Microreaction in CE for drug metabolism studies (Lecture)
| Authors | |
|---|---|
| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | Proceeding of 16th International Conference "Analytical Methods and Human Health". |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | drug metabolism; microsomes; on-capillary microreaction; electrophoresis |
| Description | Drug metabolism studies are an important part in early drug discovery. Traditionally, in vitro tests are necessary to identify biotransformation of a parent drug. Human liver microsomes, the popular in vitro model, contain predominantly cytochromes P450. These enzymes play a dominating role in the phase I metabolism of xenobiotics embracing the clearance of drug. Besides well established liquid chromatography-mass spectrometry methods, capillary electrophoretic modes offer direct automated assays for drug metabolism studies. The reaction and the final separation can take place in-capillary at the same time or consecutively. Electrophoretically mediated microanalysis (EMMA) is a well appreciated technique for enzymatic microreactions. Combined with the partial filling method, EMMA opens broad application chances to overcome individual difficulties related to the particular enzymatic system. The injection of all reaction components in appropriate order and amount permits the on-line metabolite generation at ideal reaction conditions. The drug metabolism was studied with the use of human liver microsomes and dextromethorphan, as a substrate. The most involved cytochrome P450 isoforms 3A4 and 2D6 transform the substrate to 3-methoxymorphinan, 3-hydroxymorphinan and dextrorphan. Screening reactions were performed in fused silica capillary at 37C and a powerful rinsing step was executed after each run. The background electrolyte consists of borate buffer, linear polyacrylamide, as electroosmotic flow modifier and isopropanol, selectivity additive allowing separation of all four compounds. The optimized partial filling method parameters (reaction component order and injection conditions) were utilized to monitor the drug metabolism. The presented EMMA method could be integrated in preliminary studies at first stage of drug discovery. This biotransformation study could help to screen new drug candidates without a need of radiolabeling, with minimal sample preparation and consumption and thus to speed up data throughput and to decrease the operational costs. |
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