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Method for preparing drug metabolic enzyme-porous material compound and application

A porous material and a technology for preparing drugs, applied in the field of medicine, can solve the problems of complex preparation process, large substrate mass transfer resistance, low enzyme activity, etc., and achieve the effects of simple preparation, improved stability and good reproducibility

Pending Publication Date: 2022-02-25
DALIAN UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the immobilization methods of metabolic enzymes can be divided into embedding method, covalent binding method and adsorption method. The embedding method is a method of immobilizing enzyme molecules in a carrier with a specific network structure. Chinese patent CN104342427A ( Metabolic enzyme-hydrogel system for drug metabolism, drug efficacy and toxicity evaluation) uses hydrogel to wrap and fix drug metabolizing enzymes, which can be used to evaluate drug metabolism, drug efficacy and toxicity, but because hydrogel makes metabolic enzymes It cannot be in direct contact with the substrate, and the mass transfer resistance of the substrate is relatively large, which will greatly prolong the metabolic reaction time in vitro
The covalent binding method is to form an irreversible connection between the enzyme molecule and the carrier through a covalent bond. For example, CYP1A2 and UGT1A10 can be co-immobilized by graphene nanocages. The immobilized enzyme and the carrier are stable in this method, but The preparation process is more complicated, and the enzyme activity may not be high

Method used

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  • Method for preparing drug metabolic enzyme-porous material compound and application
  • Method for preparing drug metabolic enzyme-porous material compound and application
  • Method for preparing drug metabolic enzyme-porous material compound and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation of drug-metabolizing enzyme-porous material composite

[0022] Preparation steps such as figure 1 As shown, pre-soak an appropriate amount of ZSM-48 molecular sieve with 100mM phosphate buffer (PB) with pH = 7.4 for 30min, centrifuge in a handheld centrifuge for 20s (2000g), retain the precipitate, redissolve in PB buffer, repeat the above operation 3 times , fully remove the unprecipitated impurities in the supernatant, and set aside. Dilute the microsomal stock solution with PB to 2mg / mL, add it to ZSM-48 prepared in advance, vortex 5s to mix well, then absorb at 4°C for 24h, centrifuge at 2000g for 20s, and wash the precipitate with PB buffer for more than 3 times , removing unbound enzymes in the supernatant to obtain drug-metabolizing enzyme-porous material complexes. Before and after adsorption, the BCA method was used to measure the change of protein concentration in the buffer as follows: figure 2 As shown, the protein binding amount e...

Embodiment 2

[0023] Example 2: Metabolic capacity of drug-metabolizing enzyme-porous material complex

[0024] Microsomes contain almost all phase I and phase II drug metabolizing enzymes, the most important ones are cytochrome P450 and UGT enzymes, which are involved in the metabolism of almost 65% of clinical drugs, so the broad-spectrum probe substrates of UGT enzymes are first selected 4-methylumbelliferone (4-MU) was used to verify the metabolic ability of the drug-metabolizing enzyme-porous material complex. 200μL incubation system, containing 100mMPB buffer (pH=7.4), 10mM MgCl 2 , 2.5mM UDPGA, 400μM 4-MU, 50μg / mg protein alamethicin and 0.1mg / mL microsome or drug metabolizing enzyme-porous material complex. The reaction was started by adding UDPGA last, and incubated at 37°C for 30min. The metabolic capacity of the drug-metabolizing enzyme-porous material complex was characterized by detecting the amount of the phase II metabolite 4-MUG of 4-MU in the supernatant by high performan...

Embodiment 3

[0025] Example 3: Number of repeated uses of the drug-metabolizing enzyme-porous material composite

[0026] Repeat the steps in Example 2, except that after incubation for 30 minutes, centrifuge at 2000g for 20s, separate the supernatant for HPLC detection, retain the drug-metabolizing enzyme-porous material complex precipitation, wash the buffer 3 times, and add The corresponding substances in the incubation system were catalyzed again, repeated 10 times, and the amount of catalyzed metabolites were detected 10 times respectively to characterize the reusability of the drug metabolizing enzyme-porous material complex. The results are as follows Figure 4 It can be seen that the residual activity of the enzyme remains above 60% when the catalysis is repeated 10 times continuously, realizing the repeated utilization of the metabolic enzyme.

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Abstract

The invention belongs to the technical field of medicines, and provides a method for preparing a drug metabolic enzyme-porous material compound and application. The compound is composed of a drug metabolic enzyme and a porous material, the drug metabolic enzyme is composed of macromolecular proteins with a metabolic function, such as microsomes, recombinase and an S9 mixture, and the porous material is composed of molecular sieves such as ZSM-5, ZSM-22, ZSM-23 or ZSM-48, a mesoporous material and the like. The compound is easy and convenient to prepare and good in reproducibility, the metabolic enzyme can be repeatedly used for multiple times, the stability of the metabolic enzyme is improved, and the compound can be applied to in-vitro evaluation of drug metabolism.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a method for preparing drug metabolizing enzyme-porous material compound, which is applied to in vitro evaluation of drug metabolism. Background technique [0002] The in vitro study of drug metabolism is a very important link in the process of preclinical new drug development, ignoring the biotransformation process of drug metabolism will lead to inaccurate or even wrong research results. The use of in vitro metabolic models to predict in vivo drug clearance and drug-drug interactions, and early elimination of drug candidates with poor pharmacokinetic properties will help design safer and more reliable clinical trials. Currently commonly used in vitro research models include human recombinant enzymes, human-derived microsomes, cell lines, cytosol, liver S9 (S9), transgenic cell lines, primary hepatocytes, stem cell-induced differentiation of hepatocytes, precision liver slices an...

Claims

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Application Information

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IPC IPC(8): G01N33/15
CPCG01N33/15
Inventor 刘勇王哲孟记朋姜丽丽梁长海
Owner DALIAN UNIV OF TECH
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