Screening of drug metabolism by CE

Zhang, Jie; Lou, Yijia; Hoogmartens, Jos; Schepdael, Ann Van

doi:10.1002/elps.200600163

Cited by 7 publications

(11 citation statements)

References 17 publications

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“…The proper design and development of the in-capillary assay still needs much more work than the standard off-line method. Hence it comes as no surprise that among the numerous off-line methods, one can encounter greater technical, as well as methodological versatility [3,[39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56]. Nevertheless, one can expect novel and more sophisticated EMMA-based enzymatic assays, including ones based on both Phase I and Phase II metabolism enzymes.…”

Section: Discussionmentioning

confidence: 99%

An overview of on‐line systems using drug metabolizing enzymes integrated into capillary electrophoresis

2013

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Enzymatic assays using CE are now among the most noteworthy applications of this analytical technique in pharmacology-related investigations. Studies of metabolic pathways of new chemical entities mediated by drug metabolizing enzymes are attracting particular attention. Conventional CE-based enzymatic in vitro assays are generally restricted to the separation of reagents after incubation performed off-line. EMMA represents an alternative and fully prospective approach, allowing injection, reaction, separation, and detection to be conducted in a single capillary. Such an on-line system-in contrast to the standard approach-enables automation, miniaturization, and a significant reduction in reagent volumes, resulting in a very robust and cost-efficient method. Hence, EMMA could be a method of choice for the screening of new drugs, enzymatic inhibitors, and putative drug-drug interactions. This review provides a summary of reports covering the area of EMMA-based and related methods implemented into in vitro studies of drug metabolizing enzymes. A general description of the EMMA framework, enzyme families, and a concise discussion of the prognosis for the development of this methodology are given as well.

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Section: Discussionmentioning

confidence: 99%

An overview of on‐line systems using drug metabolizing enzymes integrated into capillary electrophoresis

2013

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“…Owing to the main purpose of this work -kinetic study of recombinant human CYP3A4, based upon relative rates of NADPH depletion or NADP formation -testosterone and nifedipine were chosen as preferred substrates because they are suitable probes to assess CYP3A4 activity. Moreover, to reduce the degradation rate of NADPH as much as possible, EDTA was not included in the incubation mixture [1]. Incubation mixtures contained 4 nM CYP3A4, 20 mM NADPH and one of the probe substrates (testosterone: 6.25, 12.5, 25, 50, 100, 200 mM; nifedipine: 3.125, 6.25, 12.5, 25, 50, 100 mM) in 10 mM sodium phosphate buffer at pH 7.4.…”

Section: Off-line Approachmentioning

confidence: 99%

“…The incubation was stopped by adding 100 mL of ice-cold methanol (final composition 1:1, aqueous/methanol). Samples were analyzed by a CE method previously developed for drug metabolism-screening studies, using an ARGOS 250B fluorescence detector (Flux Instruments, Basel, Switzerland) [1]. The separation conditions were as follows: capillary, 80.5 cm (75 mm id, 64 cm effective length); 25 mM sodium phosphate buffer (pH 8.8); 28 kV (80 mA) applied voltage; fluorescence detection, Exc: 310 nm, Em: 418 nm; capillary temperature: 251C.…”

Section: Off-line Approachmentioning

confidence: 99%

“…Therefore, by monitoring the relative depletion of NADPH, or the relative production of NADP, we should be able to determine the metabolic stability of compounds toward CYP-mediated metabolism in a high-throughput format [1]. CE, viewed as an alternative and a complement to liquid chromatography, has recently emerged as a powerful and versatile separation tool for the study of minute quantities of samples.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis

2008

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An electrophoretically mediated microanalysis method for the determination of CYP3A4 activity using testosterone and nifedipine as substrates was developed. Initially, the enzymatic reaction was performed off-line and the samples were subsequently injected into the capillary by pressure. The CYP3A4 activity was determined by quantitation of the reactant cofactor, NADPH. To further optimize, speed-up and miniaturize the enzyme assay, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product cofactor, NADP, employing the plug-plug mode of electrophoretically mediated microanalysis. An amplification step was introduced by means of an on-capillary incubation of 15 min, in order to accumulate enough reaction product to detect spectrophotometrically at 260 nm. This setup resulted in a fully automated assay, which can be carried out in less than 35 min. Using the Lineweaver-Burk equation, the Michaelis constants (K(m)) for the oxidation of testosterone and nifedipine by CYP3A4 were calculated to be 58.6+/-8.3 and 19.1+/-2.4 microM, respectively, which are consistent with off-line assay and previously reported values.

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“…1. This review, which is intended as an update of two earlier reviews mentioned above [2,4], gives a summary of the development of EMMA in enzymatic assays and derivatization reactions over the last two years. Visualization of incapillary reaction of glutathione oxidation using CMOS-based absorbance detector.…”

Section: Introductionmentioning

confidence: 99%

Advances in CE‐mediated microanalysis: An update

2007

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This review, as a continuation of two earlier reports, gives an overview of the recent developments, over the period from 2005 until now, in the use of electrophoretically mediated microanalysis (EMMA) methodology for the on-line study of enzymatic reaction and derivatization. The article is divided into two parts: (i) on-line enzymatic reaction by EMMA and (ii) on-line derivatization by EMMA. Following a brief introduction, a literature overview on enzymatic reaction is provided. The second part starts with an introduction of the purpose of derivatization and the nomenclature used in the area of in-capillary derivatization based on EMMA. The development of more integrated analytical platform that combines in-capillary derivatization and sample preconcentration is discussed. Reported derivatization procedures are summarized.

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Screening of drug metabolism by CE

Cited by 7 publications

References 17 publications

An overview of on‐line systems using drug metabolizing enzymes integrated into capillary electrophoresis

An overview of on‐line systems using drug metabolizing enzymes integrated into capillary electrophoresis

Kinetic study of cytochrome P450 by capillary electrophoretically mediated microanalysis

Advances in CE‐mediated microanalysis: An update

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