Study on a metabolic profile generated by cytochrome P450 2D6 inside the separation capillary
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| Year of publication | 2010 |
| Type | Conference abstract |
| MU Faculty or unit | |
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| Description | Drug metabolism studies are essential to determine the fates of new therapeutic agents in human body. In the development phase, the metabolic profiles of drug candidates have to be determined definitively. Recombinant cytochrome P450 enzymes (rCYP) are suitable for ‘frontline’ predictive human metabolism studies in early drug discovery. rCYP are a favorite in vitro system for their availability and ease employment in high throughput assays. The cytochrome P450 2D6 (CYP2D6) isoform accounts for only a small percentage of total hepatic CYPs (<2 %), but it mediates the oxidative metabolism nearly 25 % of clinically used drugs. Those are different classes of drugs acting on the central nervous system and cardiovascular system. Dextromethorphan (DEX), a notable cough-suppressing synthetic analog of codeine, is frequently used as a probe substrate of CYP 2D6. DEX undergoes biotransformation to dextrorphan. Although, the possibility that dextrorphan maybe a substrate of CYP2D6 has been suggested too [1]. Capillary electrophoresis (CE) represents an innovative approach to perform automated enzyme assays. Different methods and procedures of enzymatic activity studies have been reported [2]. The introduction of electrophoretically mediated microanalysis methodology into CE enlarged its applicability and introduced a big advantage into the separation system enabling the performance of chemical (enzymatic) reaction right at the same place. In our work we have focused on in vitro DEX metabolite generation by rCYP 2D6 directly inside the separation capillary. The analytes were separated in 50-mum fused silica capillary (48 cm total length) at 14 kV. The capillary temperature was kept at 37C and the data were recorded at 200 nm. The background electrolyte was different from the incubation buffer and this issue was solved by the partial filling method. Namely, the background electrolyte consisted of 80 mM sodium borate, pH 9.75 containing 8 % of 2-propanol and the incubation buffer was 20 mM sodium phosphate, pH 7.4. Different injection strategies will be shown and compared in the term of reaction efficacy. As the reference we used the offline approach. |
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