The purpose of this study was to develop a method for estimating the hepatic clearance (CL(h)) without using a protein binding test. This method allows the simultaneous evaluation of the intrinsic hepatic clearance (CL(int)) with a correction for microsomal binding, and the free fraction in the serum (fu). It uses the decrease in metabolic velocity achieved by decreasing the free fraction of a compound in the incubation mixture (fu(inc)) by the addition of serum, and by changing the microsomal protein concentration. This method is denoted as the 'matrix inhibition method', because it uses the inhibition of the metabolic velocity by the incubation matrix. The metabolic rates of eight compounds (diazepam, imipramine, warfarin, and compounds A-E) were evaluated under several incubation conditions using rat serum and microsomes. The correlation of CL(int) evaluated using the method and using equilibrium dialysis after the CL(int) was corrected for microsomal binding was r = 0.968. The correlation of fu . CL(int) was r = 0.996. Although the method required a high enough fu and fu(microsomes) difference among the reaction conditions for each compound, it could evaluate CL(int) and fu simultaneously and easily by adding additional reaction conditions to the metabolic stability tests performed in ADME screening.
2007 John Wiley & Sons, Ltd