Androgen Receptor Antagonists proposed on the basis of the profiles identified metabolite

Fairly characterized by liquid Androgen Receptor Antagonists chromatography-tandem mass spectrometry and cytochrome P450 enzymes that were identified for the formation of metabolites. Been proposed on the basis of the profiles identified metabolite biotransformation pathways for 17 DMAG in HLM. Materials and Methods Materials. GA, 17 AAG and DMAG 17 were purchased from LC Laboratories. Instead of GSH reduced NADPH, MgCl 2, 0.1 M phosphate buffer, formic Acid naphthoflavone, tranylcypromine, quercetin sulphaphenazole, ticlopidine, diethyldithiocarbamate, quinidine, and ketoconazole were from Sigma Aldrich. 63 MI was obtained from Prof. S. Wang. High performance liquid chromatography grade acetonitrile was purchased from Thermo Fisher Scientific. HPLC grade water using a MilliQ-water system. HLM pooled, purified Escherichia coli expressed recombinant human P450 enzymes co-expressed with human cytochrome b5, and E. coli contr microsomes were from XenoTech LLC. Metabolic stability t test. DMAG GA, 17 or 17 AAG incubated with HLM in the absence or presence of reduced GSH to 37th The enzymes were activated by reduced NADPH. The incubation was diluted with 0.1 M phosphate buffer to 0.6. The final concentrations of the drug, microsomes, NADPH, phosphate buffer, and MgCl 2 was 5 M, 1 mg / ml, 1 mM, 0.1 M and 3.3 mM. To test GSH conjugation, was the final concentration of 5 mm GSH REDUCE. An aliquot of 40 l mixture were collected at 1, 5, 10, 15, 30, 45, 60, 120 min and proteins, Then filled with 120 l of acetonitrile containing an internal ice MI executed 63 standard. The samples were centrifuged at 14,000 rpm for 5 min and 10 l of supernatant was injected into LC-MS. To determine the apparent Km values for the formation of GA 17 AAG AAG of 17 17 different concentrations were 0.25 mg / ml HLM incubated for 15 min in triplicate. In Similar way to determine the m values for the formation of M1 and M3 of 17 DMAG, 17 different concentrations of AAG in 0.5 mg / ml HLMs for 30 min were incubated in triplicate.
Formation rates of metabolites were fitted to Michaelis-Menten equation using WinNonlin. Incubations with HLM for identification of metabolites. 17 DMAG with HLM in 0.6 ml of phosphate buffer at 37th The final Mitoxantrone concentrations of microsomes, NADPH, phosphate buffer, and MgCl 2 were the same as in the stability Tstest described. The anf Ngliche concentration of 17 DMAG was 10 M. Two different controlled Were produced were negative, while with boiled microsomes or doping M 10 17 DMAG after the F Precipitation of proteins. Both samples and controlled Negative were incubated for 2 h and the reaction was stopped with 1.2 ml of acetonitrile ice exemplary Cases the proteins. Moreover, in order to evaluate the GSH-conjugation, 17 with DMAG were eingeschr Nkter GSH and NADPH incubated in 0.1 M phosphate buffer in the presence or absence of HLM at 37 for 2 h. After Proteinf Precipitation samples have been checked and Negative centrifuged at 14,000 rpm for 5 min LC-MS analysis. LC-MS / MS. An Agilent 1200 HPLC system was used for the separation. The treated samples were loaded onto a C18-S Injected molecules Zobarx SB. A mobile phase consisting of 0.1% formic Acid and 10 mM ammonium formate in water and mobile phase.