cDNA-expressed cytochrome P450 enzyme
The metabolism of ivermectin was studied in vitro using cDNA-expressed human cytochrome P450 enzymes CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5 (1,000 pmol/mL). In separate tubes for each CYP isoform, the following reagents were added: 166 µL of 0.1 M potassium phosphate buffer (pH 7.4), 2 µL of 1.0 mM ivermectin (prepared in acetonitrile 80% (vol/vol)), and 20 µL of CYP enzyme. A separate tube was prepared for the negative control (no CYP enzyme) by adding 20 µL of insect cell microsomes. A second negative control was prepared to evaluate the effect of reductase by adding 20 µL of human P450 oxidoreductase +b5. The tubes were vortexed briefly and incubated at 37°C for 5 min in a shaking water bath. The reactions were initiated by adding 12 µL of NADPH regenerating system solution (pre-mixed 10 µL of solution A and 2 µL of solution B). Each tube was vortexed briefly and a baseline sample (0 min control) was collected by aliquoting 100 µL of the incubation to a separate tube with 100 µL of pre-chilled acetonitrile, which was held on ice until centrifugation. All tubes (reactions and controls) were incubated at 37°C for 60 min with gentle shaking. After 60 min of incubation, all tubes were removed from the water bath and cold acetonitrile was added immediately at a 1:1 ratio (v/v). Tubes were vortexed briefly and centrifuged at 10,000×g for 15 minutes at 4°C. The clear supernatant was collected into new microcentrifuge tubes and kept frozen at -80°C until LC-MS/MS analysis. The incubations and reactions described above were performed in triplicate.