Pooled human liver microsomes
Pooled human liver microsomes (containing 20 mg/mL of protein) were thawed on ice. Microsome reactions were performed in microcentrifuge tubes by adding 183 µL of 0.1 M potassium phosphate buffer (pH 7.4), 2 µL of 1.0 mM IVM (prepared in acetonitrile 80% (v/v)), and 5 µL of microsomes. The tube was vortexed briefly and incubated at 37°C for 5 min in a shaking water bath. The reaction was initiated by adding 10 µL of 20 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) prepared in 100 mM potassium phosphate buffer pH 7.4. Total reaction volume per tube was 200 µL with the final concentration of 10 µM IVM and 1 mM NADPH. Each tube was vortexed briefly and a baseline sample (0 min control) was collected by aliquoting 100 µL of the metabolite fraction mixture described above to a separate tube with 100 µL of pre-chilled acetonitrile, which was kept on ice until centrifugation. Two separate negative control tubes were prepared, one without NADPH (negative co-factor control) but with an extra 10 µL of buffer and a second tube without ivermectin substrate (negative ivermectin control) with an extra 2 µL of buffer. All remaining reactions, including negative co-factor control, and negative ivermectin control, 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 to make a final 1:1 (v/v) ratio. All tubes (0 and 60 min reactions, negative co-factor and negative IVM controls) were vortexed briefly again and centrifuged at 10,000×gfor 15 minutes at 4°C. The supernatant was collected and kept frozen at -80°C until LC-MS/MS analysis. The reactions described above were performed using IVM, pure IVM-B1a and pure IVM-B1b as substrates (biological triplicate incubations for each substrate).
For NMR analysis, sixty tubes were prepared each with 10 µL of 1.0 mM ivermectin, 915 µL of 100 mM potassium phosphate buffer (pH 7.4), 25 µL of microsomes, and 50 µL of 20 mM NADPH for a final volume of 1 mL. Reactions were stopped at 60 min with ice-cold acetonitrile and centrifuged immediately as describe above. The supernatant was collected, evaporated in speed vacuum, and kept frozen at -80°C until NMR analysis.