ABSTRACT:Edaravone was launched in Japan in 2001 and was the first neuroprotectant developed for the treatment of acute cerebral infarction. Edaravone is mainly eliminated as glucuronide conjugate in human urine (approximately 70%), but the mechanism involved in the elimination pathway remains unidentified. We investigated the glucuronidation of edaravone in human liver microsomes (HLM) and human kidney microsomes (HKM) and identified the major hepatic and renal UDP-glucuronosyltransferases (UGTs) involved. As we observed, edaravone glucuronidation in HLM and HKM exhibited biphasic kinetics. The intrinsic clearance of glucuronidation at high-affinity phase (CL int1 ) and low-affinity phase (CL int2 ) were 8. , respectively, for HLM and were 78.5 ؎ 3.9 and 3.6 ؎ 0.5 l ⅐ min ؊1 ⅐ mg
؊1, respectively, for HKM. Screening with 12 recombinant UGTs indicated that eight UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B17) produced a significant amount of glucuronide metabolite. Thus, six UGTs (UGT1A1, UGT1A6, UGT1A7, UGT1A9, UGT2B7, and UGT2B17) expressed in human liver or kidney were selected for kinetic studies. Among them, UGT1A9 exhibited the highest activity (CL int1 ؍ 42.4 ؎ 9.5 l ⅐ min ؊1 ⅐ mg
Ib is a new nonpeptide AT1 receptor antagonist, which plays an active role in cardiovascular protection. Ib monoglucuronide has been identified as its main metabolite. A detailed study of Ib glucuronidation is important for predicting potential DDI. Besides, the elucidation of the “BSA effect” in Ib glucuronidation would make obtained kinetic parameters more predictive in IVIVE. “BSA effect” means that there is a significant change in in vitro kinetic parameters when generated from incubations performed in the presence of bovine serum albumin (BSA). Five UGTs (UGT1A3, UGT2B4, UGT2B7, UGT1A9 and UGT1A8) were identified that produced abundant Ib monoglucuronide, especially UGT1A3. We investigated Ib glucuronidation in liver microsomes from different species (rat, dog, human) and in five identified major human UGTs. Ib glucuronidation in liver microsomes and recombinant human UGTs all showed substrate inhibition kinetics. DLM showed the strongest affinity and activity, HLM showed the lowest affinity, and RLM showed the weakest activity. The addition of BSA did not alter the enzyme kinetics, but significantly altered enzyme kinetic parameters resulting in a reduction in Km value and an increase in CLint value. However, high concentrations of BSA could significantly attenuate this positive effect on enzyme affinity and activity, and the effect of BSA on the Vmax of Ib glucuronidation was opposite in different enzyme sources. In conclusion, this study demonstrated the substrate inhibition kinetics of Ib glucuronidation in the liver metabolism and the effect of BSA on its kinetic parameters, in order to provide more accurate in vitro data for in vivo prediction.
A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of trans-stilbene glycoside (SG) in rat plasma. As trans-SG can be rapidly isomerized under light exposure, trans-SG plasma samples were prepared in the dark and assayed immediately. Trans-SG and internal standard were extracted by protein precipitation. Chromatographic separation was achieved on a C(18) column with a gradient elution program. The detection of analytes was performed by negative ion via multiple reaction monitoring mode. The precursor-to-product ions of m/z 405.1 → 242.9 for trans-SG and m/z 389.1 → 226.9 for polydatin (internal standard) were monitored. No interference of endogenous components was observed for any plasma samples that we studied.The method was linear over the concentration range of 1.0-1000.0 ng/mL with a good correlation coefficient. The lower limit of quantification was 1.0 ng/mL for trans-SG. The intra and inter-batch accuracy for trans-SG in stable rat plasma samples ranged from 93.3 to 102.7% and the variation was less than 8.1%. The extraction recoveries of trans-SG in rat plasma were from 102.8 to 112.4% and the matrix effects were also acceptable. This method was successfully applied to pharmacokinetic study of trans-SG in rats after intravenous administration.
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