Kit for screening CYP450 enzyme inhibition medicament with high flux and research method thereof

Abstract

The invention relates to a kit for screening a CYP450 enzyme inhibition medicament with high flux and a research method thereof, and belongs to the field of medicament safety evaluation. A kit body of the kit comprises an NADPH regeneration system, a 0.1M phosphate buffer solution, six mixed probe substrates, metabolites of the six mixed probe substrates, and 20mg / ml of liver microsomes of a mouse, rat, dog or monkey. The NADPH regeneration system, six enzyme substrate and medicaments with different concentrations are mixed, then the mixture and the liver microsomes of the mouse, rat, dog or monkey are incubated in a water bath at a temperature of 37 DEG C together, the inhibition effects of the medicaments on the activity of a CYP450 enzyme are judged by LC-MS / MS detection of the quantities of the six metabolites, and the IC50 value is calculated. The kit has the advantages of simplicity, accuracy, stability and convenience, so that the kit has broad application prospect.

Description

technical field [0001] The invention relates to a high-throughput screening kit for CYP450 enzyme inhibitory drugs and a research method thereof, belonging to the field of drug safety evaluation. Background technique [0002] Cytochrome P450 (CYP450) belongs to a hemoglobin enzyme, which widely exists in organisms and is the most important family of oxidases in the microsomal mixed-function oxidase system. There is a maximum absorption peak at 450nm. The biochemical reactions it participates in include fatty acid, sterol biosynthesis, oxidative metabolism of endogenous substrates such as steroid hormones, biological oxidation of exogenous substances and most drugs, etc.; Two directions of development: metabolic activation and metabolic detoxification, the products after metabolic activation have strong toxicity, and even produce teratogenic and carcinogenic effects. [0003] As an important enzyme related to drug metabolism, cytochrome P450 plays a very important role in d...

AI Technical Summary

Problems solved by technology

However, the evaluation of these compounds lacks corresponding rapid detection kits and technical support, so researchers need to make or prepare related reagents and microsomes by themselves, and the research methods for enzyme inhibition are not uniform and standardized, either for a single enzyme or for multiple enzymes. Enzymes, all of these are difficult for medical staff who have not been trained in drug metabolism research to carry out relevant drug interaction evaluation work. Irregularities, unprofessional calculations and other reasons lead to the inability to obtain accurate information on drug interactions, thereby underestimating the risk of drug interactions in clinical applications and potentially causing greater harm

Moreover, the enzyme activity detection kits at home and abroad are mainly for the detection of the influence of a single enzyme activity, and usually use fluorescence quantitative, HPLC detection or ELISA detection kits, the detection sensitivity is low, the error is large, the operation is complicated, and it is far from meeting the requirements of Qualcomm. Due to the requirement of rapid screening, the risk data for the evaluation of clinical drug interactions is not comprehensive, and it cannot simulate the overall situation of the real human body after drug administration. Evaluate the risk of drug interaction, especially to meet the needs of drug evaluation for scientific researchers who do not have a professional foundation in metabolism, and the evaluation task can be completed according to the kit instructions

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