| Fibrates are widely used for the treatment of dyslipidemia mediated by peroxisome proliferative activated receptorα. As a class of drug, the active form of fibrates has a carboxyl-group, and ready for metabolism by phase I and phase II enzymes. However, the contribution of phase II metabolism is always been ignored in the in vitro metabolic study of drug. Prediction of in vivo clearance for drugs metabolism by phase I and phase II enzymes simultaneously is in the initial period of development, and still challenges the in vitro screening systems. In this study, the metabolic stability of commonly used fibrates (gemfibrozil, clofibrate, fenofibrate, bezafibrate) were investigated using rat and human liver microsomes. Three reactions were initiated by adding metabolic enzymes cofactors of NADPH and UDPGA individually or simultaneously for cytochromes P450-mediated and UGT-mediated fibrate biotransformations. By substrate depletion approach, the apparent enzyme kinetics parameters michaelis constant (Km), maximum velocity (Vmax), and the intrinsic clearance (CLint) were determined in rat liver microsomes. The Michaelis constant (Km), maximum velocity (Vmax), and the intrinsic clearance (CLint) of Gem in P450-mediated metabolism were determined to be 11±4μM, 1861±967 nmol/min/mg, and 172±22μL/min/mg. Km of Gem for glucuronidation was measured to be similar with the value in phase I oxidation reaction (11±1μM). However, the Vmax was calculated to be 6993±1183 nmol/min/mg to generate the CLint of 643±26μL/min/mg, which were about fivefold higher than the value in the phase I oxidation reaction, indicating the phase II pathway was much important than that of the phase I pathway. In the combined phase I and phase II reaction, the apparent Km value was the lowest (8±3μM), the Vmax values was similar with the value in phase II reaction (6100±3041 nmol/min/mg), producing the highest CLint value of 798±103μL/min/mg in the three reactions. Similarly, the Km values of CPIB for the P450-mediated oxidation, UGT-mediated glucuronidation, and both simultaneous oxidation plus glucuronidation were measured to be 0.04±0.004, 2±1, and 2±1μM, respectively. The Vmax values for these three kinds of reactions were determined as 2.3±0.3, 163±113, and 64±49 nmol/min/mg, respectively. Therefore, the CLint was calculated to be 43±11, 88±12, and 119±15μL/min/mg for CPIB, respectively. The intrinsic clearance was too low for other fibrates studied. Referring the contributions by NADPH-driving and UDPGA-driving reactions for gemfibrozil, the ratio of P450-mediated pathway over both P450-mediated and UGT-mediated pathways is 22% and that for UGT-mediated pathway is 81%. Similarly, ratio of 36% and 74% was determined for P450-mediated pathway and UGT-mediated pathway for p-chlorophenoxyisobutyric acid, respectively. These results suggested that individual measurements either NADPH-dependent or UDPGA-dependent pathway would cause errors, and could not represent the actual biotransformation in vivo, since it is a competitive parallel reaction. It was also found that the phase II pathway in either rat liver microsomes or human liver microsomes are the major pathway for fibrates despite the species difference in term of elimination velocity.
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