Drug (variants) for treatment of solid malignancies in combination with gene-directed enzyme prodrug therapy and immunotherapy

Abstract

The invention relates to the field of biotechnology and medicine, in particular to oncology and pharmacy, and aims to provide a finished product dosage form of a pharmaceutical preparation for treating human solid malignant tumors by combining gene-directed enzyme prodrug therapy-immunotherapy. Embodiments of two two-component pharmaceutical formulations are presented. In a first embodiment, the pharmaceutical formulation comprises a first container and a second container wherein the first container comprises a first component comprising a therapeutically effective amount of pCMV-HSVtk-hGM-CSF therapeutic plasmid DNA in the form of a chilled aqueous solution; the second container contains a second component containing a therapeutically effective amount of a polyethyleneimine-polyethylene glycol-TAT peptide block copolymer in the form of a chilled aqueous solution. By unfreezing and mixing the two components, a final injection solution for a patient can be prepared immediately. In a second embodiment, the pharmaceutical formulation comprises a first container and a second container wherein the first container comprises a first component comprising a therapeutically effective amount of pCMV-HSVtk-hGM-CSF therapeutic plasmid DNA in the form of a freeze-dried product, and a second component comprising a therapeutically effective amount of pCMV-HSVtk-hGM-CSF therapeutic plasmid DNA in the form of a freeze-dried product, the second container contains a second component containing a therapeutically effective amount of a polyethyleneimine-polyethylene glycol-TAT peptide block copolymer in the form of a chilled aqueous solution. By adding water to reconstitute the first component, thawing the second component, and mixing the two components, a final injection solution for use by a patient can be prepared immediately. The pharmaceutical preparation can be used for treating human solid malignant tumors. The two proposed dosage forms have high stability and maintain the therapeutic effect within at least 18 months.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology, medicine, in particular oncology and pharmaceuticals, and aims to provide a pharmaceutical dosage form for combined gene-guided enzyme prodrug therapy-immunotherapy for the treatment of solid malignant tumors.

[0002] The disclosed pharmaceutical dosage form can be used to treat various solid malignant tumors in humans. The proposed pharmaceutical dosage form shows high stability and maintains its therapeutic efficacy for at least 18 months. Background Art

[0003] Cancer (malignant tumors) is one of the most serious problems in medicine: it ranks second in mortality worldwide and its incidence is increasing.

[0004] Neoplastic diseases combine the complexity of cellular organization with the properties of a sophisticated growth system that is able to adapt to external influences, including drugs, due to the microheterogeneity within such tumors. Cancerous tumors are heterogeneous in that each cancer cell is different from all other cells of the same tumor, and the tumor of one patient is different from the same tumor of another patient. Therefore, among the numerous cells of a tumor there are always cells that are resistant to the effects of treatment and are able to generate new tumor clones resistant to such effects. As an obvious consequence, it is quite difficult to clearly identify any universal key gene / gene product that can be used as a target for anticancer therapy.

[0005] Classic chemotherapy, established over decades as a standard treatment for cancer patients, has proven effective in slowing the progression of various types of malignancies. However, the therapeutic index of classical chemotherapy is generally low due to the lack of specificity of the drugs for cancer cells versus normal cells and the difficulty in achieving therapeutic concentrations inside the tumor without systemic toxicity. These issues are particularly relevant for solid tumors, as the lack of neoangiogenesis and associated necrotic areas often render such tumors relatively resistant to the uptake of systemically delivered drugs. Given the current situation worldwide, new strategies for cancer treatment are being sought and implemented.

[0006] One such strategy is gene-directed enzyme prodrug therapy (GDEPT) (Sverdlov ED Curr. Gene Ther. 2011, 11 (6), p. 501-531). This approach is also known as "suicide gene therapy" or "gene surgery". The method of gene surgery is to deliver genes encoding enzymes that can convert their systemically administered substrates into highly toxic agents formed in cells expressing the enzyme into target cancer cells. This strategy is similar to chemotherapy, but the difference is that the toxins in the case of gene surgery are formed inside the cancer cells, so the toxic effects on normal cells are minimal.

[0007] The GDEPT method can kill cancer cells regardless of their origin and characteristics. Another important advantage of genetic surgery is that even a small number of cells expressing the transgene is sufficient to cause the death of a large number of tumor cells. Toxins formed in cells expressing the transgene can be released from them and penetrate into neighboring cells that have not received the transgene, causing their death. This phenomenon is called the bystander effect.

[0008] Currently, the most thoroughly studied system for performing GDEPT is the herpes simplex virus thymidine kinase (HSVtk) / ganciclovir (GCV) combination, which has entered the late stages of clinical trials, during which its efficacy and safety have been confirmed (Immonen A. et al. Mol. Ther. 2004, 10(5), p.967-972; Westphal M. et al. Lancet Oncol. 2013, 14(9), p.823-833). Ganciclovir is a registered drug and is commercially available, for example as The therapeutic gene encoding thymidine kinase (e.g., herpes simplex virus thymidine kinase (HSVtk)) is delivered to tumor cells as part of an expression construct to ensure that the HSVtk enzyme is produced inside these cells. Ganciclovir is a guanosine analog and a prodrug for systemic administration. In tumor cells expressing HSVtk, ganciclovir is phosphorylated to ganciclovir monophosphate under the action of the thymidine kinase enzyme. Then, under the action of intracellular kinases, ganciclovir monophosphate is converted into ganciclovir diphosphate and ganciclovir triphosphate in turn. As a substrate and incorporated into DNA, ganciclovir triphosphate competitively inhibits DNA polymerase, which results in inhibition of DNA synthesis due to inhibition of DNA chain extension and single-stranded DNA breaks, ultimately leading to tumor cell death. In addition, the newly formed ganciclovir triphosphate can leave the tumor cells in which it is formed and penetrate into neighboring tumor cells, causing their death (above-mentioned bystander effect). Cell death mediated by the HSVtk / ganciclovir combination may be caused by various mechanisms, namely: apoptosis and necrosis, as well as immunogenic cell death (ICD) accompanied by the release of tumor-specific antigens and damage-associated molecular patterns (DAMPs), which leads to tumor infiltration of various antigen-presenting cells, including dendritic cells and T cells (Sverdlov ED Curr. Gene Ther. 2011, 11(6), p.501-531).

[0009] Because some tumor cells (or entire tumors) may be resistant to certain toxic agents used in the GDEPT system, researchers have developed ways to improve the efficacy of gene surgery. One such approach is to combine gene surgery with immunotherapy. This approach is sometimes called gene immunotherapy.

[0010] It is known that tumor cells in the early stages of carcinogenesis are almost indistinguishable from the cells of the surrounding tissue in their molecular composition; they cannot be recognized and destroyed by the immune system and are therefore non-immunogenic. As the tumor grows due to the accumulation of mutations in the DNA, so-called neoantigens are formed, which can be presented by dendritic cells to helper T cells, which are activators of specific immunity against the tumor. The appearance of neoantigens is delayed, which allows tumor cells to implement protective mechanisms against the immune system, in particular to create a microenvironment that suppresses specific anti-tumor immunity.

[0011] Many members of the cytokine family can mobilize the patient's immune system and contribute to the development of specific anti-tumor responses. For example, in Imlygic gene therapy anticancer drugs (Amgen Inc., USA), a method based on increasing the production of granulocyte-macrophage colony stimulating factor (GM-CSF) cytokines is used to enhance the presentation of tumor antigens by dendritic cells. Studies have shown that cell-based vaccines containing GM-CSF provide stronger anti-tumor responses in animals than vaccines including other cytokines (Dranoff G, Jaffee E et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43).

[0012] Granulocyte-macrophage colony-stimulating factor (GM-CSF) belongs to the family of hematopoietic cytokines. The biological activity of GM-CSF is mediated by its binding to heterologous receptors expressed on monocytes, macrophages, granulocytes, lymphocytes, endothelial cells and alveolar epithelial cells. After GM-CSF specifically binds to the receptor, signal transduction occurs along the JAK2 pathway, resulting in a functional response. GM-CSF stimulates the proliferation and differentiation of granulocytes and monocytes, enhances the phagocytic and cytotoxic activity of granulocytes and eosinophils, stimulates the proliferation, maturation and differentiation of dendritic cells, and plays an important role in the recruitment and activation of antigen-presenting cells.

[0013] It is known that the expression of GM-CSF in tumor cells increases the immunogenicity of tumors, thereby facilitating their elimination and ensuring the development of anti-tumor responses. In vivo studies have shown that the efficacy of the HSVtk / GCV system is significantly improved if the HSVtk gene is introduced into tumor cells together with the GM-CSF gene (Jones RK, Pope IM et al. (2000) Cancer Gene Ther. 7: 1519-28; Finocchiaro LM, Fiszman GL et al. (2008) Cancer Gene Ther. 15: 165-72).

[0014] It has been demonstrated in ex vivo and in vivo studies that combination therapy with the combined administration of GM-CSF and the HSVtk / GCV system is superior to gene surgery involving the administration of the HSVtk / GCV system alone.

[0015] The anti-tumor effect of GM-CSF in combination with the HSVtk / GCV system is believed to be due to the following mechanism: When the HSVtk and GM-CSF genes are simultaneously introduced into cancer cells, the cancer cells will die due to the action of the HSVtk / GCV system and will release tumor antigens therefrom. These tumor antigens will be effectively presented to the T cells of the immune system by the antigen-presenting cells activated by GM-CSF, thereby providing activation of specific anti-tumor immunity, resulting in increased tumor cell death and reduced metastasis probability. By means of synthetic GM-CSF, antigen-presenting cells can be attracted to the tumor and activated. Therefore, the antigen-presenting cells activated by GM-CSF effectively present tumor antigens to the T cells of the immune system and thus activate specific anti-tumor immunity.

[0016] One of the important factors for the success of cancer gene therapy is a perfect delivery and expression system of therapeutic genes, because these genes must work in cancer cells and must not work in normal cells. Viral and non-viral systems are used to deliver therapeutic constructs to patients' tumor cells. Compared with viral delivery systems, the main disadvantage of non-viral delivery systems is their low transfection efficiency. At the same time, non-viral delivery systems have many advantages, such as high packaging capacity, low immunogenicity, high safety and cost-effective production possibilities (Yue Y. and Wu С. (2013) Biomaterials Science 1: 152-170).

[0017] At present, polycations are widely used as non-viral DNA delivery systems for gene therapy. DNA complexes or polymer complexes (polyplexes) formed with polycations can compress DNA and protect it from external factors. Due to certain peculiarities of its chemical structure, polymer complexes provide the possibility of modification, especially by covalently linking molecules that interact with targets. Polymer complexes can overcome the endosomal membrane and enter the cytoplasm; they are non-pathogens, almost non-immunogenic and non-toxic. The relatively low production cost makes them very attractive to pharmaceutical companies, and they have very high repeatability of the production of polymer complexes with covalently linked ligands (Kang H.С., Lee M. et al. (2005) Crit. Rev. Eukaryot. Gene Expr. 15: 317-42). Polymer complexes can be used for systemic and local administration: intratumoral administration into the lungs, various tumors, brain, breast and fallopian tubes. Polymer complexes have been shown to be effective in gene therapy for hepatocellular carcinoma (Iwai M, Harada Y et al. (2002) Biochem. Biophys. Res. Commun. 291: 48-54) and disseminated pancreatic cancer (Aoki K, Furuhata S et al. (2001) Gene Ther. 8: 508-14).

[0018] This method of combined GDEPT-immunotherapy using a polycationic delivery system has been successfully implemented in a pharmaceutical composition described in, for example, RU 2 575077. The pharmaceutical composition comprises the following active substances: 1) nucleic acid encoding two therapeutic genes (killer genes and immunomodulatory genes) and regulatory elements required for expression; 2) a polymeric polycationic carrier consisting of the following three components: polyethylene glycol (PEG), polyethyleneimine (PEI) and TAT peptide. Nucleic acid is a plasmid in a high-purity supercoiled form, with the general formula pCMV-HSVtk-hGM-CSF, encoding herpes simplex virus thymidine kinase (HSVtk) and human granulocyte-macrophage colony stimulating factor (GM-CSF) under the control of a cytomegalovirus (CMV) promoter (DNA control region). The polycationic carrier is a block copolymer (BC) (collectively referred to as PPT) of PEI and PEG with a conjugated TAT peptide. The TAT peptide has a sequence characteristic of GRKKKRRQRC, is a fragment of the TAT protein, and enhances the ability of the block copolymer into which it is included to penetrate into cells and ensure plasmid delivery into tumor cells (Ulasov AV, Khramtsov YV et al. (2011) Mol. Ther. 19: 103-12; Rudolph C, Plank С et al. (2003) J. Biol. Chem. 278: 11411-8).

[0019] The recombinant plasmid DNA described in RU 2 575 077 consists of the following components: a fragment of the promoter site (581 nucleotide pairs) that drives gene transcription; the HSVtk gene, which upon expression synthesizes a form of HSVtk consisting of 374 amino acid residues; an internal ribosome entry site (IRES) that allows the expression of two therapeutic genes from a single vector (Mizuguchi, Xu et al. (2000) Mol. Ther. 1: 376-82); the hGM-CSF or mGM-CSF gene, whose expression produces the hGM-CSF (human) or mGM-CSF (mouse) form, respectively; the SV40 polyadenylation signal and the transcription terminator that provide for the synthesis of mature RNA from the gene. Since the granulocyte-macrophage colony stimulating factor protein is species-specific (Shi Y, Liu СH et al. (2006) Cell Res. 16: 126-33), and the animals most commonly used to test the efficacy of gene therapy constructs are animals with transplanted tumors (xenografts), RU 2 575 077 discloses two construct variants, one containing the mouse GM-CSF gene for mouse model experiments and the other containing the human GM-CSF gene for clinical trials and direct use in patients. In addition, the recombinant plasmid DNA described in RU 2 575 077 also contains auxiliary regions for efficient biosynthesis of plasmid DNA on an industrial scale in Escherichia coli (E. coli) production strain cells. These auxiliary regions include, in particular, f1 ori, ColE1 ori, and the ampicillin resistance selection marker AmpR.

[0020] When the molecules of the pCMV-HSVtk-hGM-CSF plasmid penetrate into tumor cells, the HSVtk enzyme is synthesized, which is able to phosphorylate the nucleoside analog of ganciclovir administered systemically into ganciclovir monophosphate, which is then further phosphorylated into the toxic form of ganciclovir triphosphate by endogenous cellular kinases. During cell division, ganciclovir triphosphate is incorporated into newly synthesized DNA chains and interrupts their further synthesis, leading to cell death. Moreover, when the plasmid molecules penetrate into tumor cells, GM-CSF protein is synthesized, which is secreted into the extracellular space and stimulates the growth, development and differentiation of granulocytes and antigen-presenting cells, attracting them to the tumor growth area to provide induction of specific anti-tumor immune response. The mechanism by which GM-CSF induces anti-tumor immunity includes the direct participation of natural killer cells and antigen-presenting cells such as dendritic cells in the tumor growth area that lead to T cell activation. Thus, due to the activity of herpes simplex virus thymidine kinase, the administered ganciclovir causes tumor cell death to release tumor antigens, which are presented to the T cells of the immune system by antigen-presenting cells activated by GM-CSF. The activation of the adaptive immune response caused by the drug can provide local and systemic immune-mediated destruction of distant tumors, thereby providing an additional anti-tumor vaccination effect and leading to the development of immunological memory and long-term control of tumor growth using immune surveillance.

[0021] The pharmaceutical composition disclosed in RU 2 575 077 contains the following excipients: 5 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 12.5 mM borate buffer, pH 7.5, and 5% glucose.

[0022] RU 2 575 077 showed high efficacy of the GDEPT-immunotherapy combination system based on the use of the pCMV-HSVtk-GM-CSF plasmid combined with systemically administered ganciclovir against a wide range of solid malignancies in mouse model experiments. In particular, the efficacy of the system was demonstrated against sarcomas, colon adenocarcinomas, cervical cancer, laryngeal cancer and fibrosarcomas.

[0023] One of the main issues with the use of gene therapy drugs in medicine is their storage stability. As mentioned above, gene therapy drugs used in clinical practice can be divided into viral and non-viral drugs based on their delivery method. Although non-viral gene therapy drugs have many advantages over viral drugs, such as low toxicity, low pathogenicity and low immunogenicity, lower production costs, easier to manufacture and / or modified in some way to achieve high specificity, both drugs are unstable when stored. The stability of this type of drug can only be achieved at negative temperatures, often requiring storage at -70°C, and improper storage temperature may result in a complete loss of drug activity.

[0024] The finished dosage form of a gene therapy drug must meet certain requirements, in particular:

[0025] 1) The production process should be operational (technological);

[0026] 2) Any finished dosage form must be stable during storage and transportation;

[0027] 3) Ideally, the shelf life should be long; and

[0028] 4) Any finished dosage form should be characterized by ease of preparation and ease of use itself.

[0029] One of the methods for developing gene therapy drug dosage forms is to obtain freeze-dried drugs. For example, WO 2009 / 021017 describes the successful development of a freeze-dried composition containing an excipient (filler), a nucleic acid and a cationic lipopolymer. The cited lipopolymer contains a main chain based on a cationic polymer, and cholesterol molecules and polyethylene glycol molecules are covalently linked to the main chain. However, this method is generally not suitable for freeze-drying of DNA complexes formed with synthetic carriers because this method changes the physicochemical properties, which causes the freeze-dried composition to aggregate and lose its transfection ability when reconstituted with a solvent. The use of cryoprotectants, such as various sugars, dextran or polyethylene glycol, may improve stability; however, the effects achieved vary and depend not only on the specific cryoprotectant used but also on the type of synthetic carrier used.

[0030] Another important factor affecting the use of lyophilization to obtain gene therapy dosage forms is that even with the most effective lyoprotectants, a very high sugar / DNA molar ratio (usually exceeding 1000:1) is still required to ensure stability. Therefore, lyophilized preparations often require a very high degree of dilution during reconstitution to achieve an acceptable osmotic pressure (ideally isotonic), resulting in a significant reduction in the DNA concentration in the reconstituted preparation compared to the original composition before lyophilization. For many preparations with cationic carriers as carriers, the final DNA concentration may be about 0.1-0.2 mg / ml or lower, which causes certain difficulties in clinical use because such a volume that is incompatible with the local administration route may be required in order to achieve the optimal administration dose. WO 2009 / 021017 reports that this drug limitation is one of the main reasons for the poor performance of synthetic gene delivery systems in human clinical trials.

[0031] Therefore, in the art, there is a need to develop new gene therapy drug dosage forms, in particular combined GDEPT-immunotherapy drugs based on the therapeutic pCMV-HSVtk-GM-CSF plasmid, combined with the possibility of long-term storage under relatively non-stringent conditions, the manufacturability of the technical process, the convenience of preparing the drug for use by patients in need of treatment, and the convenience of the administration itself. Summary of the invention

[0032] The present invention relates to a two-component drug for treating solid malignancies in combination with gene-guided enzyme prodrug therapy-immunotherapy, wherein a ready-to-use injection solution for application to a patient is obtained by mixing a first component with a second component. The first component is an aqueous solution stored frozen until use, containing pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA as an active substance, which is obtained by biosynthesis in Escherichia coli cells and has a direct therapeutic effect. The first component contains a therapeutically effective amount of active substance. The second component is an aqueous solution stored frozen until use, containing a covalently bound polyethyleneimine-polyethylene glycol-TAT peptide block copolymer (BC PPT) chemically synthesized in an aqueous phase as an active substance, which ensures that plasmid DNA penetrates into cells. The second component contains a therapeutically effective amount of active substance.

[0033] The present invention also relates to a two-component drug for combined gene-guided enzyme prodrug therapy-immunotherapy for the treatment of solid malignant tumors, wherein the first component is reconstituted by adding water and then mixed with the second component to obtain a ready-to-use injection solution for administration to a patient. The first component is a lyophilisate stored at a temperature not exceeding 4°C, obtained by freeze-drying an aqueous solution containing pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA as an active substance, which is obtained by biosynthesis in Escherichia coli cells and has a direct therapeutic effect. The first component contains a therapeutically effective amount of the active substance. The second component is an aqueous solution that is frozen and stored until use, which contains a covalently bound BC PPT chemically synthesized in an aqueous phase as an active substance, which ensures that the plasmid DNA penetrates into the cells. The second component contains a therapeutically effective amount of the active substance.

[0034] In both pharmaceutical variants, the ready-to-use injection solution for administration to the patient is prepared ex tempore, ie, immediately prior to administration to the patient in need of treatment for a solid cancer.

[0035] The mechanism of action of the claimed drug is based on the delivery of the therapeutic genes HSVtk and hGM-CSF into tumor cells by injection, preferably by intratumoral administration, using a non-viral vector, PEG-PEI-TAT (PPT) block copolymer, to ensure the production of HSVtk enzyme and hGM-CSF cytokine.

[0036] The technical result provided by the present invention is in particular that the proposed dosage form shows high stability and maintains its therapeutic efficacy for at least 18 months.

[0037] Thus, in a first aspect, the claimed invention relates to a medicament for treating solid malignancies by a combined gene-directed enzyme prodrug therapy-immunotherapy method, comprising two hermetically sealed containers, wherein:

[0038] The first container contains a first component, the first component contains a therapeutically effective amount of pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA, at least one pharmaceutically acceptable additive and water, and

[0039] The second container contains a second component, the second component contains a therapeutically effective amount of PEG-PEI-TAT peptide block copolymer, at least one pharmaceutically acceptable additive and water,

[0040] The contents of each container are sterile and the containers are stored at a temperature not higher than -18°C.

[0041] In one embodiment, the medicament is characterized by:

[0042] The first component contains:

[0043] pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 150-170 μg, glucose (dextrose) - about 100 mg,

[0044] HEPES - approximately 2.38 mg, and

[0045] Water - to a final volume of 1.0 ml,

[0046] The second component contains:

[0047] PEG-PEI-TAT peptide block copolymer - about 0.63-0.74 mg (based on PEI), borax (Sodium tetraborate) - about 2.38 mg, and

[0048] Water - to a final volume of 1.0 ml.

[0049] In one of the embodiments of the medicine, the ready-to-use injection solution is obtained by thawing the contents of the first container and the second container and mixing them.

[0050] In another embodiment, the ready-to-use injection solution is administered intratumorally.

[0051] In a further embodiment, the solid cancer is selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

[0052] In one embodiment of the drug, the PEG-PEI-TAT peptide block copolymer has a PEG / PEI molar ratio of about 1.0 to 3.0 and a TAT / PEI molar ratio of about 0.8 to 1.5. In a preferred embodiment, the PEG-PEI-TAT peptide block copolymer has a PEG / PEI molar ratio of about 2.0 to 2.5.

[0053] In another embodiment, the medicament is characterized in that:

[0054] The first component contains:

[0055] pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 160 μg,

[0056] Glucose - about 100mg,

[0057] HEPES - approximately 2.38 mg, and

[0058] Water - to a final volume of 1.0 ml,

[0059] The second component contains:

[0060] PEG-PEI-TAT peptide block copolymer - about 0.69 mg (based on PEI);

[0061] Borax - about 2.38 mg, and

[0062] Water - to a final volume of 1.0 ml,

[0063] The PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0.

[0064] In a second aspect, the present invention relates to a medicament for treating solid malignant tumors by combined gene-directed enzyme prodrug therapy-immunotherapy, comprising two hermetically sealed containers, wherein:

[0065] The first container contains a first component, which is a freeze-dried product containing a therapeutically effective amount of pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA and at least one pharmaceutically acceptable additive, and

[0066] The second container contains a second component, the second component contains a therapeutically effective amount of PEG-PEI-TAT peptide block copolymer, at least one pharmaceutically acceptable additive and water,

[0067] wherein the contents of each container are sterile, and the first container is stored at a temperature no higher than 4°C, and the second container is stored at a temperature no higher than -18°C.

[0068] In one embodiment, the drug is characterized in that:

[0069] The first component is a freeze-dried product obtained by freeze-drying a solution comprising:

[0070] pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 150-170 μg, glucose - about 100 mg,

[0071] HEPES - approximately 2.38 mg, and

[0072] Water - to a final volume of 1.0 ml,

[0073] The second component contains:

[0074] PEG-PEI-TAT peptide block copolymer - about 0.63-0.74 mg (based on PEI);

[0075] Borax - about 2.38 mg, and

[0076] Water - to a final volume of 1.0 ml.

[0077] In one embodiment of the pharmaceutical, the ready-to-use injection solution is obtained by adding 1.0 ml of water to the content in the first container for reconstitution, thawing the content in the second container and mixing them.

[0078] In another embodiment, the ready-to-use injection solution is administered intratumorally.

[0079] In a further embodiment, the solid cancer is selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

[0080] In another embodiment of the medicine, the PEG-PEI-TAT peptide block copolymer is characterized in that the PEG / PEI molar ratio is about 1.0 to 3.0 and the TAT / PEI molar ratio is about 0.8 to 1.5. In a preferred embodiment, the PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0 to 2.5.

[0081] In another embodiment, the medicament is characterized in that:

[0082] The first component is a freeze-dried product obtained by freeze-drying a solution containing:

[0083] pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 160 μg,

[0084] Glucose - about 100mg,

[0085] HEPES - approximately 2.38 mg, and

[0086] Water - to a final volume of 1.0 ml,

[0087] The second component contains:

[0088] PEG-PEI-TAT peptide block copolymer - about 0.69 mg (based on PEI);

[0089] Borax - about 2.38 mg, and

[0090] Water - to a final volume of 1.0 ml,

[0091] The PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0.

[0092] BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 -pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA expression cassette structure. CMV-cytomegalovirus promoter; HSVtk-herpes simplex virus thymidine kinase gene; hGM-CSF-human granulocyte-macrophage colony stimulating factor gene; IRES-encephalomyocarditis virus internal ribosome entry site; SV40 polyA-SV40 polyadenylation signal.

[0094] Description of the implementation plan

[0095] In numerous preliminary experiments, the inventors of the present invention studied various dosage forms of drugs for combined gene-guided enzyme prodrug therapy-immunotherapy for the treatment of solid malignancies. These studies included liquid (in the form of a single component or a two-component system) and freeze-dried (complete and partial) dosage forms. Based on their research results, the inventors concluded that a two-component system must be used because a single-component system (whether liquid or freeze-dried) cannot provide the stability required for the drug. In addition, the use of freeze-dried treatment of one of the active substances of the drug, namely PPT, inevitably leads to unsatisfactory results. Therefore, in order to further develop a drug dosage form for combined gene-guided enzyme prodrug therapy-immunotherapy for the treatment of solid malignancies, the inventors chose a two-component system, in which the first component containing the therapeutic plasmid as the active substance can be a frozen solution or a freeze-dried product, and the second component containing PPT as the active substance must be a frozen solution.

[0096] Therefore, in the first aspect of the present invention, the drug for combined GDEPT-immunotherapy for treating solid malignancies comprises two hermetically sealed containers, the content of the first container (first component) being a frozen solution containing highly pure pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA (pDNA) as an active substance.

[0097] The content of the second container (second component) is a frozen solution containing PPT as an active substance. PPT block copolymer is a covalently bonded copolymer chemically synthesized in an aqueous phase, which includes: PEG-modified polyethylene glycol MAL- -NHS ester, PEI-linear polyethyleneimine 25000 and TAT peptide. The preferred intratumoral injection solution is obtained by mixing the first and second components after preliminary thawing before use. Combining the two solutions results in the formation of a polymer complex (a complex of pCMV-HSVtk-hGM-CSF plasmid DNA and PPT block copolymer) that can penetrate into tumor cells and induce the synthesis of two therapeutic proteins HSVtk and hGM-CSF therein.

[0098] In the second aspect of the present invention, the drug for combined GDEPT-immunotherapy for treating solid malignancies comprises two hermetically sealed containers, the content of the first container (first component) being a freeze-dried product containing highly pure pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA (pDNA) as an active substance.

[0099] The content in the second container (second component) is a frozen solution containing PPT as an active substance.

[0100] In the second aspect of the present invention, the ready-to-use injection solution preferably for intratumoral administration is obtained by reconstituting the first component by adding water to the first component to the original volume and then mixing with the second component after preliminary thawing. Combining the two solutions results in the formation of a polymer complex (a complex of pCMV-HSVtk-hGM-CSF plasmid DNA and PPT block copolymer) capable of penetrating into tumor cells and inducing the synthesis of two therapeutic proteins HSVtk and hGM-CSF therein.

[0101] Structure and properties of pCMV-HSVtk-hGM-CSF plasmid DNA

[0102] The pCMV-HSVtk-hGM-CSF plasmid contains an expression cassette consisting of two therapeutic genes, the herpes simplex virus thymidine kinase gene HSVtk and the human granulocyte-macrophage colony stimulating factor gene hGM-CSF ( Figure 1 ).

[0103] The pCMV-HSVtk-hGM-CSF supercoiled pDNA is produced by biosynthesis in an E. coli host cell. Any suitable E. coli strain can be used as a host cell, which is well known to those skilled in the art. In the present invention, it is preferred to use an E. coli DH-5α strain transformed with a pCMV-HSVtk-hGM-CSF plasmid, specifically E. coli DH-5α / pCMV-HSVtk-hGM-CSF.

[0104] When the pCMV-HSVtk-hGM-CSF plasmid molecules penetrate into tumor cells, the HSVtk enzyme is synthesized, which is able to phosphorylate the systemically administered nucleoside analog, ganciclovir, into ganciclovir monophosphate, which is then phosphorylated by cellular kinases into its toxic form, ganciclovir triphosphate. In addition, when the plasmid molecules penetrate into tumor cells, human GM-CSF protein is synthesized, which is secreted into the extracellular space and stimulates the growth, development and differentiation of granulocytes and antigen-presenting cells, attracting them to the tumor growth area to provide induction of specific anti-tumor immune responses.

[0105] The drug of the present invention contains pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA in an amount of about 150-170 μg.

[0106] Structure and properties of PPT block copolymer (BC PPT)

[0107] In order to obtain BC PPT, the present invention uses modified polyethylene glycol MAL- -NHS Ester (PEG):

[0108]

[0109] Empirical formula: C 62 H 111 N3O 31 , molecular weight: 1,394.55Da.

[0110] To obtain BC PPT, the present invention uses linear polyethyleneimine 25000 (PEI):

[0111]

[0112] Empirical formula (CH2CH2NH) n , molecular weight: 25,000Da.

[0113] To obtain BC PPT, the present invention uses TAT ​​peptide having the following amino acid sequence:

[0114] Gly-Arg-Lys-Lys-Lys-Arg-Arg-Gln-Arg-Cys.

[0115] Empirical formula: C 52 H 102 N 26 O 12 S1, molecular weight: 1,315.6 Da.

[0116] PPT block copolymer is a kind of high molecular weight biopolymer with positive charge at pH 7.4-8.3 chemically synthesized in water phase. Biopolymer is based on linear PEI molecule with molecular weight of 25kDa. PEI combines the ability of high cell membrane instability with the ability of high DNA concentration, protects DNA from enzymatic degradation and increases the possibility of intact DNA entering the nucleus. The PEG molecule with molecular weight of about 1,395Da is covalently connected to the primary amine of PEI by chemical reaction, and TAT peptide is connected to PEG. Modification of PEI with PEG provides neutral surface charge, thereby reducing the aggregation of particles, reducing the toxic effects of biopolymers, and increasing the hydrophilicity of pDNA and the effect of penetrating into the nucleus of transfected tumor cells. The TAT peptide carrying nuclear localization signal ensures that the polymer complex (complex of plasmid DNA and PPT) enters the transport of the nucleus regardless of the cell cycle stage, and promotes the penetration of the entire construct into the cell.

[0117] In aqueous solution, the positively charged PPT block copolymer molecules form non-covalent spherical complexes (polymer complexes) with negatively charged pDNA molecules due to electrostatic interactions. These complexes interact with the cell membrane and are subsequently taken up by endocytosis. The PPT-pDNA complexes within the endosomes are able to buffer endosomal vesicles, causing them to swell and lyse, followed by the release of pDNA into the cytoplasm. In addition, the PPT-pDNA complexes are transported to the nucleus along the cytoskeleton pathway. In the nucleus, the polymer complexes decondense, separating pDNA from PPT. The genes contained in the pCMV-HSVtk-hGM-CSF pDNA are transcribed and translated, leading to the synthesis of two therapeutic proteins in the cell, herpes simplex virus thymidine kinase and human granulocyte-macrophage colony stimulating factor.

[0118] In a preferred embodiment of the invention, the BC PPT is characterized by a PEG / PEI molar ratio of about 1.0 to 3.0 and a TAT / PEI molar ratio of about 0.8 to 1.5. In a most preferred embodiment of the invention, the PEG / PEI molar ratio is about 2.0 to 2.5.

[0119] For efficient transfection of tumor cells, the optimal ratio of PPT and pDNA molecules in the polymer complex was experimentally selected, expressed as the ratio of N (number of nitrogen atoms) to P (number of phosphorus atoms), equal to about 20 to 40, preferably about 30. Inorganic phosphorus analysis for quantitative DNA analysis was performed according to SP 14 (State Pharmacopoeia of the Russian Federation), GPM.1.7.2.0018.15 (Analysis of nucleic acids in biopharmaceuticals by the Spirin method).

[0120] In the drug of the present invention, the content of the PEG-PEI-TAT peptide block copolymer is about 0.63-0.74 mg (calculated as PEI).

[0121] Pharmaceutically acceptable additives

[0122] When manufacturing the first component containing pCMV-HSVtk-hGM-CSF plasmid DNA, at least one pharmaceutically acceptable additive is used, and a pharmaceutically acceptable buffer is used so that the physiologically acceptable pH value of the solution is in the range of 7.0 to 8.0. HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is preferably used as a buffer, which is better at maintaining a given pH value and has minimal cytotoxicity compared to other buffer systems (phosphates and carbonates). HEPES is listed in the 14th edition of the State Pharmacopoeia of the Russian Federation (SP 14) as a component of a buffer solution (GPM.1.3.0003.15 Buffer solution, (HEPES) buffer solution pH 7.5, Volume 1, Page 1808). According to SP 14, buffer solutions are used in the following situations: (1) to achieve isotonicity in the preparation of liquid dosage forms; and (2) to maintain the stability of the dosage form (State Pharmacopoeia of the Russian Federation 14th edition, 2018).

[0123] In addition, cryoprotectants such as monosaccharides and disaccharides (lactose, glucose or sucrose) are added to the DNA to prevent it from losing its transfection activity before the solution freezing or lyophilization process. Glucose (dextrose) is preferably used to prepare the first component containing pCMV-HSVtk-hGM-CSF plasmid DNA. Glucose can be used in anhydrous form and monohydrate form, and the content of glucose in the drug should be about 100 mg in anhydrous form.

[0124] In the manufacture of the second component comprising BC PPT, at least one pharmaceutically acceptable additive is used, and a pharmaceutically acceptable buffer is used so that the physiologically acceptable pH value of the solution is in the range of 7.1 to 8.2. Preferably, a borate buffer is used as such a buffer, including borax. Borax can be expressed in anhydrous form or in any known crystalline form thereof, such as decahydrate (sodium tetraborate decahydrate). In this case, the content of borax in the drug should be about 2.38 mg in anhydrous form.

[0125] The present invention provides a drug for treating solid cancer. The tissue structure of solid tumors contains interdependent tissue regions, including parenchyma (cancer cells) and supporting stromal cells, which can provide a microenvironment in which the cancer cells are distributed. Solid malignant tumors include, but are not limited to, sarcomas, carcinomas, and lymphomas, including lung cancer, breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer (including, for example, head and neck squamous cell carcinoma-HNSCC).

[0126] The following examples are provided to illustrate the claimed invention and are not intended to limit the scope of legal protection, which shall be determined solely by the appended claims.

[0127] Example 1. Isolation, collection and storage of bacterial biomass

[0128] The biomass, i.e. the cell pellet, was separated from the culture broth by means of a floor-standing high-speed batch centrifuge with a fixed-angle rotor. For this purpose, 800 ml of the culture suspension were added to the centrifuge bottles using a measuring cylinder. The bottles were weighed alternately on a balance, the last one calibrated according to the weight of the heaviest of the six glass bottles, and the weight difference between the heaviest filled bottle and the remaining bottles was adjusted to (0.0 ± 0.5) g one by one by adding the culture suspension with a Pasteur pipette. The bottles were closed airtight with lids and placed in the rotor. Centrifugation was performed for 15 minutes at a set speed of 10,000 rpm and a centrifuge working chamber temperature of 4°C. The culture supernatant was poured into a container and transferred for sterilization by autoclaving. The bottle of the pellet was refilled with the culture suspension and the centrifugation was repeated. During the last centrifugation cycle, the weight of the bottle was adjusted with purified water in case there was insufficient culture suspension. At the end of the last centrifugation cycle, the bottle was incubated upside down on a piece of filter paper for (5 ± 1) minutes to remove the residual liquid. The biomass samples were taken with a metal spatula for authenticity control by agarose gel electrophoresis. The obtained biomass was transferred with a steel spatula into a covered container previously weighed on a balance, the filled container was closed, reweighed, labeled in the prescribed form, placed in a protective plastic bag with a zip-lock closure and stored in a low temperature freezer at a temperature below -65°C.

[0129] Example 2. Obtaining clarified lysate of bacterial biomass

[0130] The process of obtaining a clear lysate consists of suspending the bacterial cells, lysing the cells under alkaline conditions and clarifying the lysate.

[0131] The clarified lysate was obtained through three cycles, each cycle including: suspending biomass, biomass lysis and lysate clarification.

[0132] Suspended biomass

[0133] Three lysis cycles were required to process 600 g of biomass, each lysing 200 g of biomass. To resuspend each 200 g of biomass, 2 liters of solution I were consumed. Solution I included tris(hydroxymethyl)aminomethane, disodium ethylenediaminetetraacetate and glucose monohydrate.

[0134] Thaw the biomass container at +5 ± 3 ° C (if necessary) until the contents are completely thawed. Weigh 200 g of thawed biomass on a scale, add 2.0 L of solution I, and stir the container on a magnetic stirrer until a homogenous suspension with no visible cell clumps is obtained.

[0135] Cell lysis

[0136] To lyse the 200 g biomass cell suspension obtained in the suspension step, add 2 L of solution II. Pour the resulting cell suspension into a container with a screw cap, add 2.0 L of solution II, stir by inversion for 1 minute, and incubate the solution for 5 minutes.

[0137] Solution II was prepared as follows. 1.5 liters of WFI was poured into a container, 16.00 grams of sodium hydroxide and 20.00 grams of sodium lauryl sulfate were added, and the mixture was stirred with a magnetic stirrer until the reagents were completely dissolved. The volume of the solution was made up to 2.0 liters with water for injection (WFI).

[0138] Lysate clarification

[0139] To clarify the lysate of 200 g of biomass, 2 L of solution III were used.

[0140] 2.0 liters of solution III are poured into the lysate container obtained in the lysis step and mixed by multiple violent inversions. The preparation of solution III is as follows. 0.9 liters of WFI are poured into the container, 588.9 grams of potassium acetate are added, and the mixture is stirred with a magnetic stirrer until the reagent is completely dissolved. The pH is adjusted to 5.5 by adding 0.3 liters of acetic acid solution. The solution volume is supplemented to 2.0 liters with WFI. The buffer solution is filtered into the container with a filter. The solution is then incubated for 10 minutes. Cell debris is removed by centrifugation at 15,000 rpm for 30 minutes or by filtering through thick filter paper. The clarified cell lysate is concentrated by ultrafiltration using a glass fiber filter.

[0141] Example 3. Isolation of plasmid DNA from biomass

[0142] As a primary chromatographic purification step of supercoiled plasmid DNA, a gel filtration chromatography step is used, which allows the removal of ribonucleic acid impurities from the plasmid solution. Purification is performed on a column filled with Sepharose 6FF adsorbent using an Akta Pilot chromatography system. Approximately four gel filtration cycles are required to obtain the desired amount of final product and to purify the entire volume of concentrated clarified lysate: two purification cycles are required for each ultrafiltration cycle. After each cycle, the adsorbent is re-equilibrated, regenerated and then re-equilibrated after every two cycles.

[0143] An affinity chromatography step was used to purify the target DNA in supercoiled circular form from the relaxed DNA. Purification was performed using an Akta Pilot chromatography system on a column packed with Plasmid Select Xtra adsorbent.

[0144] Purification of the target combined fraction obtained from one gel filtration step requires 2-3 purification cycles on the affinity adsorbent. In order to purify the entire volume of the pDNA fraction obtained from the gel filtration step and obtain the desired amount of target material, a total of about six affinity chromatography cycles are required. The adsorbent is regenerated and re-equilibrated after each cycle.

[0145] For final purification of supercoiled circular DNA from impurities, an anion exchange chromatography step was used. Purification was performed on a column packed with Source 30Q adsorbent using an AktaPilot chromatography system. Regeneration was performed after each cycle; before each cycle, the adsorbent was depyrogenated and re-equilibrated. Approximately two cycles of chromatographic purification were required to process the entire volume of the target product.

[0146] Example 4. Filling a solution of plasmid DNA and excipients (first component) into a bottle and sealing it

[0147] In zone A of a class B cleanroom with laminar air flow switched on, the product is filled into 2R bottles (in boxes) according to the calculated filling dose (100-102.5%), which are made of first-class hydrolyzed lightweight glass and are transferred through a drying oven into the filling and capping room. The first (3±1) bottles are used to set the filling dose (volume). The filling is carried out by a peristaltic metering pump and a sterile silicone filling hose with an inner diameter of 3.2 mm. The filling dose control is carried out by weight at the beginning, middle and end of the filling. The contents of the first three bottles (corresponding to about 0.0036 liters), which are incompletely filled or have incorrect doses, are transferred for disposal or recycling. The filling is checked by the gravimetric method at least five times during the operation. 0.594 liters of solution are filled. 0.015 liters of solution remain in the pipeline (hose), which are poured into a sterile collection tank and then transferred for disposal or recycling.

[0148] Example 5. Filling the solution of PPT block copolymer and excipient (second component) into a bottle and sealing it

[0149] In Zone A of a Class B cleanroom with laminar air flow turned on, the product is filled into 2R bottles (in boxes) according to the calculated filling dose (100-102.5%), which are made of the first type of hydrolyzed lightweight glass and passed through a drying oven into the filling and capping room. The first (3±1) bottles are used to set the filling dose (volume) to 1.1-1.12. Filling is carried out by a peristaltic metering pump and a sterile silicone filling hose with an inner diameter of 3.2 mm. The filling dose control is carried out by weight at the beginning, middle and end of filling.

[0150] The contents of the first three bottles (corresponding to approximately 0.0033 litres), which are incompletely filled or have incorrect dosages, are transferred for disposal or recycling. The filling is checked by the gravimetric method at least five times during the operation.

[0151] During the entire filling process, each bottle is immediately capped with a stopper.

[0152] During the operation, the quality of the bottle capping was monitored (visual inspection).

[0153] 0.0015 liters of solution remain in the tubing (hose), which is poured into a sterile collection tank and then transferred for disposal or recycling.

[0154] The filled and capped bottles are placed into boxes and transferred to the flip-top sealing step.

[0155] Example 6. Drug for combined gene-directed enzyme prodrug therapy-immunotherapy for the treatment of solid malignancies, 2 ml (Variant 1)

[0156] During the manufacturing process of the drug, raw materials are used that meet the specifications and technical documentation through incoming control:

[0157] - Plasmid DNA material

[0158] -PPT block copolymer material

[0159] -Sodium Tetraborate Decahydrate

[0160] -HEPES 1M solution

[0161] - 40% glucose monohydrate solution (based on anhydrous glucose).

[0162] Component 1 - pCMV-HSVtk-hGM-CSF pDNA solution for injection, 1 ml.

[0163]

[0164] Description: Frozen solution. Dense and hard white mass. After thawing: Colorless and transparent solution, no visible inclusions.

[0165] Storage: Temperature not exceeding -18°C. Storage of thawed product is not allowed. Refreezing is not allowed.

[0166] Component 2 - Polyethylene glycol-polyethyleneimine-TAT peptide block copolymer solution for injection, 1 ml

[0167]

[0168] Description: Frozen solution. Dense and hard white mass. After thawing: Colorless and transparent solution, no visible inclusions.

[0169] Storage: Temperature not exceeding -18°C. Storage of thawed product is not allowed. Refreezing is not allowed.

[0170] Preparation of ready-to-use dosage forms of pharmaceuticals (Variant 1)

[0171] To prepare the ready-to-use dosage form "Injection" of this drug, it is necessary to thaw the contents of the bottle containing the first and second components. Then it is necessary to carefully mix the contents of the bottle, making circular motions and shaking them gently. When preparing the ready-to-use dosage form of this drug, vigorous mixing or shaking should be avoided. After thawing and mixing, the contents of the bottle containing the first component are transferred to the bottle with the second component and mixed by shaking. The mixture is kept at room temperature for 30 minutes. The ready-to-use drug should be used within 2 hours after preparation. Care should be taken to ensure the sterility of the prepared solution, since this drug does not contain preservatives or bacteriostatic substances.

[0172] Example 7. Obtaining a Lyophilized Form of the First Component Containing Plasmid DNA

[0173] The vials containing the first component of the drug as described in Example 6 were lyophilized in a lyophilizer according to the program shown in Table 1.

[0174] Table 1. Lyophilization conditions of the first drug component

[0175]

[0176]

[0177] After the drying process was completed, the bottles were hermetically sealed in a freeze dryer under an argon atmosphere. The bottles were stored in a dark place at a temperature not exceeding 4°C.

[0178] Example 8. Drug for combined gene-directed enzyme prodrug therapy-immunotherapy for the treatment of solid malignancies, 2 ml (Variant 2)

[0179] Composition of the first component for preparing injection, lyophilized pCMV-HSVtk-hGM-CSF plasmid DNA

[0180]

[0181] Obtain the first component: The pCMV-HSVtk-hGM-CSF plasmid DNA solution prepared as described in Example 3 was lyophilized as described in Example 7.

[0182] Description: Lyophilisate. White lumps. After reconstitution by adding 1.0 ml of water for injection: Colorless transparent solution, no visible inclusions.

[0183] Storage: Temperature not exceeding 4°C.

[0184] The second component for preparing the injection, PPT solution, is composed of 1 ml.

[0185]

[0186] Preparation of ready-to-use dosage forms of pharmaceuticals (Variant 2)

[0187] To prepare the ready-to-use dosage form "Injection" of this drug, it is necessary to thaw the contents of the bottle containing the second component. 1.1 ml of water for injection should be added to the bottle with the first component and mixed gently until the lyophilisate is completely dissolved. Then it is necessary to carefully mix the contents of the bottle, making circular motions and shaking them gently. When preparing the ready-to-use dosage form of this drug, vigorous mixing or shaking should be avoided. After thawing and mixing, the contents of the bottle containing the first component are transferred to the bottle with the second component and mixed by shaking. The mixture is kept at room temperature for 30 minutes. The ready-to-use drug should be used within 2 hours after preparation. Care should be taken to ensure the sterility of the prepared solution, since this drug does not contain preservatives or bacteriostatic substances.

[0188] Composition of the ready-to-use dosage form "Injection" after mixing the first component and the second component (Variant 2)

[0189]

[0190] Description: Colorless, transparent liquid, without foreign inclusions.

[0191] Example 9. Cytotoxicity assay to evaluate drug specific activity

[0192] The specific activity was measured by in vitro testing of C-26 cell lines (CT26.WT mouse colorectal adenocarcinoma, CRL-2638 TM The method for evaluating the activity of the drug is based on the ability of the drug to cause the death of C-26 cancer cells transfected with the drug in the presence of ganciclovir. The number of viable cells in the growth medium should not exceed 50% or 20% at a ganciclovir concentration of 2 μM or 12.5 μM, respectively.

[0193] All work was performed under sterile conditions with laminar air flow and using sterile tubes and pipettes.

[0194] Cells were grown in RPMI 1640 nutrient medium (Gibco, USA). Cells were grown in a CO2 incubator at 37°C in an atmosphere containing 5% CO2. When the monolayer confluence reached 90% (visual assessment), cells were subcultured in RPMI1640 / antibiotic nutrient medium at a ratio of 1:10. Cells from 3-10 generations were used for testing.

[0195] C-26 cells were subcultured in 12-well plates 20-24 hours before testing.

[0196] Transfection of C-26 cells

[0197] Wells of a 12-well plate (e.g., Corning-353043, Corning, USA) were inoculated with (2-3) 10 5 Cells were added to 2 ml of RPMI 1640 medium (without antibiotics) and incubated in a CO2 incubator for 24 hours. Before adding the complex solution and drug test samples for transfection, the medium was removed from the plate wells with a pipette without touching the bottom and walls of the wells. After adding the complex solution and drug test samples, the plate was incubated in a CO2 incubator for 48 hours.

[0198] Count the number of viable cells

[0199] Remove the plate from the CO2 incubator, remove the medium from all wells with a pipette, and wash the wells with 2 ml of phosphate buffered saline (PBS). Add 0.2 ml of trypsin-EDTA solution to each well and incubate in the CO2 incubator for 2 minutes. Add 1 ml of RPMI 1640 / antibiotic medium, resuspend the cells with a pipette and transfer to a sterile microtube.

[0200] The concentration of viable cells was determined using trypan blue.

[0201] Count the number of fluorescent cells

[0202] To evaluate the transfection efficiency of C-26 cells transfected with Lipofectamine 3000 and PPT containing the pEGFP-N1 indicator plasmid, the number of transfected cells was counted using a flow cytometer. A 50-microliter amount of Ccl and CGFP samples (cells transfected with pEGFP-N1-Lipofectamine complex and pEGFP-N1-PPT complex, respectively) was added to a microtube containing 50 microliters of an assay buffer containing 10 mM phosphate buffer, pH 7.4, 2.5% BSA and 0.1% sodium azide.

[0203] Transfection efficiency was calculated as the percentage of cells with fluorescence exceeding the autofluorescence level of untransfected cells.

[0204] After transfection, 100 microliters of transfected and untransfected cell suspensions were added to two 96-well plates. The plates with cell suspensions were incubated in a CO2 incubator for 24 hours. Next, ganciclovir solutions with concentrations of 0, 0.4, 4.0, 25.0, and 100.0 μM were prepared. After the cell suspensions were incubated, 100 microliters of different concentrations of ganciclovir were added to the wells.

[0205] The plate with the cell suspension was incubated in a CO2 incubator for 72 hours.

[0206] After incubation, the medium was removed from the wells and 200 μl of ganciclovir at different concentrations was added.

[0207] The plates were incubated in a CO2 incubator until a monolayer of cells formed in the wells with 0 μM ganciclovir (visual assessment).

[0208] After incubation, remove 100 μl of medium from each well. Add 20 μl of MTS reagent (CellTiter AQueous One Solution Cell Proliferation Assay (MTS, catalog number: G3582, Promega). The well contents were mixed on a shaker and incubated in a CO2 incubator for 30 minutes.

[0209] The optical density AN in the wells was measured using a plate photometer at a wavelength of 492 nm. The optical density value A of each well was calculated according to the following formula taking into account the background value. abs N:

[0210] A abs N=AN-A bg

[0211] Calculate the mean A of triplicates for each transfected C-26 cell line sample abs N av .

[0212] Calculate the percentage of viable cells at each dilution of ganciclovir in experimental and control samples for all transfected cells using the following formula:

[0213] % viable cells = A abs N av ×CN×100 / A abs 0 av ,in

[0214] A abs N av is the average optical density of triplicates of each transfected cell sample in different ganciclovir dilutions,

[0215] CN is the ganciclovir concentration in the well, and

[0216] A abs 0 av is the mean optical density of triplicates of each transfected cell sample in zero concentration of ganciclovir.

[0217] A test is considered significant if:

[0218] - The percentage of viable cells in the negative control and untransfected cells of Ccl and CGFP exceeded 90%,

[0219] A batch of drug is considered suitable for use if the percentage of viable cells in the wells with a ganciclovir concentration of 12.5 μM does not exceed 20% for the three samples from the batch.

[0220] Example 10. Determination of the properties of PPT (second component)

[0221] The PEI content in the synthetic PPT is measured spectrophotometrically, for example using a microplate reader Synergy 4 (BioTek Instruments, USA or similar instrument) at a wavelength of 620 nm. To construct a calibration curve and measure the conjugate concentration, 50 μl of an appropriate PEI or water sample, 100 μl of 40 mM Cu(CH3COO)2·H2O and 50 μl of water are added to each well of a 96-well plate. The calibration solution is measured in triplicate. The optical density of 20 mM Cu(CH3COO)2·H2O is subtracted from the average optical density value obtained. The absorption of the resulting copper complex at 620 nm is linearly correlated with the PEI concentration (С PEI = а1·Α 620 , where С PEI is the concentration of PEI in the calibration sample in μM, A 620 is the average optical density of the calibration sample minus the optical density of the sample without PEI, and а1 is the proportionality factor). The concentration of PPT is equal to the concentration of PEI in the PPT solution.

[0222] The PEG content in the synthetic PPT is measured by spectrophotometry, for example using a microplate reader Synergy 4 (BioTek Instruments, USA or similar instrument) at a wavelength of 540 nm. For this purpose, freshly prepared PEG calibration solutions with a molecular weight of 1,500 Da are used: 200 μM, 160 μM, 120 μM, 80 μM, 40 μM and 20 μM PEG, as well as 5% barium chloride solution and 12.3 g / l I2 in 2% KI. 10 μl of a known concentration of PEG solution or a sample to be tested (pre-diluted to a 20 μM PEI concentration), 10 μl of a 20 μM PEI solution or water for an unknown sample to be tested, 180 μl of water, 50 μl of a barium chloride solution and 25 μl of an iodine solution in potassium iodide are added to the wells of a 96-well plate and the absorption of the solution is measured at 540 nm after 10-13 minutes. The time is counted from the time the iodine solution is added. The measurements were performed in triplicate. The absorbance at 540 nm was linearly correlated with the PEI concentration (С PEG / С PEI = а2·Α 540 , where С PEG / С PEI is the molar ratio of PEG / PEI in the calibration sample, in μM, A 540 is the average absorbance of the calibration samples minus the absorbance of the PEG-free samples, and а2 is the proportionality factor).

[0223] The TAT peptide content in the synthetic PPT was measured by fluorescence, and the adduct content formed by the amino group of the TAT peptide in the presence of fluoresceinamine was measured. To this end, fluoresceinamine solution (1mg / ml) and 0.25M phosphate buffer, pH 9.3 in acetone were prepared. The 6mg / ml TAT peptide solution was diluted to a concentration of 100μM. A mixture of 3ml TAT peptide solution and fluoresceinamine solution was prepared so that the final concentration of the TAT peptide was 0.5μM: 15μl 100μMTAT peptide solution, 60μl 1mg / ml fluoresceinamine solution and 2,925μl 0.25M phosphate buffer, pH 9.3. A mixture of 3ml PEI-PEG-TAT block copolymer solution and fluoresceinamine solution was prepared so that the final concentration of PEI in the block copolymer was 1μM. Fluorescence measurements were performed in a glass cuvette at a fluorescence excitation wavelength of 395nm and a fluorescence wavelength of 490nm. The fluorescence value of each point was measured eight times, and the readings were averaged. The synthesis efficiency is considered to be optimal when the molar ratio of TAT / PEI is about 0.8 to 1.5.

[0224] During our preliminary experiments, we found a PEG / PEI ratio in the polymer complex that provides efficient pDNA delivery to cancer cells. For all cell lines tested, the highest transfection efficiency was achieved at a PEG / PEI molar ratio of about 1.0 to 3.0, more preferably at a ratio of about 2.0 to 2.5, and most preferably at a ratio of about 2.0.

[0225] Example 11. Stability of the pharmaceutical dosage form (Variant 1 - First component and second component in the form of a frozen solution)

[0226] The aim of our study was to evaluate the stability of the physical, chemical and biological properties of the drug of the present invention under storage conditions at -20°C ± 2°C with an expected shelf life of 18 months.

[0227] The first component and the second component are stored at -20℃±2℃ and packaged in Class 1 hydrolytic glass colorless bottles, hermetically sealed with rubber stoppers and aluminum flip caps.

[0228] The stability of the components and ready-to-use dosage forms of the drug were evaluated by a number of parameters, including: description, authenticity (restriction enzyme analysis), authenticity (PCR with specific primers), clarity, color, mechanical inclusions, residual proteins of the production strain, bacterial endotoxins, abnormal toxicity, sterility and specific activity. The results obtained within the framework of this study are shown in Tables 2-4.

[0229]

[0230]

[0231]

[0232] Therefore, in our stability study, it was found that the storage condition at a temperature of -20°C ± 2°C ensured the stability of the quality indicators of the first component and the second component, as well as the stability of the ready-to-use injection dosage form obtained after mixing the first component and the second component, with a shelf life of 18 months. The established shelf life of the drug is at least 18 months.

[0233] Example 12. Stability of the pharmaceutical dosage form (Variant 2—the first component is in the form of a lyophilisate and the second component is in the form of a frozen solution)

[0234] The objective of the study was to evaluate the stability of the physical, chemical and biological properties of the drug under storage conditions of 4°C for the first component and -20°C ± 2°C for the second component with an expected shelf life of 24 months.

[0235] The first component is stored at 4°C and packaged in a Class 1 hydrolytic glass colorless bottle, sealed with a rubber stopper and an aluminum flip cap. The second component is stored at -20°C ± 2°C and packaged in a Class 1 hydrolytic glass colorless bottle, sealed airtight with a rubber stopper and an aluminum flip cap.

[0236] The ready-to-use dosage form was prepared as described in Example 8 above.

[0237] Stability was studied by measuring multiple parameters, including the specific activity of the ready-to-use dosage form of the drug (Variant 2) at 1 day, 2 weeks, 3 months, 6 months, 12 months and 18 months after the start of the experiment. The specific activity was measured as described in Example 9 above ("Cytotoxicity test for evaluating the specific activity of the drug"). The experimental results show that the drug (Variant 2) maintains its biological activity for at least 18 months.

[0238] Example 13. Study on the anti-tumor activity of drugs

[0239] The antitumor effect of the drug was studied in a xenograft human tumor model in nude immunodeficient mice, which were obtained by inoculation of the following human tumor cells: KB oral mucosal carcinoma, HEp2 human laryngopharyngeal carcinoma, HT29 colorectal adenocarcinoma, and HeLa cervical adenocarcinoma. Animals (female, n = 9-12 animals per group) were treated with an effective regimen. Treatment was started on the 11th day (KB, HT29, HEp2) and 13th day (HeLa) of tumor growth, when the tumor volume was approximately 100 ± 10 mm 3 .

[0240] The summary results of the study are shown in Table 5.

[0241] Table 5. Antitumor activity of the combination of "drug of the present invention + ganciclovir" in a subcutaneous human tumor xenograft model

[0242] Human tumor xenografts TGI, % Τ / C,% Τ-C, days Median survival, days KB Oral mucosal cancer 82* 18 32.0 82.0** HT29 Colon Adenocarcinoma 89* 11* 49.0 89.1** HeLa cervical adenocarcinoma 57 43 21.8 71.1 HEp human pharyngeal carcinoma 2 46 64 16.6 74.8**

[0243] Note: TGI (tumor growth inhibition rate, %) and T / C (tumor inhibition degree, %) are given on day 28 (KB), day 30 (HeLa and HEp2) or day 38 (HT29) after the end of treatment. *- high antitumor activity (efficacy criteria: TGI ≥ 70%, T / C ≤ 15%, ILE ≥ 50% (increased life expectancy, %). **- statistically significant difference compared with the group of animals injected with PBS (control group) (p < 0.05; Student's t test). TC, tumor growth delay.

[0244] From the results presented in Table 5, it can be seen that a high (according to the efficacy criteria) antitumor effect was detected in nude mice with KB human oral mucosal cancer: its TGI increased with the observation period, reaching 66-82% on days 46-60 after tumor cell inoculation, tumor growth was delayed by 32 days, and the median survival time of animals in the experimental and control groups differed by 24 days (82 days and 58 days, respectively).

[0245] Significant antitumor effects were also observed against HT29 human colon adenocarcinoma: TGI exceeded 70% during the 32-67 day observation period, with the maximum TGI (89%) observed on day 67; a delay in tumor growth (49 days) was also observed. The median survival of animals in the experimental group was 14 days longer than that of animals in the control group (89 days and 75 days, respectively).

[0246] In HeLa xenograft mice, the efficacy of the combination of "the drug of the present invention + ganciclovir" was significantly lower than that in HT29 and KB tumor models. In the HEp2 xenograft model of immunodeficient animals, the lowest anti-tumor therapeutic effect was observed. However, the median survival of animals in the experimental group and the control group differed significantly by 13.6 days (74.8 days and 61.2 days, respectively).

[0247] In vivo studies using a variety of animal models of human solid malignancies showed that KB oral mucosal carcinoma and HT29 human colorectal carcinoma were the most susceptible.

Claims

1. A drug for treating solid malignant tumors by a combined gene-directed enzyme prodrug therapy-immunotherapy method, comprising two hermetically sealed containers, wherein: The first container contains a first component, which contains a therapeutically effective amount of pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA, at least one pharmaceutically acceptable additive and water. The second container contains a second component, the second component contains a therapeutically effective amount of PEG-PEI-TAT peptide block copolymer, at least one pharmaceutically acceptable additive and water, The contents of each container are sterile and the containers are stored at a temperature not higher than -18°C.

2. The drug according to claim 1, wherein: The first component contains: pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 150-170 μg, Glucose - about 100mg, HEPES - approximately 2.38 mg, and Water - to a final volume of 1.0 ml, The second component contains: PEG-PEI-TAT peptide block copolymer - about 0.63-0.74 mg (based on PEI), Borax - about 2.38 mg, and Water - to a final volume of 1.0 ml.

3. The medicine according to claim 1, wherein the ready-to-use injection solution is obtained by thawing the contents of the first container and the second container and mixing them. The medicament according to claim 1 , wherein the ready-to-use injection solution is administered intratumorally.

5. The medicament according to claim 1, wherein the solid cancer is selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

6. The drug of claim 1, wherein the PEG-PEI-TAT peptide block copolymer has a PEG / PEI molar ratio of about 1.0 to 3.0 and a TAT / PEI molar ratio of about 0.8 to 1.

5.

7. The drug according to claim 6, wherein the PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0 to 2.

5.

8. The medicament according to any one of claims 1 to 7, wherein: The first component contains: pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 160 μg, Glucose - about 100mg, HEPES - approximately 2.38 mg, and Water - to a final volume of 1.0 ml, The second component contains: PEG-PEI-TAT peptide block copolymer - about 0.69 mg (based on PEI), Borax - about 2.38 mg, and Water - to a final volume of 1.0 ml, The PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.

0.

9. A medicament for treating solid malignant tumors by a combined gene-directed enzyme prodrug therapy-immunotherapy method, comprising two hermetically sealed containers, wherein: The first container contains a first component, which is a freeze-dried product containing a therapeutically effective amount of pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA and at least one pharmaceutically acceptable additive. The second container contains a second component, the second component contains a therapeutically effective amount of PEG-PEI-TAT peptide block copolymer, at least one pharmaceutically acceptable additive and water, wherein the contents of each container are sterile, and the first container is stored at a temperature no higher than 4°C, and the second container is stored at a temperature no higher than -18°C.

10. The drug according to claim 9, wherein: The first component is a freeze-dried product obtained by freeze-drying a solution containing: pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 150-170 μg, Glucose - about 100mg, HEPES - about 2.38 mg, and Water - to a final volume of 1.0 ml, The second component contains: PEG-PEI-TAT peptide block copolymer - about 0.63-0.74 mg (based on PEI); Borax - about 2.38 mg, and Water - to a final volume of 1.0 ml.

11. The medicament according to claim 9, wherein the ready-to-use injection solution is obtained by adding 1.0 ml of water to the content in the first container for reconstitution, thawing the content in the second container and mixing them.

12. The medicament according to claim 9, wherein the ready-to-use injection solution is administered intratumorally.

13. The medicament according to claim 9, wherein the solid cancer is selected from the group comprising lung cancer, pancreatic cancer, melanoma, fibrosarcoma, sarcoma, head and neck cancer.

14. The drug according to claim 9, wherein the PEG-PEI-TAT peptide block copolymer has a PEG / PEI molar ratio of about 1.0 to 3.0 and a TAT / PEI molar ratio of about 0.8 to 1.

5.

15. The drug according to claim 14, wherein the PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0 to 2.

5.

16. The medicament according to any one of claims 9 to 15, wherein: The first component is a freeze-dried product obtained by freeze-drying a solution containing: pCMV-HSVtk-hGM-CSF supercoiled plasmid DNA - about 160 μg, Glucose - about 100mg, HEPES - about 2.38 mg, and Water - to a final volume of 1.0 ml, The second component contains: PEG-PEI-TAT peptide block copolymer - about 0.69 mg (based on PEI); Borax - about 2.38 mg, and Water - to a final volume of 1.0 ml, The PEG / PEI molar ratio of the PEG-PEI-TAT peptide block copolymer is about 2.0.

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