Optimization of the in vitro oxidative biotransformation of glimepiride as a model substrate for cytochrome p450 using factorial design

Linearity of enzyme reactions in microsomal incubations was assessed by monitoring the effect of incubation time (from 5 to 60 min) and HLM concentration (from 0.2 to 0.75 mg/ml) on metabolite formation of GLM. The ideal conditions for turnover of GLM were justified using 3x3 factorial design. F value was calculated to confirm the omission of insignificant terms from the full-model to derive a reduced- model polynomial equation. The regression equation was used to develop a contour plot that showed turnover rate within the limits of this design. The optimized reaction velocity data was extrapolated to carry out the kinetic studies in vitro to generate a saturation curve for the determination of Km and Vmax values.The reaction was found to be linear with respect to both incubation time between 24 and 50 min and HLM concentration between 0.3 to 0.65 mg/ml. The Km and Vmax values obtained by nonlinear least squares regression method was found to be 28.9？±？2.97 μMole and 0.559？±？0.017 μMole respectively. Lineweaver-Burk plot was also used to estimate Km and Vmax which yield value of 29.411？±？1.25 μMole and 0.571？±？0.020 μMole/min/mg protein respectively.The statistical approach successfully allows for the optimization of reaction time course experiments. The results obtained with linear as well as the nonlinear transformation were found to be in close agreement with each other which shows the best precision for estimates of Km and Vmax.For most drugs, biotransformation is the major route of elimination, and oxidative metabolism by cytochrome P450 (CYP450) enzymes is a common metabolic pathway [1]. More than 90% of oxidative metabolic reactions (phase I) of drugs are catalyzed by enzymes of the CYP450 family present in liver [2]. In order to investigate drug metabolism prior to human exposure, there are a number of options ranging from in vitro screening with human enzymes to in vivo assessment in experimental animals. Although animal models can provide information about