In Vitro Screening of Metabolic Clearance Using Two Concentration Points

Eric      T. Williams; Nadia      Farah; Robert      D. Pelletier; W.      George Lai

Abstract

For high throughput screens, the quickest methodology possible is desirable, but a substantial amount of potentially useful information is lacking since most screens for metabolic stability are conducted at one concentration, and sometimes at one time point. Information that would benefit projects during the discovery phase are to know the metabolic rate linearity (Km value) and projected hepatic clearance (CLh value), which is possible by the addition of one more concentration. This study used the FDA-preferred probe cytochrome P450 substrates to determine Km, Vmax, and CLint values. The results showed that compounds with relatively high metabolic rates produced more accurate and reproducible results that match well with predicted Km values according to the FDA. On the other hand, compounds with relatively low metabolic rates yielded more variable results. Thus, the use of two substrate concentrations should be useful with screening assays for assessing the kinetic values for other compounds.

Keywords: Cytochrome P450, metabolic stability, Michaelis-Menten, drug discovery, screening, FDA-preferred probe, hepatic intrinsic clear-ance, liver microsomes, pharmacokinetic, drug-drug interaction, Michaelis-Menten kinetics, intra-operator variability, pseudo-first order kinetics, Michaelis-Menten Curves, nebulizing, phenacetin, nicotine, taxol, diclofenac, testosterone, (S)-warfarin, dextromethorphan, LC/MS/MS, coumarin, efavirenz, bupropion, tolbutamide, (S)-mephenytoin, bufuralol, chlorzoxazone, midazolam, ADME, Reproducibility, Midazolam Metabolism, Potential P450 Inhibition