Stable oncolytic adenovirus composition

Abstract

The invention discloses a stable oncolytic adenovirus composition, which contains adenovirus or recombinant oncolytic adenovirus modified based on the adenovirus, and also contains a nonionic surfactant (virus stabilizer), a cryoprotectant, an osmotic pressure regulator and a buffer substance. The composition can still keep stable activity in low-temperature storage (lower than-70 DEG C), cold-chain transportation, repeated freezing and thawing and temporary storage at room temperature (10-30 DEG C). The invention also relates to application of the composition in medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a stable oncolytic adenovirus composition. Background Art

[0002] According to the latest national cancer statistics released by the National Cancer Center in 2022, there were 4.064 million new cancer cases and 2.414 million cancer deaths in my country in 2016. The latest global cancer burden estimates for 2020 released by the World Health Organization's International Agency for Research on Cancer (IARC) show that there were 19.29 million new cancer cases and 9.96 million cancer deaths in 2020. Among them, there were 4.57 million new cancer cases and 3 million cancer deaths in my country, accounting for 23.7% and 30% respectively, and the incidence and mortality rates ranked first in the world. Although in recent years, with the development of medicine and science and technology, the annual standardized 5-year survival rate of cancer treatment in my country has increased from 30.9% (2003) to 40.5% (2015), the overall survival rate of cancer patients is still low, and the 5-year survival rate of some cancers is less than 20%. Malignant tumors seriously endanger human health and bring heavy burdens to individuals and society.

[0003] Oncolytic viruses are a class of natural or genetically modified RNA or DNA viruses that can selectively replicate in tumor cells and then lyse tumor cells without affecting the survival of normal cells. Oncolytic viruses mainly exert their anti-tumor effects by directly lysing cells through proliferation and activating local and systemic immune responses. Compared with traditional treatments, oncolytic virus therapy has the advantages of good targeting, few adverse reactions, multiple tumor killing pathways, and low resistance. Many clinical studies have shown that oncolytic viruses can bring clinical benefits to patients with different types, different stages of progression, and even metastatic and incurable tumors.

[0004] In 2005, the State Food and Drug Administration approved the oncolytic adenovirus drug H101 (recombinant human adenovirus type 5, ) combined with chemotherapy drugs for the treatment of advanced nasopharyngeal carcinoma. This is also the only oncolytic virus therapy drug currently approved in my country. In 2015, the United States and Europe successively approved T-VEC (talimogene laherparepvec, ) for the treatment of advanced melanoma, further promoting the rapid development of oncolytic virus therapy. In June 2021, Teserpaturev was approved for marketing in Japan for the treatment of malignant glioma, once again attracting high attention to oncolytic virus therapy.

[0005] However, as a live viral biological agent, oncolytic virus has stability problems such as decreased infection titer, severe aggregation of viral particles, decreased expression of introduced immune regulatory factors, and reduced activity during storage, transportation, freeze-thaw, and short-term exposure to low temperature conditions. Summary of the invention

[0006] In order to solve the above-mentioned technical problems existing in the prior art, the present invention discloses a stable oncolytic adenovirus composition by preferably using non-ionic surfactants (virus stabilizers), cryoprotectants, osmotic pressure regulators and suitable buffer substances, etc., and its activity can still be maintained at a certain level after repeated freezing and thawing or short-term storage out of low temperature.

[0007] The technical solution of the present invention is as follows:

[0008] The first objective of the present invention is to provide a stable oncolytic adenovirus composition, wherein the composition contains an oncolytic adenovirus or a recombinant oncolytic adenovirus modified based on an adenovirus as an active substance, and the composition further comprises a non-ionic surfactant, which serves as a virus stabilizer, a cryoprotectant, an osmotic pressure regulator, and a buffer substance. The composition is a colorless clear solution, and the pH value of the composition is 7.0 to 9.0.

[0009] In a particular embodiment, the composition is a colorless clear solution.

[0010] Furthermore, the recombinant oncolytic adenovirus based on adenovirus modification is a recombinant oncolytic adenovirus based on adenovirus backbone modification and introduced with immune regulatory factors.

[0011] Furthermore, the immunomodulatory factor is one or a combination of GM-CSF, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-23, IL-24, CCL20, CCL21, and CXCL10. Preferably, the immunomodulatory factor is GM-CSF.

[0012] Furthermore, the nonionic surfactant is any one of Tween 80 and Tween 20 or a combination thereof.

[0013] Furthermore, the cryoprotectant is any one of glycerol, sucrose, sorbitol, mannitol, trehalose, or a combination thereof.

[0014] Furthermore, the osmotic pressure regulator is sodium chloride or glucose.

[0015] Furthermore, the buffer substance is selected from one of a sodium phosphate buffer system, a potassium phosphate buffer system, a Tris-HCl buffer system, a sodium acetate buffer system, and an arginine hydrochloride buffer system.

[0016] Furthermore, the composition comprises 2.0×10 10 PFU / ml~8.0×10 10PFU / ml active substance, 0.01% to 0.8% (w / v) non-ionic surfactant, 1% to 10% (w / v) cryoprotectant, 25 to 140 mM osmotic pressure regulator, buffer substance, and the balance is water.

[0017] In a specific embodiment, the amount of the buffer substance is 10 mM to 25 mM.

[0018] The second object of the present invention is to provide a method for preparing the aforementioned oncolytic adenovirus composition, the method comprising the following steps: taking a prescribed amount of nonionic surfactant, cryoprotectant, osmotic pressure regulator and buffer substance, dissolving them with water for injection, and after dissolution is complete, adjusting the pH to 7.0-9.0 with 0.5M NaOH or 0.5M HCL, adding water for injection to the required volume, and diluting the oncolytic adenovirus stock solution to 2.0×10 10 PFU / ml~8.0×10 10 PFU / ml, sterilized and filtered using a disposable sterile filter.

[0019] The third object of the present invention is to provide the use of the aforementioned oncolytic adenovirus composition in the preparation of a drug for treating solid tumors.

[0020] Preferably, the solid tumor is head and neck squamous cell carcinoma, breast cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer and melanoma.

[0021] Preferably, the dosage form of the drug is an injection.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] (1) A nonionic surfactant (virus stabilizer) is added to the composition, which effectively reduces interfacial adsorption, improves physical stability, inhibits the aggregation of adenovirus, reduces filtration losses during product preparation, and improves product stability during storage and transportation.

[0024] (2) The combination contains one or more virus cryoprotectants and virus stabilizers, and is matched with a buffer system with the optimal pH for virus activity, which ensures the maintenance of the infection titer, virus activity, and activity of the introduced immune regulatory factors during repeated freezing and thawing, storage, transportation, and short-term removal from low temperature. DETAILED DESCRIPTION

[0025] The following examples are provided to further illustrate the present invention and are not intended to limit the present invention. Equivalent replacements or substitutions made on the basis of the present invention all fall within the protection scope of the present invention.

[0026] Example 1 Preparation of recombinant oncolytic adenovirus compositions with different prescriptions

[0027] Weigh the selected materials according to the prescription amount in Table 1, dissolve the weighed materials with water for injection, and after the materials are dissolved, use 0.5M NaOH or 0.5M HCL to adjust to the corresponding pH. After adding water for injection to the required volume, use a 0.22μm filter to filter and sterilize, and dilute the recombinant oncolytic adenovirus stock solution (recombinant oncolytic adenovirus (multiple clone insertion sites of exogenous genes in the E3 region of adenovirus type 5 introduced into GM-CSF)) to 4.0×10 10 PFU / ml, sterilized and filtered using a disposable sterile filter.

[0028] Table 1 Recombinant oncolytic adenovirus compositions with different prescriptions

[0029] prescription Preparation formula composition Prescription 1 10 mM Tris, 120 mM NaCl, 2.5% (w / v) glycerol, 0.4% Tween 80, pH 8.0 Prescription 2 15mM Tris, 120mM NaCl, 2.5% (w / v) sucrose 0.2% Tween 80, pH 8.0 Prescription 3 20 mM Tris, 70 mM NaCl, 5% (w / v) glycerol, 0.4% Tween 80, pH 8.5 Prescription 4 25 mM Tris, 70 mM NaCl, 5% (w / v) sucrose, 0.2% Tween 80, pH 8.5 Prescription 5 <![CDATA[2 mM NaH2PO4, 8 mM Na2HPO4, 100 mM NaCl, 2.5% (w / v) glycerol, 0.4% Tween 80, pH 8.0]]> Prescription 6 <![CDATA[2 mM NaH2PO4, 8 mM Na2HPO4, 100 mM NaCl, 2.5% (w / v) sucrose, 0.2% Tween 80, pH 8.0]]> Prescription 7 <![CDATA[2 mM NaH2PO4, 8 mM Na2HPO4, 90 mM NaCl, 5% (w / v) glycerol, 0.4% Tween 80, pH 7.0]]> Prescription 8 <![CDATA[2 mM NaH2PO4, 8 mM Na2HPO4, 90 mM NaCl, 5% (w / v) sucrose, 0.2% Tween 80, pH 7.0]]>

[0030] Example 2 Stability study of adenovirus compositions with different formulations

[0031] The stability of the above formulations was investigated by freeze-thawing 5 times below -70°C, storing at 4°C and room temperature (10°C to 30°C) for 14 days. The results of the stability investigation are shown in Table 2.

[0032] Table 2 Results of different formulation compositions and stability studies

[0033]

[0034] As shown in Table 2, various formulations can maintain good biological activity after repeated freezing and thawing, and storage at 4°C and room temperature for 14 days. This provides a guarantee for maintaining the activity of the product during storage, transportation, and short-term storage out of low temperature during actual use.

[0035] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A stable oncolytic adenovirus composition, characterized in that: The composition contains oncolytic adenovirus or recombinant oncolytic adenovirus modified based on adenovirus as an active substance. The composition also contains a non-ionic surfactant, a cryoprotectant, an osmotic pressure regulator, and a buffer substance. The pH of the composition is 7.0 to 9.

0.

2. The oncolytic adenovirus composition according to claim 1, characterized in that The recombinant oncolytic adenovirus based on adenovirus modification is a recombinant oncolytic adenovirus based on adenovirus backbone modification and introduced with immunomodulatory factors.

3. The oncolytic adenovirus composition according to claim 2, characterized in that The immunomodulatory factor is one or a combination of GM-CSF, IFN-γ, IL-2, IL-12, IL-15, IL-18, IL-23, IL-24, CCL20, CCL21, and CXCL10. Preferably, the immunomodulatory factor is GM-CSF.

4. The oncolytic adenovirus composition according to claim 1, characterized in that The nonionic surfactant is any one of Tween 80 and Tween 20 or a combination thereof.

5. The oncolytic adenovirus composition according to claim 1, characterized in that The cryoprotectant is any one of glycerol, sucrose, sorbitol, mannitol, trehalose or a combination thereof.

6. The oncolytic adenovirus composition according to claim 1, characterized in that The osmotic pressure regulator is sodium chloride or glucose.

7. The oncolytic adenovirus composition according to claim 1, characterized in that The buffer substance is selected from a combination of one or more of a sodium phosphate buffer system, a potassium phosphate buffer system, a Tris-HCl buffer system, a sodium acetate buffer system, and an arginine hydrochloride buffer system.

8. The oncolytic adenovirus composition according to claim 1, characterized in that The composition contains 2.0×10 10 PFU / ml~8.0×10 10 PFU / ml active substance, 0.01% to 0.8% (w / v) non-ionic surfactant, 1% to 10% (w / v) cryoprotectant, 25 to 140 mM osmotic pressure regulator, buffer substance, and the balance is water.

9. The method for preparing the oncolytic adenovirus composition according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: taking a prescribed amount of nonionic surfactant, cryoprotectant, osmotic pressure regulator and buffer substance, dissolving them with water for injection, adjusting the pH to 7.0-9.0 with 0.5M NaOH or 0.5M HCL after dissolution, adding water to the required volume, and diluting the oncolytic adenovirus stock solution to 2.0×10 10 PFU / ml~8.0×10 10 PFU / ml, sterilize and filter.

10. Use of the oncolytic adenovirus composition according to any one of claims 1 to 8 in the preparation of a medicament for treating solid tumors, preferably, the solid tumor is head and neck squamous cell carcinoma, breast cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer and melanoma; preferably, the dosage form of the medicament is an injection.

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