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Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation

A hydrogel and metabolic enzyme technology, applied in the field of metabolic enzyme-hydrogel system, to achieve accurate evaluation of drug efficacy or toxicity, no effect on cell viability, and suitable spatial structure

Active Publication Date: 2015-02-11
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] So far, there is no information about using the above two materials at the same time to make a new polymer with the advantages of the two materials, as a carrier of drug-metabolizing enzymes, and after wrapping drug-metabolizing enzymes, a metabolic enzyme-hydrogel system is formed. , and then use this system to study drug metabolism, drug efficacy and toxicity reports

Method used

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  • Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation
  • Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation
  • Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Construction of metabolic enzyme-hydrogel system

[0028] Preparation steps such as figure 1 as shown,

[0029] (1) Through the condensation reaction of acryloyl chloride and F127 under alkaline conditions, F127 was successfully modified with ester bonds to have double bonds at both ends; the macromonomer was dissolved in deionized water to prepare 30% Concentration mother solution, stored at 4°C;

[0030] (2) To prepare 100 ul hydrogel carrier, absorb 33 ul double bond F127 mother solution, 1 ul purchased 30% acrylamide:bisacrylamide (29:1) mixed solution, 1 ul 10% ammonium persulfate solution, 0.5 ul TEMED, and the rest with 64.5 ul PBS phosphate buffer solution; transfer the mixture to a 96-well plate, carry out cross-linking at room temperature or 37°C, and obtain a hydrogel after about 30 min;

[0031] (3) Place the hydrogel obtained in the above step (2) into 5% mannitol solution and shake and wash it repeatedly, about 5 times, 20 min each time;

...

Embodiment 2

[0034] Example 2 Metabolism of microsome-hydrogel system

[0035] Such as figure 2 As shown, microsomes contain almost all of a metabolic enzyme, and a metabolic enzyme is responsible for the biotransformation process of most drugs. Therefore, several representative metabolic enzymes including CYP3A, CYP2D6, and CYP2E1 are selected for verification Metabolic capacity of microsome-hydrogel systems.

[0036]The standard substrates of the three enzymes certified by the FDA, 90μM testosterone (CYP3A), 100μM dextromethorphan (CYP2D6) and 200μM chlorzoxazone (CYP2E1), were used for incubation at 37°C; in the 500ul incubation system, 50ul of the above concentration Substrate, 100ul metabolic reaction initiation system NADPH (final concentration 1mM), 350ul PBS buffer and 100ul microsome-hydrogel; 50 ul samples were drawn at 6 h, 8 h and 24 h, and the content of the corresponding substrates 6β-OH testosterone, nordextromethorphan and 6-OH testosterone in the sample was analyzed b...

Embodiment 3

[0037] Example 3 Drug efficacy and toxicity prediction ability of microsome-hydrogel system

[0038] Such as image 3 As shown, cyclophosphamide is a classic anti-tumor prodrug, which can be transformed into active nitrogen mustard only after being metabolized in the body, so as to exert its anti-tumor ability. Cyclophosphamide was selected as the model drug, and human breast cancer MCF-7 cells were used as the test cells to investigate the influence of the presence or absence of the microsome-hydrogel system on its antitumor efficacy.

[0039] MCF-7 cells were treated with 5 mM, 10 mM, 15 mM and 20 mM cyclophosphamide for 24 hours, and the microsome-hydrogel system was added to one group of the culture system, and the control group without the system was set up, and the collected Cells were stained with Annexin V / PI, and finally the degree of apoptosis was analyzed by flow cytometry; the results were as follows image 3 As shown, the number of apoptotic cells added to the...

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Abstract

The invention belongs to the technical field of medicine, and relates to a metabolic enzyme-hydrogel system for drug metabolism, drug effect, and toxicity evaluation. The system is composed of drug metabolic enzymes and hydrogel carriers, wherein the drug metabolic enzymes are composed of protein macromolecules having metabolism functions such as microsomes, recombinant enzymes, and the like; and the hydrogel carriers are prepared by carrying out reactions among pluronics, amine acrylate, and diamine acrylate in the presence of a catalyst namely N,N,N',N'-tetramethyldiethylamine. The metabolic enzyme-hydrogel system is prepared by the following steps: subjecting the interlinked hydrogel carriers to pretreatments of cleaning and freeze-drying, and absorbing drug metabolism enzymes with controllable concentration by the hydrogel carriers so as to form the metabolism-hydrogel system. The metabolism enzyme-hydrogel system has the advantages of proper mechanical strength, non-toxicity, good repeatability, convenient operation, and thermal sensitivity, is capable of comprehensively predicting the effects of drugs and metabolic substances thereof on cells, and thus precisely evaluates the drug effect and toxicity of the drugs.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to the evaluation of drug metabolism, drug efficacy and toxicity, in particular to a metabolic enzyme-hydrogel system used for drug metabolism, drug efficacy and toxicity evaluation; the system can be used to predict drug metabolism, drug Efficacy and toxicity evaluation. Background technique [0002] Cell culture is an important in vitro pharmaceutical research method; since most cells, even liver cells, usually do not express or low express many important drug metabolizing enzymes, this method cannot fully simulate the real in vivo environment. The biotransformation process of drug metabolism is ignored in pharmaceutical research, which will lead to inaccurate, incomplete or even wrong research results; for example, the antipyretic, analgesic and anti-inflammatory drug acetaminophen can only be transformed in the liver by cytochrome enzyme CYP 2E1 After NAPQI becomes an active int...

Claims

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Application Information

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IPC IPC(8): C12N11/08C12N11/04C12Q1/25
Inventor 杨慧莹蔡卫民郑媛婷
Owner FUDAN UNIV
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