应用特异性探针药物分析双峰驼CYP2D6酶体外活性

doi:10.11843/j.issn.0366-6964.2016.12.021

Abstract

Abstract:

Cytochrome P450 is a superfamily of enzymes responsible for the metabolism of drugs, xenobiotics and endogenous compounds. In them, the member of the CYP2D6 constitutes only about 2% of total hepatic CYPs, however, it is responsible for the metabolism of about 20% of commonly prescribed therapeutic compounds. The purpose of this research was to systematically study the activity of bactrian camel CYP2D6 enzyme and its sensitivity to the specific inhibitors in vitro. Liver microsomes of bactrian camels was prepared by modified differential centrifugation method and the protein concentration, total content of CYP enzyme were measured by BCA method and CO reduction method, respectively. Then the characteristics of Bactrian camel CYP2D6 enzyme kinetics, substrate metabolic rate and inhibitory effect of quinidine on the enzyme were studied respectively on the basis of optimization of liver microsomal system and the establishment of the HPLC method for testing the specific substrate dextromethorphan hydrobromide of CYP2D6 enzyme and its active metabolite dextrophan. The results showed that the both liver microsomal protein concentration and the total CYP enzyme content could meet the study requirements. The established HPLC method for determining the specific substrate of CYP2D6 enzyme and its metabolite was high sensitivity and stability, as well as all the peaks of each component were separated completely with no interference. The V_max value and K_m value of CYP2D6 enzyme were 0.141 6±0.052 7 nmol•(min•mg)^－1 and 12.986 0±0.357 2 μmol•L^－1, respectively. The IC₅₀ value of quinidine was 2.779 0±0.063 9 μmol•L^－1. Therefore, the incubation system of Bactrian camel liver microsomal was established and optimized successfully for the further studying of the CYP2D6 enzyme activities in vitro, and found out the kinetic parameters of Bactrian camel CYP2D6 enzyme and the specific inhibitor's IC₅₀ value of the enzyme. Hence, the experimental results filled the blank in research area of CYP2D6 enzyme activities in vitro.

CLC Number:

S859.7

WANG Yan, GAO Fei, HASI Surong. Use Cocktail Probe Drug to Analyse the Activity of Bactrian Camel CYP2D6 Enzyme in vitro[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(12): 2501-2509.

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Use Cocktail Probe Drug to Analyse the Activity of Bactrian Camel CYP2D6 Enzyme in vitro

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