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.