CN114088898A - Method for preparing drug metabolizing enzyme-porous material compound and application - Google Patents
Method for preparing drug metabolizing enzyme-porous material compound and application Download PDFInfo
- Publication number
- CN114088898A CN114088898A CN202111286602.4A CN202111286602A CN114088898A CN 114088898 A CN114088898 A CN 114088898A CN 202111286602 A CN202111286602 A CN 202111286602A CN 114088898 A CN114088898 A CN 114088898A
- Authority
- CN
- China
- Prior art keywords
- drug
- metabolizing enzyme
- enzyme
- porous material
- zsm
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of medicines, and provides a method for preparing a drug metabolizing enzyme-porous material compound and application thereof. The compound consists of drug metabolizing enzyme and porous material, wherein the drug metabolizing enzyme consists of macromolecular protein with a metabolizing function, such as microsomes, recombinase, S9 mixture and the like, and the porous material consists of molecular sieve, mesoporous material and the like such as ZSM-5, ZSM-22, ZSM-23 or ZSM-48 and the like. The compound has simple preparation and good reproducibility, realizes repeated use of metabolic enzyme, improves the stability of the metabolic enzyme, and can be applied to in vitro evaluation of drug metabolism.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a method for preparing a drug metabolizing enzyme-porous material compound, which is applied to in-vitro evaluation of drug metabolism.
Background
In vitro research on drug metabolism is a very important link in the process of developing new drugs in preclinical, and the biological transformation process of drug metabolism is neglected to lead to inaccurate and even wrong research results. The in vitro metabolism model is used for predicting the in vivo drug clearance rate and the drug-drug interaction, candidate drugs with poor pharmacokinetic properties are eliminated as early as possible, and the design of safer and more reliable clinical tests is facilitated. 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 differentiated hepatocytes, precision liver slices, liver perfusion, and the like. These models all have many disadvantages due to source limitations, in vitro activity, and model complexity. Human microsomes, liver S9 or recombinant enzymes have become the most common in vitro metabolic studies because the types and activities of their metabolic enzymes (e.g., cytochrome P450 and UDP-glucuronic acid transferase UGTs) are most similar to those of the human body. Considering that the sources of human microsomes, liver S9 or recombinant enzymes are limited and the recombinant enzymes are expensive, how to immobilize the recombinant enzymes to realize the recycling of the recombinant enzymes in metabolic research and apply the recombinant enzymes to the in vitro evaluation of drug metabolism is an improvement and innovation for the in vitro metabolic research of drugs.
The current immobilization methods of related metabolic enzymes can be divided into an embedding method, a covalent bonding method and an adsorption method, the embedding method is an immobilization method for trapping enzyme molecules in a carrier with a specific net-shaped structure, and Chinese patent CN104342427A (a metabolic enzyme-hydrogel system for evaluating drug metabolism, drug effect and toxicity) adopts hydrogel to wrap and fix drug metabolic enzymes, and can be used for evaluating drug metabolism, drug effect and toxicity. The covalent binding method is to form irreversible linkage between an enzyme molecule and a carrier through a covalent bond, for example, CYP1a2 and UGT1a10 can be co-immobilized by a graphene nanocage through double enzymes, and the immobilized enzyme and the carrier are stably bound by the method, but the preparation process is complex and the enzyme activity may not be high. The immobilized metabolic enzyme by the adsorption method mainly selects porous nano materials, enzyme molecules are adsorbed on the surface of the water-insoluble porous nano material through hydrogen bonds, ionic bonds and the like, so that the immobilization effect is realized, and most researches are mainly carried out on porous SiO2Porous Al2O3、TiO2Nanotube array and nanoporous grapheneAnd the adsorption method is simple and convenient to operate, the metabolic enzyme activity is completely preserved, and the application is most extensive.
According to the invention, a series of molecular sieves with high specific surface area and regular and ordered pore channel structures and mesoporous materials are used as carriers for immobilizing drug metabolizing enzyme for the first time to form a novel metabolizing enzyme-porous material compound, so that repeated use of the metabolizing enzyme is realized, the stability of the metabolizing enzyme is improved, and the compound is successfully applied to in-vitro evaluation of drug metabolism.
Disclosure of Invention
The invention aims to provide a method for preparing a drug metabolizing enzyme-porous material compound and application thereof in drug metabolism.
The technical scheme adopted by the invention is as follows:
a method for preparing a drug metabolizing enzyme-porous material compound comprises the steps of adding a drug metabolizing enzyme into a porous material which is washed by a buffer solution in advance, fully and uniformly mixing, then refrigerating and adsorbing for 0.5-48h, centrifuging for 1-20min at 1000-20000g, washing precipitates by using the buffer solution, and removing the enzyme which is not combined with the supernatant to obtain the drug metabolizing enzyme-porous material compound.
The concentration range of the drug metabolizing enzyme is 0.5-20 mg/mL.
The drug metabolizing enzyme is macromolecular protein with a metabolizing function, such as microsomes, recombinase, S9 mixture and the like.
The porous material is a molecular sieve and a mesoporous material, and comprises ZSM-5, ZSM-22, ZSM-23, ZSM-48, SAPO-34, HY, Beta, MOR, SBA-15, SBA-16, MCM-41, KIT-6, MCM-48 and the like.
The buffer solution is 10-100mM phosphate buffer solution with pH value of 6-8 or 10-100mM Tris-HCl buffer solution with pH value of 6-8.
In a typical in vitro metabolic system, after a substrate is metabolized by a metabolic enzyme to generate a product, the enzyme activity is required to be ensured, and the enzyme and the product can be separated only by adopting ultra-high speed centrifugation, so that the enzyme is usually denatured and precipitated by an organic reagent in actual operation and is difficult to realize recycling, and after the metabolic enzyme is immobilized by a porous material, the repeated use of the metabolic enzyme is realized, the stability of the metabolic enzyme is improved, and the current situations of limited sources and high price of the metabolic enzyme are greatly relieved. The metabolizing enzyme-porous material composite can be applied to the evaluation of the metabolic property of the drug.
The invention has the beneficial effects that: the compound has simple preparation and good reproducibility, realizes repeated use of metabolic enzyme, improves the stability of the metabolic enzyme, and can be applied to in vitro evaluation of drug metabolism.
Drawings
FIG. 1 is a preparation route of the drug-metabolizing enzyme-porous material composite according to example 1.
FIG. 2 is a graph showing the protein content in the buffer before and after adsorption of the drug-metabolizing enzyme-porous material complex described in example 1.
FIG. 3 is a chromatogram of the metabolic capability product of the drug metabolizing enzyme-porous material composite described in example 2.
FIG. 4 is a graph showing the number of times of reuse of the drug-metabolizing enzyme-porous material composite according to example 3.
FIG. 5 shows the catalytic stability of the drug metabolizing enzyme-porous material complex described in example 4.
Detailed Description
The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
To specifically illustrate the present invention, the following examples were carried out using human liver microsomes as drug metabolizing enzymes and ZSM-48 molecular sieves as carriers.
Example 1: preparation of drug metabolizing enzyme-porous material composite
The preparation process is shown in figure 1, pre-soaking a proper amount of ZSM-48 molecular sieve in 100mM Phosphate Buffer (PB) with pH 7.4 for 30min, centrifuging for 20s (2000g) by a palm centrifuge, retaining the precipitate, re-dissolving the PB buffer, repeating the above operation for 3 times, and sufficiently removing the impurities which are not precipitated in the supernatant for later use. Diluting the microsome stock solution to 2mg/mL by using PB, adding the diluted microsome stock solution into ZSM-48 prepared in advance, fully and uniformly mixing by vortex for 5s, adsorbing for 24h at 4 ℃, centrifuging for 20s at 2000g, washing the precipitate for more than 3 times by using PB buffer solution, and removing the unbound enzyme of the supernatant to obtain the drug metabolizing enzyme-porous material compound. Before and after adsorption, the results of measuring the change of the protein concentration in the buffer solution by using the BCA method are shown in figure 2, the protein binding amount exceeds 60%, after 2 times of elution, the protein concentration measured in the 3 rd time is below the quantitative limit, the protein is basically determined to be no longer eluted, and the binding between the human liver microsomes and the HZSM-48 is firm.
Example 2: metabolizing ability of drug-metabolizing enzyme-porous material composite
The microsome contains almost all phase I and phase II drug metabolizing enzymes, the most important are cytochrome P450 and UGT enzyme respectively, and almost participate in 65% of clinical drug metabolism, so that a broad-spectrum probe substrate 4-methylumbelliferone (4-MU) of the UGT enzyme is selected to verify the metabolizing capacity of the drug metabolizing enzyme-porous material complex. The incubation system contained 100mM PB buffer (pH 7.4) and 10mM MgCl in 200 μ L22.5mM UDPGA, 400. mu.M 4-MU, 50. mu.g/mg protein of promethrin and 0.1mg/mL microsome or drug metabolizing enzyme-porous material complex. The reaction was initiated by the final addition of UDPGA and incubated at 37 ℃ for 30 min. Detecting the amount of II-phase metabolite 4-MUG of 4-MU in the supernatant by high performance liquid chromatography to characterize the metabolic capability of the drug metabolizing enzyme-porous material compound. The result is shown in fig. 3, under the same condition, the peak height of the product of the drug metabolizing enzyme-porous material compound is obviously higher than that of the free microsome group, and the calculated product 4-MUG amount is about 2.26 times of that of the free microsome group, so that the catalytic efficiency is improved, and the compound can be efficiently used for developing related metabolic studies.
Example 3: reuse times of drug metabolizing enzyme-porous material composite
The steps in the example 2 are repeated, except that after incubation for 30min, centrifugation is carried out for 20s at 2000g, the supernatant is separated and subjected to high-phase liquid chromatography detection, the drug metabolizing enzyme-porous material composite precipitate is retained, buffer solution is washed for 3 times, corresponding substances in the incubation system are added again, re-catalysis is carried out, the repetition is carried out for 10 times, the amount of catalytic metabolites is detected for 10 times respectively to represent the repeated use capacity of the drug metabolizing enzyme-porous material composite, the result is shown in fig. 4, continuous repeated catalysis is carried out for 10 times, the residual activity of the enzyme is still retained above 60%, and the repeated use of the metabolizing enzyme is realized.
Example 4: catalytic stability of drug metabolizing enzyme-porous material composite
The steps in example 2 are repeated, except that after incubation for 30min, centrifugation is carried out for 20s at 2000g, the supernatant is separated and subjected to high-phase liquid chromatography detection, the drug metabolizing enzyme-porous material composite precipitate is retained, buffer solution is washed for 3 times, the mixture is stored at 4 ℃, corresponding substances in the incubation system are added again in days 2, 3, 4, 5, 10, 15 and 30 respectively, re-catalysis is carried out, the amount of catalytic metabolites at different times is detected respectively to characterize the catalytic stability of the drug metabolizing enzyme-porous material composite, and the result is shown in fig. 5, and at day 30, the enzyme residual activity of the drug metabolizing enzyme-porous material composite is still retained at about 75% and is higher than that of a free microsome group, which indicates that the drug metabolizing enzyme-porous material composite has high catalytic stability.
Claims (10)
1. A method for preparing a drug-metabolizing enzyme-porous material compound is characterized in that the drug-metabolizing enzyme is added into a porous material which is washed by a buffer solution in advance, the mixture is fully and evenly mixed, then refrigerated and adsorbed for 0.5 to 48 hours, centrifugation is carried out for 1 to 20min at 1000-.
2. The method for preparing a drug-metabolizing enzyme-porous material complex as defined in claim 1, wherein the concentration of the drug-metabolizing enzyme is in the range of 0.5-20 mg/mL.
3. The method for preparing a drug metabolizing enzyme-porous material complex according to claim 1 or 2, wherein the drug metabolizing enzyme is a macromolecular protein having a metabolic function.
4. The method for preparing a drug-metabolizing enzyme-porous-material complex as defined in claim 3, wherein the macromolecular protein is microsome, recombinant enzyme or S9 mixture.
5. The method for preparing the drug-metabolizing enzyme-porous-material complex according to claim 1 or 2, wherein the porous material is molecular sieve and mesoporous material, including ZSM-5, ZSM-22, ZSM-23, ZSM-48, SAPO-34, HY, Beta, MOR, SBA-15, SBA-16, MCM-41, KIT-6, MCM-48.
6. The method for preparing the drug-metabolizing enzyme-porous-material complex according to claim 3, wherein the porous material is molecular sieve and mesoporous material, including ZSM-5, ZSM-22, ZSM-23, ZSM-48, SAPO-34, HY, Beta, MOR, SBA-15, SBA-16, MCM-41, KIT-6, MCM-48.
7. The method for preparing a drug-metabolizing enzyme-porous-material complex according to claim 1, 2, 4 or 6, wherein the buffer solution is 10-100mM phosphate buffer pH 6-8 or 10-100mM Tris-HCl buffer pH 6-8.
8. The method for preparing a drug-metabolizing enzyme-porous-material complex according to claim 3, wherein the buffer solution is 10-100mM phosphate buffer solution having a pH of 6-8 or 10-100mM Tris-HCl buffer solution having a pH of 6-8.
9. The method for preparing a drug-metabolizing enzyme-porous-material complex according to claim 5, wherein the buffer solution is 10-100mM phosphate buffer solution having a pH of 6-8 or 10-100mM Tris-HCl buffer solution having a pH of 6-8.
10. The drug metabolizing enzyme-porous material composite prepared by the method of any one of claims 1 to 9 is applied to the research of drug metabolism in vitro.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111286602.4A CN114088898A (en) | 2021-11-02 | 2021-11-02 | Method for preparing drug metabolizing enzyme-porous material compound and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111286602.4A CN114088898A (en) | 2021-11-02 | 2021-11-02 | Method for preparing drug metabolizing enzyme-porous material compound and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114088898A true CN114088898A (en) | 2022-02-25 |
Family
ID=80298569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111286602.4A Pending CN114088898A (en) | 2021-11-02 | 2021-11-02 | Method for preparing drug metabolizing enzyme-porous material compound and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114088898A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1161553A1 (en) * | 1983-07-28 | 1985-06-15 | Физико-Химический Институт Академии Наук Усср | Method of immobilization of liver microsoma |
| JP2009294037A (en) * | 2008-06-04 | 2009-12-17 | Funai Electric Advanced Applied Technology Research Institute Inc | Enzyme sensor |
| CN101914437A (en) * | 2010-05-31 | 2010-12-15 | 广西师范大学 | Capillary enzyme microreactor capable of immobilizing enzyme by embedding and preparation method thereof |
| CN102937631A (en) * | 2012-11-19 | 2013-02-20 | 北京理工大学 | Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein |
| CN104342427A (en) * | 2013-07-23 | 2015-02-11 | 复旦大学 | Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation |
| CN104698108A (en) * | 2015-03-26 | 2015-06-10 | 中国药科大学 | Method for screening I type 17 beta-hydroxysteroid dehydrogenase inhibitor through immobilized enzyme |
| CN105713892A (en) * | 2016-04-22 | 2016-06-29 | 中国药科大学 | Method for preparing immobilized aromatase |
| US20180044657A1 (en) * | 2015-02-26 | 2018-02-15 | The Board Of Regents For Oklahoma State University | Microsomal bioreactor for synthesis of drug metabolites |
| CN111320760A (en) * | 2020-02-28 | 2020-06-23 | 南开大学 | Porous framework material, enzyme preparation containing porous framework material, preparation method and application |
| CN111778295A (en) * | 2019-04-04 | 2020-10-16 | 南通厚元生物科技有限公司 | Method for synthesizing phosphatidylserine by using immobilized biocatalyst |
| CN112805091A (en) * | 2018-09-05 | 2021-05-14 | 齐姆特罗尼克斯催化系统股份有限公司 | Enzyme and microsomes immobilized on magnetic scaffolds |
-
2021
- 2021-11-02 CN CN202111286602.4A patent/CN114088898A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1161553A1 (en) * | 1983-07-28 | 1985-06-15 | Физико-Химический Институт Академии Наук Усср | Method of immobilization of liver microsoma |
| JP2009294037A (en) * | 2008-06-04 | 2009-12-17 | Funai Electric Advanced Applied Technology Research Institute Inc | Enzyme sensor |
| CN101914437A (en) * | 2010-05-31 | 2010-12-15 | 广西师范大学 | Capillary enzyme microreactor capable of immobilizing enzyme by embedding and preparation method thereof |
| CN102937631A (en) * | 2012-11-19 | 2013-02-20 | 北京理工大学 | Method for screening active ingredients of traditional Chinese medicine by nano mesoporous material immobilized target protein |
| CN104342427A (en) * | 2013-07-23 | 2015-02-11 | 复旦大学 | Metabolic enzyme-hydrogel system for drug metabolism, drug effect and toxicity evaluation |
| US20180044657A1 (en) * | 2015-02-26 | 2018-02-15 | The Board Of Regents For Oklahoma State University | Microsomal bioreactor for synthesis of drug metabolites |
| CN104698108A (en) * | 2015-03-26 | 2015-06-10 | 中国药科大学 | Method for screening I type 17 beta-hydroxysteroid dehydrogenase inhibitor through immobilized enzyme |
| CN105713892A (en) * | 2016-04-22 | 2016-06-29 | 中国药科大学 | Method for preparing immobilized aromatase |
| CN112805091A (en) * | 2018-09-05 | 2021-05-14 | 齐姆特罗尼克斯催化系统股份有限公司 | Enzyme and microsomes immobilized on magnetic scaffolds |
| CN111778295A (en) * | 2019-04-04 | 2020-10-16 | 南通厚元生物科技有限公司 | Method for synthesizing phosphatidylserine by using immobilized biocatalyst |
| CN111320760A (en) * | 2020-02-28 | 2020-06-23 | 南开大学 | Porous framework material, enzyme preparation containing porous framework material, preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 李丽 等: "介孔分子筛在生物酶固定化中的应用", 《化学研究与应用》 * |
| 肖宁 等: "介孔分子筛MCM241固定化木瓜蛋白酶的研究", 《北京化工大学学报》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Chemiluminescence flow biosensor for hydrogen peroxide using DNAzyme immobilized on eggshell membrane as a thermally stable biocatalyst | |
| Williams et al. | Adipocyte beta-adrenergic receptors. Identification and subcellular localization by (-)-[3H] dihydroalprenolol binding. | |
| Alberti et al. | Functional immobilization of signaling proteins enables control of stem cell fate | |
| Deng et al. | Autonomous and growth factor–induced hypertrophy in cultured neonatal mouse cardiac myocytes: comparison with rat | |
| Niederkofler et al. | Determination of β-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system | |
| Abd El‐Aleem et al. | Cellular and physiological upregulation of inducible nitric oxide synthase, arginase, and inducible cyclooxygenase in wound healing | |
| Ponomareva et al. | Monolithic bioreactors: effect of chymotrypsin immobilization on its biocatalytic properties | |
| Vagaska et al. | Modelling human CNS injury with human neural stem cells in 2-and 3-Dimensional cultures | |
| Le et al. | Recent advances in the engineering and application of streptavidin-like molecules | |
| DOHENY et al. | Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor–cellulose-binding domain fusion protein | |
| Li et al. | Integrated proteomic sample preparation with combination of on-line high-abundance protein depletion, denaturation, reduction, desalting and digestion to achieve high throughput plasma proteome quantification | |
| CN114088898A (en) | Method for preparing drug metabolizing enzyme-porous material compound and application | |
| Bruno et al. | Development of DNA aptamers for cytochemical detection of acetylcholine | |
| Zhang et al. | Boronate avidity assisted by dendrimer-like polyhedral oligomeric silsesquioxanes for a microfluidic platform for selective enrichment of ubiquitination and glycosylation | |
| Morelli et al. | Human hepatocyte functions in a galactosylated membrane bioreactor | |
| US11280789B2 (en) | Compositions, kits, and methods for cell separation | |
| Wei et al. | Cell-based 3D bionic screening by mimicking the drug–receptor interaction environment in vivo | |
| Horwitz et al. | Immobilized IL-2 preserves the viability of an IL-2 dependent cell line | |
| Chen et al. | Development of the reversible PGA immobilization by using the immobilized metal ion affinity membrane | |
| NO320104B1 (en) | Process for the preparation of baccatin in the presence of an enzyme and an acetyl donor, the enzyme, a process for its preparation and a process for the preparation of taxol and / or taxol derivatives. | |
| CN108107209B (en) | Optimization method for detecting content of secretory reporter protein and biological material used by optimization method | |
| Li et al. | Human induced pluripotent stem cell-derived closed-loop cardiac tissue for drug assessment | |
| CN113999889A (en) | Dry-type glucose test strip adopting Prussian blue nanoenzyme and preparation method thereof | |
| Mueggler et al. | Malate dehydrogenase, anticooperative NADH, and L-malate binding in ternary complexes with supernatant pig heart enzyme | |
| Watanabe et al. | Activity and subunit functions of immobilized bacterial luciferase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |