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474644 
Journal Article 
Direct determination of unbound intrinsic drug clearance in the microsomal stability assay 
Giuliano, C; Jairaj, M; Zafiu, CM; Laufer, R 
2005 
Yes 
Drug Metabolism and Disposition
ISSN: 0090-9556
EISSN: 1521-009X 
33 
9 
1319-1324 
English 
The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CLin, u) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CLin, u from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CLin, u of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay. 
in-vitro metabolism; nonspecific-binding; prediction; propranolol; inhibition; impact