Application of schizandrin A in detection of cytochrome P4503A enzymatic activity and detection method thereof

A technology of schisandrin and cytochrome, which is applied in the field of detection of cytochrome P4503A enzyme activity by schisandrin, can solve problems such as blood drug concentration fluctuations, adverse reactions, and potential safety hazards in clinical medication, and achieve easy drug content and high conversion rate Effect

Active Publication Date: 2013-02-20
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difference in the metabolism of cytochrome P4503A between different patients, the blood drug concentration will fluctuate greatly. Once the blood drug concentration in the body exceeds the minimum toxic dose, it is easy to cause serious adverse reactions, which will bring safety hazards to clinical medication.

Method used

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  • Application of schizandrin A in detection of cytochrome P4503A enzymatic activity and detection method thereof
  • Application of schizandrin A in detection of cytochrome P4503A enzymatic activity and detection method thereof
  • Application of schizandrin A in detection of cytochrome P4503A enzymatic activity and detection method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The specificity of schisandrin as a cytochrome P4503A substrate was verified by recombinant cytochrome P4503A single enzyme and mammalian subcellular component system:

[0042] Twelve samples from different human livers were selected to prepare human liver microsomes. Purchase BD company in vitro recombinant human liver single enzyme cytochrome P450 3A4, 3A5, 3A7. Liver samples from 10 Sprague Dawley rats, 6 Bama miniature pig mice, 5 beagle dogs, 10 rabbits, and 3 monkeys were selected to prepare animal liver microsomes respectively. Through enzyme reaction kinetics, correlation analysis, specific inhibition experiments, and evidences of recombinant single enzyme metabolic reactions, it is proved that schisandrin A is specifically metabolized by the enzymes of the cytochrome P4503A subfamily to generate schisandrin A as its metabolism. product.

[0043] (1) Under the three temperature conditions of 10°C, 37°C, and 60°C, human (containing cytochrome P4503A4, 3A5), mou...

Embodiment 2

[0048] Liver microsomes, S9, freshly extracted hepatocytes, primary cultured hepatocytes, and liver slice system were used to verify the specificity of Schizandrin A metabolic response:

[0049] Liver microsomes, S9, freshly extracted hepatocytes, primary cultured hepatocytes, and liver slice system were selected to verify whether other major drug-metabolizing enzymes were involved in the metabolism of schisandrin, and it was proved that only cytochrome P4503A was involved in its metabolism. The reaction conditions and results are listed as follows:

[0050] Table 1 Different in vitro metabolic incubation systems were used to verify the specificity of metabolic reactions of schisandrin A

[0051]

Embodiment 3

[0053] The human liver microsomes were prepared from the liver samples of 12 patients, and the ability of schisandrin A to standardize the activity of cytochrome P4503A enzyme was verified:

[0054] (1) Twelve liver samples from different adults were collected, and the non-cancerous part was selected, washed with 100mM potassium phosphate buffer (pH 7.4) containing 0.15M KCl, and processed into about 2 grams of liver with tissue scissors. Small piece;

[0055] (2) Homogenize the above-mentioned obtained tissue block by using an in-cut type homogenizer;

[0056] (3) Using a Hitachi CR-21G high-speed refrigerated centrifuge, under the condition of 9000×g, after high-speed centrifugation for 20 minutes, the supernatant was taken to obtain the human liver S9 fraction;

[0057] (4) Under the condition of an ultracentrifuge at 105,000×g, after ultracentrifugation for 60 minutes, remove the supernatant, wash with 100 mM potassium phosphate buffer (pH7.4), and resuspend;

[0058] (5...

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Abstract

The invention provides application of schizandrin A in detection of cytochrome P4503A enzymatic activity and a detection method thereof. The schizandrin A can be used as probe substrate to detect the cytochrome P4503A enzymatic activity, and the schizandrin A or other medicines are used as specific probe substrate to obtain schizandrin to be used as metabolite at the reaction temperature between 10 DEG C and 60 DEG C. The decrement of the schizandrin A substrate or the metabolite generation amount of the schizandrin A is used as an evaluation index of cytochrome P4503A subfamily enzymatic activity. Or a mammal to be detected is enabled to take the schizandrin A or other medicines with 0.01-1000mg / kg of weight of the mammal through an intravenous injection, muscle injection or oral way; a time point is selected within 0-72h, and then a biological sample of blood plasma, urine, excrement or bile of the mammal to be detected is collected; and the decrement of the schizandrin A substrate or the metabolite generation amount of the schizandrin A is measured to be used as the evaluation index of the cytochrome P4503A subfamily enzymatic activity.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to the use and detection method of schisandrin for detecting cytochrome P4503A enzyme activity, which can be used to evaluate the body's ability to dispose of drugs during clinical treatment. Background technique [0002] The cytochrome P450 enzyme (Cytochrome P450, CYP) superfamily includes the most important drug-metabolizing enzymes in the body, and participates in the metabolism of more than 80% of marketed drugs, and the drugs metabolized by the cytochrome P4503A subfamily account for about 50%, including a variety of Clinical first-line drug with narrow treatment threshold. Four subtypes of cytochrome P4503A are expressed in the human body, namely cytochrome P4503A4, P4503A5, P4503A7 and P4503A43, which are widely distributed in liver, intestine, kidney, spleen, prostate and heart. Cytochrome P450 3A4 is very significantly expressed in the liver and intestinal tra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26A61K49/00
Inventor 杨凌张延延吴敬敬葛广波
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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