Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis

Zhang, Jie; Ha, Pham Thi Thanh; Lou, Yijia; Hoogmartens, Jos; Schepdael, Ann Van

doi:10.1016/j.jpba.2005.04.019

Cited by 15 publications

(7 citation statements)

References 24 publications

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“…Verapamil is a commonly used marker substrate of CYP3A4 activity, its predominant metabolites being 2‐(3,4‐dimethoxyphenyl)‐5‐methylamino‐2‐isopropylvaleronitrile (D‐617) and norverapamil via N ‐dealkylation and N ‐demethylation, respectively 34–37. An off‐line CE assay assessing CYP3A4 activity with verapamil as a probe substrate was developed in our lab previously 38 and adopted in this study with some small changes. Capillary length was shortened to reduce analysis time.…”

Section: Resultsmentioning

confidence: 99%

Electrostatic Complexation and Photoinduced Electron Transfer between Zn‐Cytochrome c and Polyanionic Fullerene Dendrimers

Braun

Atalick

Guldi

et al. 2003

Chemistry A European J

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Two dendritic fullerene (DF) monoadducts, 2 and 3, which can carry up to 9 and 18 negative charges, respectively, were examined with respect to electrostatic complexation with Cytochrome c (Cytc). To facilitate comprehensive photophysical investigations, the zinc analogue of Cytc (ZnCytc) was prepared according to a novel, modified procedure. The association of ZnCytc and DF, and consequential photoinduced electron transfer within ZnCytc-DF from the photoexcited protein to the fullerene, was proven by fluorescence spectroscopy and transient absorption spectroscopy. These findings were also supported by circular dichroism as well as by extensive molecular dynamics (MD) simulations.

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Section: Resultsmentioning

confidence: 99%

Electrostatic Complexation and Photoinduced Electron Transfer between Zn‐Cytochrome c and Polyanionic Fullerene Dendrimers

Braun

Atalick

Guldi

et al. 2003

Chemistry A European J

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“…However, the Michaelis parameters can only be compared with values obtained using the same enzyme source and identical conditions in the enzymatic assay. Even using the same source of enzyme, different batches can produce different results . In that work, baculovirus‐expressed human CYP enzymes, prepared following a published procedure, were used and the experimental conditions of the assay were very different from ours (see Section ).…”

Section: Resultsmentioning

confidence: 99%

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: Application to fluoxetine metabolism by CYP2D6

et al. 2013

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In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 μM for S-FLX and 39 ± 5 μM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX.

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“…Preliminary experiments were performed with the classic CYP metabolic assay approach, by measuring the formation of reaction products, the metabolites [4,6]. R-Warfarin and verapamil were chosen as model substrates for CYP3A4.…”

Section: Preliminary Conditionsmentioning

confidence: 99%

“…The separation conditions were as follows: capillary, 80. In the case of verapamil, CYP3A4 activity was determined by the quantitation of the product, norverapamil, based on separation by CE with UV detection [6]. The separation conditions were as follows: capillary, 80.5 cm (75 mm id, 72 cm effective length); 50 mM sodium phosphate buffer (pH 8.8); 20 kV (100 mA) applied voltage; UV, 200 nm; capillary temperature, 257C.…”

Section: Preliminary Conditionsmentioning

confidence: 99%

Screening of drug metabolism by CE

et al. 2006

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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.

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Kinetic study of CYP3A4 activity on verapamil by capillary electrophoresis

Cited by 15 publications

References 24 publications

Electrostatic Complexation and Photoinduced Electron Transfer between Zn‐Cytochrome c and Polyanionic Fullerene Dendrimers

Electrostatic Complexation and Photoinduced Electron Transfer between Zn‐Cytochrome c and Polyanionic Fullerene Dendrimers

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: Application to fluoxetine metabolism by CYP2D6

Screening of drug metabolism by CE

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