[go: up one dir, main page]

CN110960484A - Recombinant adenovirus sustained-release preparation, preparation method and application thereof - Google Patents

Recombinant adenovirus sustained-release preparation, preparation method and application thereof Download PDF

Info

Publication number
CN110960484A
CN110960484A CN201911069414.9A CN201911069414A CN110960484A CN 110960484 A CN110960484 A CN 110960484A CN 201911069414 A CN201911069414 A CN 201911069414A CN 110960484 A CN110960484 A CN 110960484A
Authority
CN
China
Prior art keywords
adenovirus
sustained
release
preparation
solution
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
Application number
CN201911069414.9A
Other languages
Chinese (zh)
Inventor
马丁
卢运萍
王世宣
周剑锋
李震中
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Tdk Gene Eng Co Ltd
Shenzhen Tdk Gene Engineering Co Ltd
Original Assignee
Shenzhen Tdk Gene Eng Co Ltd
Shenzhen Tdk Gene Engineering Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shenzhen Tdk Gene Eng Co Ltd, Shenzhen Tdk Gene Engineering Co Ltd filed Critical Shenzhen Tdk Gene Eng Co Ltd
Priority to CN201911069414.9A priority Critical patent/CN110960484A/en
Publication of CN110960484A publication Critical patent/CN110960484A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01021—Thymidine kinase (2.7.1.21)

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Inorganic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to a recombinant adenovirus sustained-release preparation, a preparation method and application thereof. The sustained release preparation comprises an adenovirus anti-tumor gene drug and a sustained release protective agent; the anti-tumor gene medicine is a gene medicine of herpes simplex virus thymidine kinase transduced on the conditional replication adenovirus; the slow release protective agent comprises at least one of sodium alginate, calcium chloride, lactose, mannitol and dextran. The adenovirus sustained-release preparation is more stable, solves the problem that the traditional adenovirus liquid preparation is easy to inactivate during storage, prolongs the storage time of the adenovirus preparation, can effectively prolong the action time of the adenovirus in a tumor microenvironment in an in-situ malignant melanoma mouse model, obviously inhibits tumor growth, prolongs the survival time of a mouse, and has a curative effect obviously superior to that of the liquid preparation.

Description

Recombinant adenovirus sustained-release preparation, preparation method and application thereof
Technical Field
The invention relates to the field of adenovirus preparations, in particular to a recombinant adenovirus sustained-release preparation, a preparation method and application thereof in preparing antitumor drugs.
Background
Gene therapy (gene therapy) refers to the introduction of foreign genes into target cells to correct or compensate for diseases caused by gene defects or abnormal expression of genes. Technological innovation and clinical trials of gene therapy are emerging in recent years as spring shoots after rain, and the subsidies of various countries in the world in the field of gene therapy are continuously increasing. A plurality of gene therapy projects are approved to be put on the market in the United states, European Union, China and other countries. By 4 months in 2017, 2463 clinical trial protocols for gene therapy are registered on clinical trial websites all over the world, wherein nearly 500 clinical trial protocols for gene therapy entering the II/III stage are provided, and 7 gene therapy products are listed in the United states, European Union, China and other countries.
As a gene therapy vector, the development prospect of the oncolytic virus for treating malignant tumor is good. In recent years, various oncolytic virus projects such as cross-country medicine enterprises-Ancheng, Huifeng, Aliskikang and the like are laid out. The gene therapy research and clinical test in China are almost started in synchronization with developed countries in the world, and tumors are the main direction of attack. Nearly 20 gene therapy products aiming at malignant tumors enter clinical trials in China. Among the vectors for oncolytic virus therapy, recombinant adenovirus vectors are most widely used, and the clinical feasibility and safety of the recombinant adenovirus vectors are well recognized, and adenovirus gene therapy vectors have the following outstanding biological advantages and clinical application advantages: 1. the infection spectrum is wide, and the cells of various tissue sources, whether in the division phase or the stationary phase, can be effectively attacked, so the anti-cancer spectrum is wide; 2. after the adenovirus enters cells, the adenovirus is not integrated into host chromosomes, has no risk of mutation and carcinogenesis, is safe in clinical application, only produces cold-like symptoms after being used, and has no hematopoietic function and immunologic function inhibition; 3. the clinical application is convenient, and the medicine can be applied by the ways of cavity administration (abdominal cavity, thoracic cavity and cranial cavity), local or tumor in-vivo direct injection (one point or a plurality of points), interventional therapy and the like; 4. intravenous or topical application produces only a mild inflammatory response with minor side effects; 5. the adenovirus of clinical grade quantity is easy to produce and purify. Based on the advantages, more and more adenovirus vectors are constructed and generated, and the good clinical application prospect is shown.
Although adenovirus has obvious advantages compared with other various gene therapy vectors, the existing adenovirus therapy vectors at home and abroad gradually make some breakthrough progress, but still have some insurmountable defects to limit the wide application of adenovirus. The existing adenovirus carrier medicine mainly adopts the form of liquid injection with traditional formula in the preparation process, the stability of the adenovirus in the liquid preparation is poor and the inactivation is easy, the preservation period is only 6-12 months at the temperature of 2-8 ℃, therefore, most adenovirus preparations need to be preserved at-20 ℃ or even-80 ℃ for a long time, and in the production, transportation and use processes of the adenovirus preparations, if the operation is improper, the temperature of the preservation environment of the adenovirus preparations is increased, which often causes the efficacy reduction of the adenovirus preparations; in addition, the virus can only be expressed continuously for two weeks when entering into the organism, if the concentration of the adenovirus at the tumor part is required to be maintained for a certain concentration, the injection for the next treatment course is needed after two weeks, the problems of toxic and side effects, high cost and the like caused by the impact therapy also limit the wide clinical application of the virus, and a heavy burden is caused to patients.
Although the prior adenovirus gene therapy preparations at home and abroad are obviously improved and have breakthrough progress, the technical problems can not be solved, and the invention is especially provided in view of the above.
Disclosure of Invention
Based on the technical background and important medical requirements, the invention relates to a novel Ad5 gene therapy slow-release protective agent, a preparation method and application thereof in preparing anti-tumor gene drugs, the adenovirus slow-release protective agent obtained by the invention has the obvious advantages of strong stability, long validity period, long-term specific expression of exogenous therapeutic proteins in tumor local, strong tumor specific bystander effect and the like, has high therapeutic index, and can solve the key defects in the current tumor therapy technology and practice.
Therefore, the invention overcomes the defects in the prior art and provides an adenovirus sustained-release preparation, a preparation method thereof and application thereof in preparing anti-tumor gene medicaments.
In order to solve the technical problems, the invention provides the following technical scheme:
in one aspect of the present invention, there is provided an adenovirus sustained release formulation comprising: (1) adenovirus anti-tumor gene medicine; and (2) a slow release protective agent; the slow release protective agent comprises at least one of sodium alginate, calcium chloride, lactose, mannitol and dextran. The adenovirus sustained-release preparation is more stable, solves the problem that the traditional adenovirus liquid preparation is easy to inactivate during storage, prolongs the storage time of the adenovirus preparation, can effectively prolong the action time of the adenovirus in a tumor microenvironment in an in-situ malignant melanoma mouse model, obviously inhibits tumor growth, prolongs the survival time of a mouse, and has a curative effect obviously superior to that of the liquid preparation.
Preferably, the adenovirus anti-tumor gene drug is a gene drug of herpes simplex virus thymidine kinase (HSV-TK) transduced on a conditionally replicative adenovirus. Preferably, the adenovirus anti-tumor gene drug is a recombinant adenovirus-thymidine kinase construct, the length of the whole construct is 43Kb, and the adenovirus anti-tumor gene drug comprises: the total genomic DNA of type 5 adenoviruses except the regions E1a, E1b, E3; thymidine kinase gene expression fragment, about 2.8Kb in length, comprising RSV-LTR promoter, a start fragment of length 18bp and sequence ATGGCTTCGTACCCCTGC and a thymidine kinase cDNA of length 2.3Kb and having the sequence of FIG. 3 in ZL 98124960.4. The adenovirus anti-tumor gene medicine is prepared by referring to the method described in the patent of recombinant adenovirus-thymidine kinase construction body and the obtaining method and the application thereof (patent number: ZL 98124960.4).
Preferably, the adenovirusThe concentration of the anti-tumor gene medicine in the adenovirus sustained release preparation is 1-5 multiplied by 1011pfu/mL; most preferably 2.5X 1011pfu/mL。
Preferably, the slow release protective agent comprises five components of sodium alginate, calcium chloride, lactose, mannitol and dextran.
Preferably, the buffer protectant comprises:
0.5-2 parts by weight of sodium alginate, preferably 1 part by weight;
0.5-2 parts by weight of calcium chloride, preferably 1 part by weight;
1-5 parts by weight of lactose, preferably 2 parts by weight;
1-5 parts by weight of mannitol, preferably 2 parts by weight;
1-5 parts by weight of dextran, preferably 2 parts by weight.
Preferably, the concentration of sodium alginate is 0.5-2% (w/w) calculated on the final formulation; most preferably 1% (w/w).
Preferably, the calcium chloride is present in a concentration of 0.5-2% (w/w), based on the final formulation; most preferably 1% (w/w).
Preferably, the lactose is present in a concentration of 1-5% (w/w), calculated on the final formulation; most preferably 2% (w/w).
Preferably, the concentration of mannitol is 1-5% (w/w) calculated on the final formulation; most preferably 2% (w/w).
Preferably, the dextran concentration is 1-5% (w/w) calculated on the final formulation; most preferably 2% (w/w).
In another aspect, the invention relates to a method of preparing an adenovirus sustained release formulation, the method comprising: providing a first solution as a slow release protectant; providing a second solution containing adenovirus anti-tumor gene drugs; mixing the first solution and the second solution, and filtering and collecting formed gel substances to obtain adenovirus sustained-release hydrogel; and sequentially carrying out pre-freezing control, cold well setting, primary drying and analysis drying on the slow-release hydrogel to obtain the adenovirus slow-release preparation.
Preferably, the first solution is cooled to a temperature of 1-10 ℃ in step (3) before mixing the first and second solutions; preferably 3-5 deg.C, most preferably 4 deg.C.
Preferably, in step (3), the first solution and the second solution are mixed in equal volumes.
The first solution contains one or more of sodium alginate, calcium chloride, lactose, mannitol and dextran, and preferably contains five components of sodium alginate, calcium chloride, lactose, mannitol and dextran.
The second solution contains ADV-TK virus and Tris buffer solution.
Preferably, the Tris buffer comprises Tris-HCl, MgCl2, CaCl2 and glycerol.
Preferably, the Tris buffer is 1 × Tris buffer.
Preferably, the concentration of each component in the 1 × Tris buffer is as follows: 10mM Tris-HCl,1mM MgCl2,150 mM CaCl210% glycerol.
The second solution may be prepared as described in "recombinant adenovirus-thymidine kinase construct, methods of obtaining and use thereof" patent application No. ZL 98124960.4.
The second solution is 1ml per bottle and contains ADV-TK 1-5 × 1011pfu, diluted with Tris buffer. Preferably the second solution contains ADV-TK 2.5X 1011pfu/mL. The Tris buffer solution comprises Tris-HCl and MgCl2、 CaCl2And glycerol.
Preferably, the Tris buffer is 1 × Tris buffer.
Preferably, the concentration of each component in the 1 × Tris buffer is as follows: 10mM Tris-HCl,1mM MgCl2,150 mM CaCl2, 10% glycerol.
Preferably, the concentration of sodium alginate is 0.5-2% (w/w) calculated on the final formulation; most preferably 1% (w/w).
Preferably, the calcium chloride is present in a concentration of 0.5-2% (w/w), based on the final formulation; most preferably 1% (w/w).
Preferably, the lactose is present in a concentration of 1-5% (w/w), calculated on the final formulation; most preferably 2% (w/w).
Preferably, the concentration of mannitol is 1-5% (w/w) calculated on the final formulation; most preferably 2% (w/w).
Preferably, the dextran concentration is 1-5% (w/w) calculated on the final formulation; most preferably 2%.
More preferably, a first solution is taken wherein the sodium alginate concentration is 2% (w/w), the calcium chloride concentration is 4% (w/w), the lactose concentration is 4% (w/w), the mannitol concentration is 4% (w/w) and the dextran concentration is 4% (w/w). Cooling to 4 deg.C, mixing with virus solution at 4 deg.C in equal volume to obtain virus mixed solution. The mixed solution (i.e., the final preparation) had a sodium alginate concentration of 1% (w/w), a calcium chloride concentration of 2% (w/w), a lactose concentration of 2% (w/w), a mannitol concentration of 2% (w/w), and a dextran concentration of 2% (w/w). The gel material formed was collected by filtration.
On the other hand, the invention also relates to the application of the adenovirus sustained-release preparation in preparing anti-tumor drugs, preferably, the tumor is malignant melanoma.
As a sustained release protective agent in the adenovirus sustained release preparation, sodium alginate is a byproduct after iodine and mannitol are extracted from kelp or gulfweed of brown algae, molecules of the sodium alginate are connected by β -D-mannuronic acid (β -D-mannuronic, M) and α -L-guluronic acid (α -L-guluronic acid, G) according to (1 → 4) bonds, the sodium alginate is a natural polysaccharide, has stability, solubility, viscosity and safety required by pharmaceutical preparation auxiliary materials, and has rapid ion exchange reaction with divalent calcium ions in calcium chloride to generate alginate gel with high film strength, and the calcium alginate gel generated after mixing can wrap and stabilize adenovirus and other protective agent components.
Saccharides such as lactose, mannitol and dextran can protect adenovirus, avoid loss of adenovirus in the preparation process and increase the stability of adenovirus under the condition of preservation temperature.
As a preferred scheme of the application of the adenovirus sustained release preparation in preparing the antitumor drugs, the gene drug has the action mode that thymidine kinase gene (TK) is transferred into tumor cells through a recombinant adenovirus vector (ADV) to be expressed, and then, thymidine kinase converts nucleoside triphosphate into nucleoside triphosphate with the participation of Ganciclovir (GCV), and the nucleoside triphosphate is combined with a newborn DNA chain of the tumor cells to interfere DNA synthesis to generate a suicide effect, so that the tumor cells are killed; in addition, due to the 'bystander effect' of ADV-TK, even if only a few tumor cells are transfected by the TK during treatment, the surrounding uninfected tumor cells can be killed through mechanisms such as gap connection, the TK released by phagocytosis of dead cells through intercellular channels, immune-mediated killing effect and the like, so that the killing efficiency is greatly enhanced.
The research of the invention finds that the adenovirus sustained-release preparation (hereinafter referred to as S1/Adv-TK) is more stable, the problem that the traditional adenovirus liquid preparation is easy to inactivate when being stored is solved, the storage time of the adenovirus preparation is prolonged, and in a malignant melanoma subcutaneous tumor mouse model, the novel adenovirus sustained-release preparation can effectively prolong the survival time of adenovirus in a tumor microenvironment, obviously inhibit tumor growth, prolong the survival time of a mouse, and has a curative effect obviously superior to that of the Adv-TK of the liquid preparation.
Drawings
FIG. 1 is a graph showing the results of sustained release of Adv-TK liquid solution and S1/Adv-TK sustained release formulation in mice.
FIG. 2 is a graph of tumor growth in situ models of malignant melanoma with Adv-TK liquid solution and S1/Adv-TK sustained release formulation.
FIG. 3 is a graph showing the survival time of mice bearing tumors with Adv-TK liquid solution and S1/Adv-TK sustained release formulation.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are described in the following for clear and complete description. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Comparative example: preparation of Adv-TK liquid solution
ADV-TK-containing solution in 1 XTTris buffer (10mM Tris-HCl,1mM MgCl)2,150mM CaCl210% glycerol) to 2.5X 1011pfu/ml。
EXAMPLE 1 preparation of Adv-TK Slow Release hydrogel
This example 1 provides a method for preparing an adenovirus sustained-release hydrogel, comprising: a first solution used as a slow-release protective agent and a second solution containing adenovirus anti-tumor gene drugs.
The first solution contains sodium alginate, calcium chloride, lactose, mannitol and dextran. Wherein the first solution comprises alginic acid sodium 2% (w/w), calcium chloride 4% (w/w), lactose 4% (w/w), mannitol 4% (w/w) and dextran 4% (w/w).
The second solution is 1ml each, and contains ADV-TK 2.5 × 1011pfu, 1 XTTris buffer (10mM Tris-HCl,1mM MgCl)2，150mM CaCl210% glycerol). The specific preparation method is described in recombinant adenovirus-thymidine kinase construction body, obtaining method and use thereof, patent No. ZL 98124960.4;
accurately weighing sodium alginate, calcium chloride, lactose, mannitol and dextran, adding appropriate amount of water for injection, stirring until completely dissolving, allowing each component to reach target final concentration, sterilizing at 116 deg.C for 45min to obtain sterile delayed release protective agent, i.e. first solution. And (3) taking the first solution, cooling the first solution to 4 ℃, mixing the first solution and the second solution at 4 ℃ in an equal volume uniformly, and collecting the formed gel substance in a filtering manner to obtain the adenovirus sustained-release hydrogel. Wherein the concentration of the sodium alginate in the adenovirus sustained-release hydrogel is 1% (w/w), the concentration of the calcium chloride is 2% (w/w), the concentration of the lactose is 2% (w/w), the concentration of the mannitol is 2% (w/w), and the concentration of the dextran is 2% (w/w).
The formed adenovirus sustained-release hydrogel only contains sodium alginate and calcium chloride with low concentration, so that the formed gel has thinner texture, can smoothly pass through a standard 1ml syringe needle in the implementation process, and has no influence on the use mode of local virus injection.
Example 2 preparation of S1/Adv-TK
In the preparation process of the adenovirus sustained release preparation provided in this example 2, 2ml of the adenovirus sustained release hydrogel solution is filled in each preparation bottle, and then the following freeze-drying operation steps are performed:
1) pre-freezing control: setting the temperature at 4 ℃, setting the time for 2min and the duration for 10 min; setting the temperature to be-45 ℃, the time to be 2min and the duration to be 120 min;
2) and (3) cold well setting: the refrigeration temperature of the water catcher is set to be 60 ℃ below zero, and the pre-vacuum is 0.14 mBar;
3) primary drying: setting the temperature at-25 deg.C, setting time for 60min, duration for 150min, and vacuum controlling for 0.14 mBar; setting the temperature at-10 deg.C, setting time 150min, duration 900min, and vacuum controlling 0.14 mBar; setting the temperature at 4 ℃, setting the time at 120min, keeping the time at 60min, and controlling the vacuum at 0.14 mBar;
4) and (3) resolving and drying: setting the temperature at 20 ℃, setting the time for 20min, keeping the time for 60min, and controlling the vacuum at 0.14 mBar; setting the temperature at 25 deg.C, setting the time at 30min, maintaining the time at 0min, and controlling the vacuum at 0.0 mBar.
S1/Adv-TK freeze-dried powder is prepared.
Example 3 different shelf-life aging test
Samples of the adenovirus sustained-release preparation of example 2 and Adv-TK of a conventional liquid preparation were stored at 4 ℃, -20 ℃, -80 ℃ and taken once after one month, three months, six months, and one year, respectively, wherein S1/Adv-TK was dissolved in physiological saline to 2ml per preparation, and the multiplicity of infection of adenovirus (multiplicity of infection) was detected using a kit (purchased from clontech, Inc., adono-x Rapid kit, cat # 632250).
The results showed that the virus titer of the sample prepared in example 2 was maintained at 1.25X 10 in one year cycle at-80 ℃ in the storage at11pfu/ml, with hardly any loss. The reduction in viral titer after one year storage at 4 ℃ was 0.8X 1011pfu/ml, only 36% of viral activity was lost.While the titer of the adv-tk virus solution of the traditional liquid preparation is reduced to 1.0 multiplied by 10 after being stored for one month at 4 DEG C9pfu/ml, decreased to 1.0X 10 after three months5pfu/ml, decreased to 1.5X 10 after six months3The drug efficacy cannot be guaranteed when the pfu/ml is less than or equal to.
The above experiment shows that the virus titer of the adenovirus sustained-release protective agent prepared in example 2 is less likely to decrease at different temperatures than that of the virus solution of the conventional formula, thereby demonstrating that the sustained-release protective agent contained in the adenovirus sustained-release preparation prepared in example 2 improves the stability of the sample at different temperatures.
Example 4: detecting slow release in the body of the mouse;
conventional culture of B16-F10 melanoma cell lines, culturing with DMEM + 10% FBS +100u/ml penicillin and 100mg/ml streptomycin (invitrogen) in a 37 ℃ and 50% carbon dioxide incubator, stably passaging for 3 and 4 generations, taking cells in logarithmic phase, digesting with trypsin, and then re-suspending and collecting the cells with serum-free culture solution.
Establishing and grouping animal models:
40 female C57BL mice of 5 weeks in size were purchased from Beijing Huafukang Biotech Co., Ltd, and the dorsal subcutaneous inoculation of malignant melanoma B16-F10 cells in mice was 1X 105The experimental animals were observed daily for weight and general condition with free access to water and food. After two weeks, the tumor reached 70mm3On the left and right, the mice were randomly divided into 2 groups (hereinafter referred to as a, b groups), each containing 20 mice. On the day of the group, all mice were intratumorally injected with 1x 107pfu adenovirus, wherein the group a is a control group, and Adv-TK virus liquid with a traditional formula is injected; group b is the experimental group, injected with the adenovirus sustained release protectant prepared in example 1.
After injection, mice were sacrificed at 6h, 3d, 7d, 14d, 30d, respectively, and tumor tissues were removed. Extracting tumor tissue DNA by using a kit (purchased from qiagen company, mini DNA kit), performing real-time quantitative PCR by using an adenovirus capsid protein fiber region primer, and comparing the content of adenovirus in a tumor microenvironment at different times.
F：ACTATATGGACAACGTCAACCCATT
R：ACCTTCTGAGGCACCTGGATGT
The results showed that the virus titer gradually increased from 6 hours to 3 days after the injection of S1/Adv-TK, and after 30 days, the virus titer remained 3.3X 10 in the tumor local microenvironment3pfu/ml; in the Adv-TK liquid preparation, the virus titer reaches the peak value 3 days after injection and gradually decreases, and the virus titer is 1.2 multiplied by 10 after 14 days2pfu/ml, whereas after 30 days Adv-TK was barely detectable in the tumor part, see FIG. 1.
The experiment shows that S1/Adv-TK can be slowly released in local tumor, the action time of Adv-TK and tumor cells is prolonged, and the therapeutic effect of Adv-TK is enhanced.
Example 5: in vivo experiments prove that S1/Adv-TK has a tumor killing effect.
The specific experimental steps are as follows:
subcutaneous tumor animal model: BALB/c mice of 4-6 weeks size, mouse malignant melanoma 1X 10 subcutaneously inoculated on unilateral dorsal side5B16-F10 cells, two weeks later, at tumor soybean size, mice were divided into three groups (n-6) of: a-blank group; b-injection of Adv-TK; c-injection of S1/Adv-TK. Wherein, in groups b and c, 1X 10 is injected into tumor7pfu, once every other day, was injected three times in total. Tumor volume was measured every three days, starting after tumor implantation, and the change in tumor volume was shown in fig. 2.
In the blank group a, the tumor volume is increased continuously, in the experimental group b, the injection of Adv-TK cannot well control the tumor growth, but in the experimental group c, the injection of S1/Adv-TK gradually reduces the tumor volume and can basically control the disease progression.
In addition, mice were observed every three days for survival starting after tumor implantation, and the survival curves are shown in fig. 3: in the experimental group c, the survival time of the mice is obviously longer than that of the other groups, and 80% of the mice in the group c still exist after 70 days of tumor cell inoculation, while the mice in the control group die.
The results show that the adenovirus sustained-release preparation S1/Adv-TK is more stable, the problem that the traditional adenovirus liquid preparation is easy to inactivate when stored is solved, the storage time of the adenovirus preparation is prolonged, and in a malignant melanoma subcutaneous tumor mouse model, the novel adenovirus sustained-release preparation can effectively prolong the survival time of adenovirus in a tumor microenvironment, obviously inhibit tumor growth, prolong the survival time of a mouse, and has a curative effect obviously superior to that of the Adv-TK of the liquid preparation.
Although the present invention has been described with respect to the preferred embodiments, it is not intended to be limited to the embodiments disclosed, and many modifications and variations are possible to those skilled in the art without departing from the spirit of the invention.

Claims (10)

1. A recombinant adenovirus sustained release formulation, wherein the adenovirus buffer formulation comprises:
(1) adenovirus anti-tumor gene medicine; and
(2) the slow release protective agent comprises at least one of sodium alginate, calcium chloride, lactose, mannitol or dextran.
2. The adenovirus sustained-release preparation according to claim 1, wherein the adenovirus anti-tumor gene drug is a gene drug of herpes simplex virus thymidine kinase (HSV-TK) transduced on a conditionally replicating adenovirus.
3. The adenovirus sustained-release preparation according to claim 1, wherein the concentration of the adenovirus anti-tumor gene drug in the adenovirus sustained-release preparation is 1-5 x 1011pfu/mL, preferably 2.5X 1011pfu/mL。
4. The adenovirus sustained-release formulation according to claim 1, wherein the sustained-release protective agent comprises sodium alginate, calcium chloride, lactose, mannitol, and dextran.
5. The adenovirus sustained-release formulation according to claim 4, wherein the buffer protectant comprises:
0.5-2 parts by weight of sodium alginate, preferably 1 part by weight;
0.5-2 parts by weight of calcium chloride, preferably 1 part by weight;
1-5 parts by weight of lactose, preferably 2 parts by weight;
1-5 parts by weight of mannitol, preferably 2 parts by weight;
1-5 parts by weight of dextran, preferably 2 parts by weight.
6. The adenovirus sustained-release preparation according to claim 4, wherein the sustained-release preparation comprises, based on the total mass of the adenovirus sustained-release preparation,
the mass fraction of the sodium alginate is 0.5-2%, preferably 1%;
the mass fraction of the calcium chloride is 0.5-2%, preferably 1%;
the mass fraction of the lactose is 1-5%, preferably 2%;
the mass fraction of the mannitol is 1-5%, preferably 2%;
the mass fraction of the dextran is 1-5%, preferably 2%.
7. A method for preparing an adenovirus sustained release formulation according to any one of claims 1 to 6, comprising the steps of:
(1) providing a first solution as a slow release protectant;
(2) providing a second solution containing adenovirus anti-tumor gene drugs;
(3) mixing the first solution and the second solution, and filtering and collecting formed gel substances to obtain adenovirus sustained-release hydrogel;
(4) sequentially carrying out pre-freezing control, cold well setting, primary drying and analysis drying on the adenovirus sustained-release hydrogel to obtain the adenovirus sustained-release preparation;
optionally, the second solution comprises ADV-TK virus and Tris buffer.
8. The method of claim 7, wherein the first solution is cooled to a temperature of 1-10 ℃, preferably 3-5 ℃, most preferably 4 ℃ prior to mixing the first solution and the second solution in step (3);
optionally, the first solution and the second solution in step (3) are mixed in equal volumes.
9. Use of the adenovirus sustained-release preparation according to any one of claims 1 to 6 or the adenovirus sustained-release preparation prepared by the preparation method according to claim 7 or 8 in the preparation of an anti-tumor medicament.
10. Use according to claim 9, wherein the tumour is a melanoma, preferably a malignant melanoma.
CN201911069414.9A 2019-11-05 2019-11-05 Recombinant adenovirus sustained-release preparation, preparation method and application thereof Pending CN110960484A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911069414.9A CN110960484A (en) 2019-11-05 2019-11-05 Recombinant adenovirus sustained-release preparation, preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911069414.9A CN110960484A (en) 2019-11-05 2019-11-05 Recombinant adenovirus sustained-release preparation, preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN110960484A true CN110960484A (en) 2020-04-07

Family

ID=70030126

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911069414.9A Pending CN110960484A (en) 2019-11-05 2019-11-05 Recombinant adenovirus sustained-release preparation, preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110960484A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022101640A1 (en) * 2020-11-16 2022-05-19 Atelerix Limited Storing and/or transporting extracellular nucleic acids

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009675A1 (en) * 1998-08-14 2000-02-24 Aventis Pharmaceuticals Products Inc. Adenovirus formulations for gene therapy
US20010006629A1 (en) * 1993-10-25 2001-07-05 Richard J. Gregory Recombinant adenoviral vector and methods of use
CN1640496A (en) * 2004-01-17 2005-07-20 上海三维生物技术有限公司 Recombinant adenovirus lyophilized preparation and its preparing method
US20100143304A1 (en) * 2007-01-30 2010-06-10 Cedars-Sinai Medical Center Adenoviral vector comprising herpes simplex virus type 1 thymidine kinase and a transgene for increasing the expression of the transgene
CN101768576A (en) * 2010-01-04 2010-07-07 中国人民解放军第三军医大学 HSV1-TK (herpes simplex virus type 1-thymidine kinase) gene recombination oncolytic adenovirus, preparation method and application thereof
CN102206613A (en) * 2010-12-26 2011-10-05 周剑峰 Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct
CN102286433A (en) * 2010-12-26 2011-12-21 马丁 Obtainment and application of novel oncolytic adenovirus-thymidine kinase genetic construct
CN106492213A (en) * 2016-12-05 2017-03-15 天津康希诺生物技术有限公司 A kind of adenoviruss lyophilization additive and adenoviruss lyophilized formulations
CN108175774A (en) * 2018-01-26 2018-06-19 成都远睿生物技术有限公司 A kind of freeze-drying sustained release protective agent for being used to prepare adenovirus preparation and adenovirus preparation and preparation method

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010006629A1 (en) * 1993-10-25 2001-07-05 Richard J. Gregory Recombinant adenoviral vector and methods of use
WO2000009675A1 (en) * 1998-08-14 2000-02-24 Aventis Pharmaceuticals Products Inc. Adenovirus formulations for gene therapy
CN1640496A (en) * 2004-01-17 2005-07-20 上海三维生物技术有限公司 Recombinant adenovirus lyophilized preparation and its preparing method
US20100143304A1 (en) * 2007-01-30 2010-06-10 Cedars-Sinai Medical Center Adenoviral vector comprising herpes simplex virus type 1 thymidine kinase and a transgene for increasing the expression of the transgene
CN101768576A (en) * 2010-01-04 2010-07-07 中国人民解放军第三军医大学 HSV1-TK (herpes simplex virus type 1-thymidine kinase) gene recombination oncolytic adenovirus, preparation method and application thereof
CN102206613A (en) * 2010-12-26 2011-10-05 周剑峰 Acquisition and use of tumor-selective replicative adenovirus - thymidine kinase gene construct
CN102286433A (en) * 2010-12-26 2011-12-21 马丁 Obtainment and application of novel oncolytic adenovirus-thymidine kinase genetic construct
CN106492213A (en) * 2016-12-05 2017-03-15 天津康希诺生物技术有限公司 A kind of adenoviruss lyophilization additive and adenoviruss lyophilized formulations
CN108175774A (en) * 2018-01-26 2018-06-19 成都远睿生物技术有限公司 A kind of freeze-drying sustained release protective agent for being used to prepare adenovirus preparation and adenovirus preparation and preparation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
杨树源等: "《神经外科学》", 31 January 2008, 人民卫生出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022101640A1 (en) * 2020-11-16 2022-05-19 Atelerix Limited Storing and/or transporting extracellular nucleic acids

Similar Documents

Publication Publication Date Title
ES2393748T3 (en) Compositions and methods of using ABCI chondroitinase mutants
CN111588859B (en) Freeze-drying protective agent and application thereof, freeze-dried seedling and preparation method thereof
CN113388563A (en) Escherichia coli Nissle1917 genetically engineered bacterium with hypoglycemic effect and preparation method and application thereof
CN105308173B (en) Pharmaceutical composition for stimulating angiogenesis
CN110960484A (en) Recombinant adenovirus sustained-release preparation, preparation method and application thereof
CN110772477A (en) Recombinant adenovirus sustained-release hydrogel, preparation method and application thereof
WO2017016430A1 (en) Tumour inhibitory peptide
CN116057071B (en) Novel mutant of recombinant ganoderma lucidum immunomodulatory protein and application thereof
AU2022204563B2 (en) Application and method of superoxide dismutase in preparing medicine for treating psoriasis
CN111094324B (en) Oncolytic virus, synthetic DNA sequence and application thereof
CN109593725A (en) A kind of recombination mescenchymal stem cell and its application
AU2004240968B2 (en) Recombinant gene medicine of adenovirus vector and gene p53 for treating proliferative diseases
CN115068632A (en) Application of Ago2 in preparation of medicine for treating heart failure, protein, gene, transformant, medicine and preparation method thereof
CN114712393A (en) Application of Hnf-1 alpha gene modified mesenchymal stem cells in prevention and treatment of liver cancer
CN115197157B (en) Reduction-responsive nucleic acid delivery vector, and preparation method and application thereof
CN104771753B (en) A kind of Brucellosis treatment preparation
CN111979203B (en) Oncolytic vaccinia virus carrying CTTNBP2NL gene, construction method and application in preparation of antitumor drugs
CN102226186B (en) Activated Bax gene for treating malignant tumor by taking adenovirus as carrier
Lin et al. Stem Cell Therapy for Glioblastoma
AU2004217830B2 (en) A recombinant constructed by a virus vector and a human tumor suppressor gene and its use
Iyer et al. Molecular insights and promise of oncolytic virus based immunotherapy
CN113755455A (en) Oncolytic vaccinia virus carrying SIKE gene, construction method and application
RU2489168C1 (en) Pharmaceutical composition producing antioxidant, antimicrobial, antitoxic human lactoferrin protein, method for preparing it, and method of therapy
WO2021087851A1 (en) Recombinant viral vector, immunogenic composition comprising same and use thereof
AU2005326331B2 (en) Recombined adenovirus p53 preparation for treating tumor

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
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20200407

WD01 Invention patent application deemed withdrawn after publication