Screening of drug metabolism by CE.
The use of CE for rapid assessment of metabolic stability of drugs with cytochrome P450 (CYP) enzymes, based on relative rates of reduced nicotinamide adenine dinucleotide phosphate (NADPH) consumption and nicotinamide adenine dinucleotide phosphate (NADP) production, was investigated. The separation conditions were as follows: capillary, 80.5 cm (75 microm id, 72 cm effective length for UV detection, 58 cm effective length for fluorescence detection); 25 mM sodium phosphate buffer (pH 8.8); 28 kV (80 microA) applied voltage; UV, 260 nm; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 25 degrees C. For UV detection, the incubation conditions were as follows: CYP3A4: 20 pmol/mL; NADPH: 1 mM; EDTA: 1 mM; concentration of the substrate: 5-10 times its reported literature K(m) value; temperature: 37 degrees C; incubation time: 15 min. For fluorescence detection, the concentrations were reduced to CYP3A4: 4 pmol/mL, NADPH: 20 microM, EDTA: 20 microM and substrate: 10 microM. Blank incubations were performed in the absence of substrate. Compared with the blank, significant differences were found for the consumption of NADPH and the production of NADP. The development of this assay system allows rapid assessment of metabolic stability relative to standard compounds, as well as potential identification of the major CYP involved in the metabolism. It would reduce the backlog of compounds that require LC/MS analysis, and thereby expedite the process of metabolic stability screening.
Zhang
Jie
J
Laboratory of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
Lou
Yijia
Y
Hoogmartens
Jos
J
Van Schepdael
Ann
A
D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004355:Drug Stability; D019075:Electrophoresis, Capillary / Q000379:methods*; D009249:NADP / Q000032:analysis / Q000737:chemistry; D010084:Oxidation-Reduction; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D004364:Pharmaceutical Preparations; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human