Reduced nicotinamide adenine dinucleotide phosphate

Mouse or human liver
microsomes and reduced nicotinamide adenine dinucleotide phosphate
(NADPH) were obtained from Fisher Scientific SL. This assay gives
information on the metabolic stability of early drug discovery compounds
based on liver microsomes. Microsome stability was tested by incubating
10 μM of test compounds (11 and 13) and verapamil (as positive metabolized control) with 1.0 mg/mL
hepatic microsomes (pooled human liver microsomes and pooled mouse
(CD-1) liver microsomes) in 0.1 M potassium phosphate buffer (pH 7.4)
with MgCl₂ 5 mM. The reaction was initiated by adding NADPH
(1 mM final concentration). Aliquots of 150 μL were collected
at defined time points (0, 5, 15, 30, 45, and 60 min) and added to
cold acetonitrile (150 μL) containing an internal standard (5
μg/mL warfarin) to stop the reaction and precipitate the protein.
After stopping the reaction, the samples were centrifuged at 4 °C
for 15 min and the loss of parent compounds was analyzed by HPLC–MS
using single ion mode (SIM) detection. Data were log transformed and
represented as half-life. All experiments were conducted by duplicate.

Human
liver microsomes and reduced nicotinamide adenine dinucleotide phosphate
(NADPH) were purchased from Fisher Scientific SL. This assay provides
information on the metabolic stability of early drug discovery compounds
based on liver microsomes. Microsome stability was tested by incubating
8 μM of the test compound and verapamil (as control) with 1.0
mg/mL hepatic microsomes (pooled human liver microsomes) in 0.1 M
potassium phosphate buffer (pH 7.4) with 5 mM MgCl₂. The
reaction was initiated by adding NADPH (1 mM final concentration).
Aliquots of 150 μL were collected at defined time points (0,
5, 15, 30, and 60 min) and added to cold acetonitrile (150 μL)
containing an internal standard (5 μg/mL warfarin) to stop the
reaction and precipitate the protein. After stopping the reaction,
the samples were centrifuged at 4 °C for 15 min, and the loss
of the parent compound was analyzed by high-pressure liquid chromatography
coupled to mass spectrometry (HPLC-MS). Data were log transformed
and represented as half-life. All experiments were conducted in duplicates.