The inhibitors were common for Pimobendan Vetmedin naphthoflavone 1A2, 2A6 for tranylcypromine, 2B6 and 2C19 to ticlopidine, quercetin for 2C8, 2C9 for sulphaphenazole, quinidine used for 2D6, 2E1 for diethyldithiocarbamate and ketoconazole for 3A. In controlled experiments Positive, 10 M naphthoflavone the formation of acetaminophen inhibition of phenacetin, 1 M tranylcypromine the formation of 7 hydroxycoumarin coumarin inhibited, inhibits 10 M ticlopidine, the formation of hydroxybupropion to bupropion and training hydroxyomprazole the omprazole, 1 M quercetin, the formation of 6 hydroxypaclitaxel with paclitaxel inhibited the formation of 1 M sulphaphenazole hydroxytolbutamide tolbutamide inhibited 1 M quinidine inhibits the formation of dextrorphan, dextromethorphan, 10 M diethyldithiocarbamate to inhibit formation of hydroxychlorzoxazone of chlorzoxazone, and 1 M ketoconazole inhibited the formation of a hydroxymidazolam to midazolam. The reactions were terminated by addition of 400 l of acetonitrile ice with 100 ng / ml MI 63rd The samples were vortexed for 30 s and centrifuged at 14,000 rpm for 5 min. The supernatant was analyzed by LC-MS / MS to monitor the rate of formation of 17 metabolites DMAG. Ion Trnsfer length For MRM determination of M1, M2, M3, M4, M6 and were 3,175,633, 633 3322, 603 3510, 603 3524, and 6,653,157 respectively. The percentage of inhibition were by the ratio Ratio of the amounts of metabolites with and without the specific inhibitor was formed. Incubation of recombinant CYP450 enzymes. The incubation mixtures containing 20 pmol recombinant enzyme CYP450, 0.2 mg of reduced NADPH and 3.3 mM MgCl 2 were pre-incubated in 0.1 M phosphate buffer at 37 in a stirred bath water. E. coli microsomes controlled control without the cDNA of human P450 as were used negative. Reactions were initiated by addition of 17 DMAG.
After 30 minutes incubation, the reactions HA-1077 were terminated by addition of 400 l of acetonitrile ice with 100 ng / ml MI 63rd LC-MS / MS was used to monitor the rate of formation of 17 metabolites DMAG. Formation rates of metabolites in each CYP450 isoform were expressed as a percentage of their formation rate in CYP3A4. Results for shares of the metabolites in the incubations. The Km values for the formation of 17 of 17 AAG and AG M1 and M3 of 17 DMAG in HLM were as 29.1, 6.61, and 7.83 m respectively, the h Ago were those determined to anf Nglichen concentrations of 17 and 17 AAG DMAG in stability tsassays. The percentages were determined tze Of GA and 17 AAG and the corresponding hydroquinone and 19 glutathionyl conjugates of stability t in Figure 2 Values were expressed as percentages Tze normalized Peakfl Surface of GA or AAG 17 and the internal standard, expressed on a 1 min incubation. GA has a high metabolic stability of t in HLM in the absence of reduced GSH. Sixty-three percent of the GA was metabolized after 2 h incubation of 1 mg / ml HLM. A total of 6.4 to 9.3% of GAH2 has been w Received during the incubation. A Similar amount of GAH2 was also detected in the incubation with HLM in the shell, suggesting that the formation GAH2 not dependent on CYP450 enzymes Nts. However, the presence of 5 mM GSH reduced entered Born in the rapid metabolism of GA in the fir.