantly affect the observed kinetics of metabolism and hamper the accurate prediction of clearance, and there are now several examples where knowledge of the extent of microsomal binding can lead to a better understanding of the relationship between in vitro metabolism and in vivo pharmacokinet...

A Unified Model for Predicting Human Hepatic, Metabolic Clearance From in Vitro Intrinsic Clearance Data in Hepatocytes and Microsomes

“…Some controversy also still exists over use of fu inc , with some laboratories having suggested that fu inc and fu b may cancel, negating their inclusion in liver models (Obach et al, 1997). Recent reports have challenged this assumption (Obach, 1999;Austin et al, 2002) and perhaps suggest that consideration of CL h rather than CL int, in vivo may desensitize such analyses, particularly to errors associated with higher enzyme activities (Ito and Houston, 2004).…”

“…Some controversy also still exists over use of fu inc , with some laboratories having suggested that fu inc and fu b may cancel, negating their inclusion in liver models (Obach et al, 1997). Recent reports have challenged this assumption (Obach, 1999;Austin et al, 2002) and perhaps suggest that consideration of CL h rather than CL int, in vivo may desensitize such analyses, particularly to errors associated with higher enzyme activities (Ito and Houston, 2004).The aim of this study was to investigate direct in vitro-in vivo scaling of human in vitro data generated in hepatocytes and microsomes for predicting human clearance in vivo by applying recently described models for estimating fu inc (Austin et al, 2002(Austin et al, , 2005. To provide a more mechanistic insight, in vitro human CL int data were compiled from recent in-house and published studies, and associated in vivo CL int values were derived from published CL h data.…”

“…The aim of this study was to investigate direct in vitro-in vivo scaling of human in vitro data generated in hepatocytes and microsomes for predicting human clearance in vivo by applying recently described models for estimating fu inc (Austin et al, 2002(Austin et al, , 2005. To provide a more mechanistic insight, in vitro human CL int data were compiled from recent in-house and published studies, and associated in vivo CL int values were derived from published CL h data.…”

Substrate-dependent modulation of the catalytic activity of CYP3A by erlotinib

“…The unbound K I (K I, u ) was calculated according to equation (6), where f u, m is the free fraction of erlotinib in the microsomes. f u, m is predicted according to equation (7) as previously reported [28] . The terms are defined as follows: C mic is the microsomal protein concentration used in the preincubation, and logP is the log of the octanol-water (pH 7.4) partition (P) coefficient of the erlotinib.…”

Time‐dependent inhibition (TDI) of CYP3A4 and CYP2C9 by noscapine potentially explains clinical noscapine–warfarin interaction

“…To determine kobs (observed inactivation rate) values, the decrease in natural logarithm of activity over time was plotted for each noscapine concentration, and kobs values were described as the negative slopes of the line. Inactivation kinetic parameters were calculated using nonlinear regression of the data according to the Equation 1: as previously reported [14]. The terms are defined as follows: Cmic is the microsomal protein concentration used in the preincubation, and LogP is the log of the octanolbuffer (pH = 7.4) partition (P) coefficient of the noscapine.…”