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Metabolic enzyme inhibition tests were conducted to evaluate the inhibitory effects of the tested products on major subenzymes such as CYP450, UGT, SULT or CES, and to understand their direct inhibition or time-dependent inhibition (TDI) effects, so as to evaluate their possible metabolic interactions.
Incubation system | Cell component: liver cells Subcellular components: microsomes, S9 or homogenates of tissues such as liver, intestine, lung or kidney Gene recombination enzyme system |
Species | Man |
Tested enzymes | CYP450 enzyme: CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A, etc UGT enzymes: UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7 and 2B15, etc SULT enzymes: SULT1A1, 1A2, 1A3, 1B1, 1E1 and 2A1, etc CES enzymes: CES1 and CES2 Other: Other enzymes or subtypes such as AADAC, FMO are available upon request |
Test concentrations | 5 to 7 samples were designed according to known efficacy and toxicity data |
Probe substrate concentration | 1 or 1-5 (direct inhibition constant determination), based on the probe substrate Km value design |
Incubation system Protein concentration | Subcellular component: 0.1-1mg /mL (different concentrations available on request) Cell composition: 0.1-2×106 cells/mL Recombinant enzyme system: 250-500 pmol/mL or 0.1-1 mg/mL |
Incubation time | Direct inhibition or TDI IC50 shift: One pre-incubation time point, one incubation time point TDI KI and Kinact measurements: 2 to 5 time points for pre-incubation, one time point for formal incubation Available on request |
Coenzyme factor | NADPH or UDPGA (Other Coenzyme factors are available upon request) |
Analytical method | LC-MS/MS |
Data presentation | Relative production of probe substrate metabolites Relative activity (% of NC) Median inhibitory concentration (IC50) Inhibitory activity (%) IC50 migration Inhibition constant (Ki) Maximum deactivation rate Constant (Kinact) Concentration of free inhibitors (KI) leading to 50% maximum inactivation |