Pharmaceutical composition for treating cancer comprising vaccinia virus and hydroxyurea as active ingredients

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising vaccinia virus and hydroxyurea as active ingredients. The pharmaceutical composition for treating cancer, comprising vaccinia virus and hydroxyurea as active ingredients according to the present invention, exhibits a higher anti-cancer effect and safety than conventional vaccinia virus alone administration. Therefore, the pharmaceutical composition comprising vaccinia virus and hydroxyurea as active ingredients according to the present invention can be advantageously used for treating cancer.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises vaccinia virus and hydroxyurea as active ingredients. Background Art

[0002] Oncolytic viruses have excellent tumor-specific targeting ability, proliferation ability in cancer cells, and cancer cell killing ability. Recently, various clinical studies based on oncolytic viruses have been conducted. In 2015, the United States and Europe opened the era of oncolytic viruses, and the oncolytic virus talimogene laherparepvec (T-Vec) based on herpes simplex virus was successfully commercialized as a therapeutic agent for advanced melanoma.

[0003] Recently, the usefulness of oncolytic viruses has exceeded their own efficacy, and the viruses have activated tumor immunity, thus showing their potential as therapeutic agents used in combination with another immunotherapeutic agent. Until 2000, which was the early stage of the development of oncolytic viruses, the direct killing effect of the virus caused by the cancer cell-specific proliferation of the virus was relatively more important. However, subsequent clinical studies found that the activation of tumor immunity is a key mechanism rather than the direct cancer cell killing effect. Based on this discovery, recently, therapeutic agents including the combined administration of oncolytic viruses and immunotherapeutic agents such as immune checkpoint inhibitors have been developed. This is because it is known that oncolytic viruses transform the immunosuppressive tumor microenvironment into a tumor microenvironment suitable for immunotherapy.

[0004] In a number of clinical studies on oncolytic viruses based on vaccinia virus, oncolytic virus therapy may lead to acute tumor necrosis, durable responses, or complete responses, but in some cases, it may lead to unpredictable results (pharmacodynamic variability), such as progressive disease or premature death. For example, for Pexa-vec based on vaccinia virus, in a phase 1 clinical trial, some patients died prematurely within one month after oncolytic virus therapy, which was related to persistent systemic inflammatory response and major organ dysfunction. In addition, the transient influenza-like symptoms (high fever) and hypotension observed after oncolytic virus therapy are the most common adverse events after oncolytic virus therapy.

[0005] Therefore, in order to enhance the therapeutic effect of oncolytic viruses, it is necessary to understand the interaction between cancer cells, the immune status of patients, and oncolytic viruses; and based on this understanding, it is necessary to study technologies that can improve the clinical efficacy of oncolytic viruses. Summary of the Invention

[0006] Technical Problem

[0007] Therefore, studies were conducted to enhance the anti-cancer effect of vaccinia virus used as an oncolytic virus. As a result, the present inventors found that, compared with the conventional case of administering only vaccinia virus, when vaccinia virus and hydroxyurea are co-administered to an individual with cancer, the systemic inflammatory response is significantly reduced to ensure safe use. In addition, the present inventors found that, when vaccinia virus is administered systemically, excellent cancer cell-specific selectivity and proliferative ability can be obtained in the case of co-administering hydroxyurea.

[0008] Solution

[0009] In order to achieve the above object, in one aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, which comprises vaccinia virus and hydroxyurea as active ingredients.

[0010] In another aspect of the present invention, there is provided a method for treating cancer, which comprises administering vaccinia virus and hydroxyurea to an individual with cancer.

[0011] In still another aspect of the present invention, there is provided the use of a composition comprising vaccinia virus and hydroxyurea for preventing or treating cancer.

[0012] In yet another aspect of the present invention, there is provided the use of a composition comprising vaccinia virus and hydroxyurea in the preparation of a drug for preventing or treating cancer.

[0013] In still another aspect of the present invention, there is provided an anti-cancer adjuvant, which comprises hydroxyurea as an active ingredient.

[0014] Advantageous Effects

[0015] Compared with the conventional case of administering only vaccinia virus, the pharmaceutical composition for treating cancer according to the present invention, which comprises vaccinia virus and hydroxyurea as active ingredients, has excellent anti-cancer effects and safety. Therefore, the pharmaceutical composition according to the present invention, which comprises vaccinia virus and hydroxyurea as active ingredients, can be effectively used for treating cancer. Description of the Drawings

[0016] Figure 1 Shows the results obtained by administering wild-type vaccinia virus (Vaccinia virus Western Reserve strain, WR) and hydroxyurea (HU) to mouse renal cancer cell-transplanted mice (Renca), and then measuring the tumor volume on days 0, 3, 7, 10, and 14;

[0017] Figure 2 Shows the results obtained by administering wild-type vaccinia virus (WR) and HU to mouse renal cancer cell-transplanted mice (Renca), and then measuring the body weight on days 0, 3, 7, 10, and 14;

[0018] Figure 3 Shows the results obtained by administering recombinant vaccinia virus (WR VV tk- ) and HU (60 mg / kg) to mice transplanted with mouse renal carcinoma cells (Renca), and then measuring the tumor volume on days 0, 3, 7, 10, 14, 17, and 21. The recombinant vaccinia virus (WR VV tk- ) was obtained by deleting the TK gene in WR;

[0019] Figure 4 Shows the results obtained by administering recombinant vaccinia virus (WR VV tk- ) and HU (30 mg / kg) to mice transplanted with mouse renal carcinoma cells (Renca), and then measuring the tumor volume on days 0, 3, 7, 10, and 14;

[0020] Figure 5 Shows the results obtained by measuring the tumor volume on day -1 before and days 4 and 7 after administering recombinant vaccinia virus (VV_DD) and HU to mice transplanted with mouse melanoma cells (B16F10). The recombinant vaccinia virus (VV_DD) was obtained by simultaneously deleting the TK gene and the virus growth factor (VGF) gene in WR;

[0021] Figure 6 Shows the results obtained by administering recombinant vaccinia virus (WOTS-418) and HU to mice transplanted with human colorectal cancer cells (CT-26), and then measuring the tumor volume on days -1, 5, 10, 12, and 15;

[0022] Figure 7 Shows the results obtained by administering recombinant vaccinia virus (WOTS-418) and HU to mice transplanted with human lung cancer cells (NCI-H460), and then measuring the survival rate;

[0023] Figure 8 Shows the results obtained by administering recombinant vaccinia virus (VV tk- ) and human granulocyte colony-stimulating factor (rhG-CSF) or HU to mice transplanted with mouse renal carcinoma cells (Renca), and then measuring the mouse tumor volume;

[0024] Figure 9 Shows the results obtained by isolating lymphocytes from the spleen of mice transplanted with mouse renal carcinoma cells (Renca) that had been administered recombinant vaccinia virus (VV tk- ) and human granulocyte colony-stimulating factor (rhG-CSF) or HU, administering the lymphocytes to new mice, and then measuring the tumor volume of the new mice;

[0025] Figure 10Shows the results obtained by administering recombinant vaccinia virus (WyethVV tk- ) and HU to mouse renal cell carcinoma transplanted mice (Renca), and then measuring the tumor volume of the mice;

[0026] Figure 11 Shows the results obtained by isolating T lymphocytes from mouse renal cell carcinoma transplanted mice (Renca) that have been administered recombinant vaccinia virus (Wyeth VV tk- ) and HU, administering the T lymphocytes to new mice, and then measuring the tumor volume of the new mice;

[0027] Figure 12 Shows the results obtained by isolating splenocytes from mouse renal cell carcinoma transplanted mice (Renca) that have been administered recombinant vaccinia virus (Wyeth VV tk- ) and HU, administering the splenocytes to new mice, and then measuring the tumor volume of the new mice;

[0028] Figure 13 Shows the results obtained by administering recombinant vaccinia virus (WyethVV tk- ) and HU to mouse renal cell carcinoma transplanted mice (Renca), and then measuring the tumor volume on day 22;

[0029] Figure 14 Shows the results obtained by administering recombinant vaccinia virus (WyethVV tk- ) and HU to mouse renal cell carcinoma transplanted mice (Renca), and then observing the proliferation of CD4+ T cells or CD8+ T cells in the spleen tissue;

[0030] Figure 15 Shows the results obtained by administering recombinant vaccinia virus (OTS-412) and HU to mouse breast cancer cell transplanted mice (4T1), and then observing the proliferation of CD4+ T cells or CD8+ T cells in the blood and spleen tissue;

[0031] Figure 16 Shows the results obtained by administering recombinant vaccinia virus (WR VV tk- ) and HU to the left tumor in mouse breast cancer cell transplanted mice (4T1), and then measuring the volume of the left tumor;

[0032] Figure 17 Shows the results obtained by administering recombinant vaccinia virus (WR VV tk- ) and HU to the left tumor in mouse breast cancer cell transplanted mice (4T1), and then measuring the volume of the right tumor;

[0033] Figure 18Shows the results obtained by transplanting recombinant vaccinia virus (WR VV) into murine renal carcinoma cells (Renca) of mice and then staining on day 22 to determine the distribution of recombinant vaccinia virus in murine tumor tissues; tk- ) and HU, and then staining on day 22 to determine the distribution of recombinant vaccinia virus in murine tumor tissues;

[0034] Figure 19 Shows the results obtained by administering wild-type vaccinia virus (WR), or wild-type vaccinia virus (WR) and HU to normal mice, and then identifying the distribution of wild-type vaccinia virus in liver tissues and kidney tissues.

[0035] Best Mode

[0036] The present invention will be specifically described below.

[0037] In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises vaccinia virus and hydroxyurea as active ingredients.

[0038] The vaccinia virus and hydroxyurea contained in the pharmaceutical composition can be administered in combination simultaneously, sequentially, or in the reverse order. Specifically, the vaccinia virus and hydroxyurea can be administered simultaneously. In addition, hydroxyurea can be administered first, and then the vaccinia virus. In addition, the vaccinia virus can be administered first, and then hydroxyurea. Additionally, hydroxyurea can be administered first, then the vaccinia virus, and then hydroxyurea again.

[0039] The vaccinia virus can belong to but is not limited to: Western Reserve (WR), New York vaccinia virus (NYVAC), Wyeth (The New York City Board of Health (NYCBOH)), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), or International Health Division-White (IHD-W) vaccinia virus strains. In one embodiment of the present invention, Western Reserve strain vaccinia virus and Wyeth strain vaccinia virus are used.

[0040] The vaccinia virus can be a wild-type vaccinia virus or a recombinant vaccinia virus. Specifically, the recombinant vaccinia virus can be obtained by deleting a gene or inserting a foreign gene into the wild-type vaccinia virus. In this article, in the genes of the wild-type vaccinia virus, any one of the virus virulence-related genes selected from the group consisting of: thymidine kinase (TK), vaccinia growth factor (VGF), WR53.5, F13.5L, F14.5, A56R, B18R, or a combination thereof can be deleted.

[0041] In addition, the inserted foreign gene can be a gene that promotes immunity and encodes any one selected from the group consisting of: herpes simplex virus thymidine kinase (HSV-TK), mutant HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), cytosine deaminase (CD), carboxylesterase 1, carboxylesterase 2, interferon beta (INF-β), somatostatin receptor 2, and combinations thereof.

[0042] Specifically, the recombinant vaccinia virus can be obtained by deleting the TK gene in a vaccinia virus belonging to the following: Western Reserve (WR), New York vaccinia virus (NYVAC), Wyeth (New York City Board of Health (NYCBOH)), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, International Health Division-J (IHD-J), or International Health Division-White (IHD-W) vaccinia virus strains. In one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene in the Western Reserve strain vaccinia virus is used, and this virus is named "WR VV" tk- ". Additionally, in one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene in the Wyeth strain vaccinia virus is used, and this virus is named "Wyeth VV" tk- ".

[0043] In addition, recombinant vaccinia virus can be obtained by deleting the TK gene and the VGF gene in vaccinia viruses belonging to the following: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains. In one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene and the VGF gene in the Western Reserve strain vaccinia virus is used, and this virus is named "VV_DD".

[0044] In addition, recombinant vaccinia virus can be obtained by deleting the TK gene in vaccinia viruses belonging to the following and inserting the HSV-TK gene into the vaccinia virus: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains.

[0045] In addition, recombinant vaccinia virus can be obtained by deleting the TK gene in vaccinia viruses belonging to the following and inserting a mutant HSV-TK gene into the vaccinia virus: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains. In one embodiment of the present invention, the recombinant vaccinia virus is obtained by deleting the TK gene in the Wyeth strain vaccinia virus and inserting a gene encoding the HSV-TK fragment (1-330aa) of SEQ ID NO: 1 at the deletion position, and this virus is named "OTS-412". In addition, in one embodiment of the present invention, a recombinant vaccinia virus obtained by deleting the TK gene from the Western Reserve strain vaccinia virus and inserting a gene encoding the HSV-TK variant of SEQ ID NO: 2 of the HSV-TK gene at the deletion position is used, and this virus is named "WOTS-418".

[0046] In addition, recombinant vaccinia virus can be obtained by deleting the TK gene in vaccinia viruses belonging to the following and inserting the GM-CSF gene into the vaccinia virus: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains.

[0047] In addition, a recombinant vaccinia virus can be obtained by deleting the TK gene in a vaccinia virus belonging to the following: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains and inserting the C-CSF gene into the vaccinia virus.

[0048] In addition, a recombinant vaccinia virus can be obtained by deleting the TK gene in a vaccinia virus belonging to the following: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains and inserting the cytosine deaminase (CD) gene into the vaccinia virus.

[0049] In addition, a recombinant vaccinia virus can be obtained by deleting the TK gene in a vaccinia virus belonging to the following: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains and inserting the somatostatin receptor 2 gene into the vaccinia virus.

[0050] In addition, a recombinant vaccinia virus can be obtained by deleting the TK gene in a vaccinia virus and inserting any two or more genes selected from the group consisting of genes encoding herpes simplex virus thymidine kinase (HSV-TK), mutant HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), cytosine deaminase (CD) or somatostatin receptor 2 into the vaccinia virus, and the vaccinia virus belongs to: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains.

[0051] In addition, a recombinant vaccinia virus can be obtained by deleting the TK gene and the VGF gene in a vaccinia virus and inserting any one gene selected from the group consisting of genes encoding herpes simplex virus thymidine kinase (HSV-TK), mutant HSV-TK, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), cytosine deaminase (CD), or somatostatin receptor 2, and combinations thereof into the vaccinia virus, and the vaccinia virus belongs to: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J or IHD-W vaccinia virus strains.

[0052] As used herein, the term "gene deletion" refers to the non-expression of a gene due to partial or complete deletion of the gene or insertion of a foreign gene therein. In the case of a partial deletion occurring in a gene, some amino acids at the N-terminus or C-terminus of the polypeptide expressed by the gene may be deleted.

[0053] As used herein, the term "thymidine kinase (TK)" refers to an enzyme called thymidine kinase that is involved in nucleotide biosynthesis. TK is an enzyme used for nucleotide biosynthesis in cells and viruses. Here, for cells, normal cells no longer divide, so TK does not exist therein; even for rapidly dividing cells such as hair follicle cells, the amount of TK present is not sufficient for the virus to utilize. From these viewpoints, the virus can proliferate only in cancer cells where TK is present by deleting the TK gene therein, thereby selectively killing cancer cells.

[0054] As used herein, the term "vaccinia growth factor (VGF)" refers to a polypeptide that has sequence homology with epidermal growth factor and stimulates the proliferation of cells surrounding infected cells. Vaccinia virus replicates better in proliferating cells and can thus be advantageously used for in vivo virus replication. In order to make the oncolytic virus proliferate more specifically only in cancer cells, in addition to the TK gene deletion, the virus can also be additionally subjected to VGF gene deletion.

[0055] As used herein, the term "GM-CSF", also known as granulocyte-macrophage colony-stimulating factor, refers to a protein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, basophils, eosinophils) and monocytes. In addition, GM-CSF rapidly increases the number of macrophages, thereby inducing an immune response. GM-CSF can be of human origin and can be a protein having the sequence of GenBank: AAA52578.1.

[0056] As used herein, the term "CD", also known as cytosine deaminase, refers to an enzyme that catalyzes the hydrolytic deamination of cytosine to uracil and ammonia.

[0057] As used herein, the term "G-CSF", also known as granulocyte colony-stimulating factor, refers to a cytokine produced by macrophages, fibroblasts, endothelial cells, etc. when stimulated by inflammation or endotoxin. G-CSF promotes the production of neutrophils. G-CSF can be of human origin (rhGCSF) and can be a protein having the sequence of GenBank: AAA03056.1.

[0058] As used herein, the term "somatostatin receptor 2" refers to a protein encoded by the SSTR2 gene in humans. Somatostatin receptor 2 is mainly expressed in tumors, and patients with neuroendocrine tumors overexpressing somatostatin receptor 2 show an improved prognosis. Somatostatin receptor 2 has the ability to stimulate apoptosis in many cells, including cancer cells.

[0059] As used herein, the term "hydroxyurea" refers to a compound having the following formula.

[0060] [Formula 1]

[0061]

[0062] Hydroxyurea is considered an anticancer agent that inhibits DNA synthesis. However, its exact mechanism of action has not been elucidated. In addition, hydroxyurea can be included in a pharmaceutical composition in the form of a commercial drug containing hydroxyurea. Examples of commercial drugs containing hydroxyurea can include, but are not limited to Droxia TM 、Mylocel TM 、 and capsules. Hydroxyurea can be administered orally or parenterally.

[0063] The dose of vaccinia virus varies according to the individual's condition and body weight, disease severity, type of drug, route of administration, and time, and can be appropriately selected by those skilled in the art. The dose can be such that the patient receives 1×10 5 to 1×10 18 of viral particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu) of vaccinia virus. Specifically, the dose can be such that the patient receives 1×10 5 、2×10 5 、5×10 5 、1×10 6 、2×10 6 、5×10 6 、1×10 7, 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2××10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or higher of vaccinia virus in terms of viral particles, infectious virus units, or plaque-forming units, and may also include each value and range between the above values. Preferably, vaccinia virus can be administered at a dose of 1×10 5 pfu to 1×10 10 pfu. More preferably, vaccinia virus can be administered at a dose equal to or greater than 1×10 5 pfu and less than 1×10 9 pfu. In one embodiment of the present invention, vaccinia virus is administered at 1×10 5 pfu or 1×10 7 pfu.

[0064] In addition, hydroxyurea can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day or 10 mg / kg / day to 90 mg / kg / day. Specifically, hydroxyurea can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In one embodiment of the present invention, hydroxyurea is administered at 30 mg / kg / day or 60 mg / kg / day. Depending on the dose, hydroxyurea can be administered in divided doses several times a day. Specifically, hydroxyurea can be administered 1 to 4 times a day or 1 to 2 times a day.

[0065] Cancer can be solid cancer or blood cancer. Specifically, blood cancer can be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. Solid cancer can be any one selected from the group consisting of: lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0066] In addition, the pharmaceutical composition of the present invention may further comprise a physiologically acceptable carrier. In addition, the pharmaceutical composition of the present invention may further comprise suitable excipients and diluents commonly used in the preparation of pharmaceutical compositions. In addition, the pharmaceutical composition can be formulated in the form of an injection by conventional methods.

[0067] In the case of being formulated into a preparation for parenteral administration, the pharmaceutical composition can be formulated into a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried preparation, a suppository, etc. As the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injection esters such as ethyl oleate, etc. can be used. As the matrix of the suppository, Witepsol TM , polyethylene glycol, Tween TM 61, cocoa butter, laurel fat, glycerol gelatin, etc. can be used.

[0068] Regarding the route of administration, dosage, and frequency of administration, the pharmaceutical composition can be administered to the subject in various ways and amounts according to the condition of the patient and the presence or absence of side effects; and those skilled in the art can select the optimal route of administration, dosage, and frequency of administration within a suitable range. In addition, the pharmaceutical composition can be administered in combination with another drug or physiologically active substance known for the therapeutic effect on the disease to be treated, or can be formulated in the form of a combined preparation with other drugs.

[0069] The pharmaceutical composition can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intravenous, or intraarterial administration. Among them, intratumoral, intraperitoneal, or intravenous administration can be preferably used. On the other hand, the dosage of the pharmaceutical composition can be determined according to the administration regimen, total dosage, and the health condition of the patient.

[0070] The pharmaceutical composition for treating cancer can be characterized by an increased cancer selectivity of vaccinia virus.

[0071] In another aspect of the present invention, there is provided a kit for preventing or treating cancer, which comprises a first composition containing vaccinia virus as an active ingredient and a second composition containing hydroxyurea as an active ingredient.

[0072] The vaccinia virus is as described above for the pharmaceutical composition.

[0073] The second composition containing hydroxyurea as an active ingredient can be a commercial drug. Examples of commercial drugs containing hydroxyurea can include Droxia TM 、Mylocel TM 、 and capsules. The second composition can be administered orally or parenterally.

[0074] The dose of vaccinia virus varies according to the individual's condition and body weight, disease severity, type of drug, route and time of administration, and can be appropriately selected by those skilled in the art. The dose can be such that the patient receives 1×10 5 to 1×10 18 virus particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu) of vaccinia virus. Specifically, the dose can be such that the patient receives 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2××10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or higher virus particles, infectious virus units, or plaque-forming units of vaccinia virus, and can also include all values and ranges between the above values. Preferably, the vaccinia virus can be administered at a dose of 1×10 5 pfu to 1×10 10 pfu. More preferably, the vaccinia virus can be equal to or greater than 1×10 5 pfu and less than 1×10 9Administration of the dose of pfu. In one embodiment of the present invention, vaccinia virus is administered at 1×10 5 pfu or 1×10 7 pfu.

[0075] In addition, the second composition can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day or 10 mg / kg / day to 90 mg / kg / day. Specifically, the second composition can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In one embodiment of the present invention, the second composition is administered at 30 mg / kg / day or 60 mg / kg / day. Depending on the dose, the second composition can be administered in divided doses several times a day. Specifically, the second composition can be administered 1 to 4 times a day or 1 to 2 times a day.

[0076] The cancer can be solid cancer or hematological cancer. Specifically, the hematological cancer can be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. The solid cancer can be any one selected from the group consisting of: lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0077] The first composition and the second composition can also contain a physiologically acceptable carrier. In addition, the compositions included in the kit of the present invention can also contain suitable excipients and diluents commonly used in the preparation of pharmaceutical compositions. In addition, the compositions can be formulated in the form of injections according to conventional methods.

[0078] In the case of a preparation formulated for parenteral administration, the first composition and the second composition can be formulated into a sterile aqueous solution, non-aqueous solution, suspension, emulsion, freeze-dried preparation, suppository, etc. As the non-aqueous solution or suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injection esters such as ethyl oleate, etc. can be used. As the matrix of the suppository, Witepsol TM 、 polyethylene glycol, Tween TM 61, cocoa butter, laurel fat, glycerogelatin, etc. can be used.

[0079] Regarding the route of administration, dosage, and frequency of administration, the first composition and the second composition can be administered to a subject in various ways and amounts according to the condition of the patient and the presence or absence of side effects; and those skilled in the art can select the optimal route of administration, dosage, and frequency of administration within a suitable range. In addition, the pharmaceutical composition can be administered in combination with another drug or physiologically active substance known to have a therapeutic effect on the disease to be treated, or can be formulated in the form of a combined preparation with other drugs.

[0080] The second composition can be administered orally or parenterally. Specifically, the second composition can be administered parenterally, and such administration can be carried out by intraperitoneal, intraarterial, or intravenous administration.

[0081] The first composition can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intraarterial, or intravenous administration. Among them, intratumoral, intraperitoneal, or intravenous administration can be preferred. On the other hand, the dosage of the first composition and the dosage of the second composition can be determined according to the administration protocol, total dosage, and the health condition of the patient.

[0082] In addition, the first composition can be administered 1 to 10 times or 2 to 5 times, and it can be administered to an individual at intervals of 7 to 30 days. Specifically, the first composition can be administered at intervals of 7 days, 14 days, 21 days, or 30 days.

[0083] The second composition can be administered before or after the administration of the first composition. Specifically, the second composition can be continuously administered once a day starting from 3 to 5 days before the administration of the first composition, and can be continuously administered once a day starting from within 24 hours or 24 hours after the administration of the first composition for 9 to 28 days. In one embodiment of the present invention, the second composition can be continuously administered once a day starting from 1 to 3 days before the administration of the first composition, and can be continuously administered once a day for 13 days, 17 days, 18 days, or 28 days after the administration of the first composition.

[0084] In yet another aspect of the present invention, a method for treating cancer is provided, which includes administering vaccinia virus and hydroxyurea to an individual suffering from cancer.

[0085] The vaccinia virus can belong to but is not limited to: Western Reserve, NYVAC, Wyeth, LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, IHD-J, or IHD-W vaccinia virus strains.

[0086] Vaccinia virus and hydroxyurea can be administered in combination simultaneously, sequentially, or in the reverse order. Specifically, vaccinia virus and hydroxyurea can be administered simultaneously. In addition, hydroxyurea can be administered first, followed by vaccinia virus. In addition, vaccinia virus can be administered first, followed by hydroxyurea. Additionally, hydroxyurea can be administered first, followed by vaccinia virus, and then hydroxyurea can be administered again.

[0087] The dose of vaccinia virus varies according to the individual's condition and body weight, disease severity, type of drug, route of administration, and time, and can be appropriately selected by those skilled in the art. The dose can be such that the patient receives from 1×10 5 to 1×10 18 virus particles, infectious virus units (TCID 50 ), or plaque-forming units (pfu) of vaccinia virus. Specifically, the dose can be such that the patient receives 1×10 5 , 2×10 5 , 5×10 5 , 1×10 6 , 2×10 6 , 5×10 6 , 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2××10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 or higher virus particles, infectious virus units, or plaque-forming units of vaccinia virus, and various values and ranges between the above values can also be included. Preferably, vaccinia virus can be administered at a dose of 1×10 5 pfu to 1×10 10 pfu. More preferably, vaccinia virus can be administered at a dose equal to or greater than 1×10 5 pfu and less than 1×10 9 pfu. In one embodiment of the present invention, vaccinia virus is administered at 1×10 5 pfu or 1×10 7Administration of pfu.

[0088] In addition, hydroxyurea can be administered at a dose of 1 mg / kg / day to 100 mg / kg / day or 10 mg / kg / day to 90 mg / kg / day. Specifically, hydroxyurea can be administered at a dose of 10 mg / kg / day to 90 mg / kg / day, 15 mg / kg / day to 80 mg / kg / day, 20 mg / kg / day to 70 mg / kg / day, 25 mg / kg / day to 65 mg / kg / day, or 30 mg / kg / day to 60 mg / kg / day. In one embodiment of the present invention, hydroxyurea is administered at 30 mg / kg / day or 60 mg / kg / day. Depending on the dose, hydroxyurea can be administered in divided doses several times a day. Specifically, hydroxyurea can be administered 1 to 4 times a day or 1 to 2 times a day.

[0089] In addition, vaccinia virus can be administered 1 to 10 times or 2 to 5 times, and it can be administered to an individual at intervals of 7 to 30 days. Specifically, vaccinia virus can be administered at intervals of 7 days, 14 days, 21 days, or 30 days.

[0090] Hydroxyurea can be administered before, during, or after the administration of vaccinia virus. Specifically, hydroxyurea can be administered before or after the administration of vaccinia virus. Hydroxyurea can be continuously administered once a day starting 3 to 5 days before the administration of vaccinia virus, and can be continuously administered once a day starting 24 hours after the administration of vaccinia virus for 9 to 28 days. In one embodiment of the present invention, hydroxyurea can be continuously administered once a day starting 1 to 3 days before the administration of vaccinia virus, and can be continuously administered once a day for 13 days, 17 days, 18 days, or 28 days after the administration of vaccinia virus.

[0091] The cancer can be solid cancer or blood cancer. Specifically, the blood cancer can be any one selected from the group consisting of lymphoma, acute leukemia, and multiple myeloma. The solid cancer can be any one selected from the group consisting of: lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, and combinations thereof.

[0092] Hydroxyurea can be administered orally or parenterally. Specifically, hydroxyurea can be administered parenterally, and such administration can be carried out by intraperitoneal, intraarterial, or intravenous administration.

[0093] Vaccinia virus and hydroxyurea can be administered parenterally, and such administration can be carried out by any suitable method, such as intratumoral, intraperitoneal, subcutaneous, intradermal, intranodal, intravenous or intraarterial administration. Among them, intratumoral, intraperitoneal or intravenous administration can be preferably used. On the other hand, the doses of vaccinia virus and hydroxyurea can be determined according to the administration protocol, the total dose and the health condition of the patient.

[0094] As used herein, the term "individual" refers to a human being suffering from or being afflicted with a disease that can be alleviated, inhibited or treated by administering the pharmaceutical composition of the present invention.

[0095] As used herein, the term "administration" refers to introducing an effective amount of a substance into an individual by a suitable method, and the administration of vaccinia virus and hydroxyurea can be carried out by a general route that enables the substance to reach the target tissue.

[0096] In addition, vaccinia virus and hydroxyurea can be administered in combination with another drug or physiologically active substance known for its therapeutic effect on the disease to be treated, or can be formulated in the form of a combined preparation with other drugs.

[0097] In yet another aspect of the present invention, there is provided the use of a composition comprising vaccinia virus and hydroxyurea for the prevention or treatment of cancer.

[0098] In yet another aspect of the present invention, there is provided the use of a composition comprising vaccinia virus and hydroxyurea in the preparation of a drug for the prevention or treatment of cancer.

[0099] In yet another aspect of the present invention, there is provided an anti-cancer adjuvant comprising hydroxyurea as an active ingredient. Here, hydroxyurea is as described above for the pharmaceutical composition. In addition, the anti-cancer adjuvant can be characterized in that it is used as an anti-cancer adjuvant for an anti-cancer agent comprising vaccinia virus as an active ingredient. The anti-cancer adjuvant can be characterized in that it improves, enhances or increases the anti-cancer activity of vaccinia virus. The anti-cancer adjuvant can be characterized in that it increases the cancer selectivity of vaccinia virus. Detailed Description of the Invention

[0100] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0101] Preparation Example 1. Production of recombinant vaccinia virus (Wyeth VV tk- 、WR VV tk- )

[0102] Preparation Example 1.1. Construction of a shuttle plasmid vector

[0103] To produce recombinant vaccinia viruses lacking the thymidine kinase (TK) gene, wild-type vaccinia viruses, namely the Wyeth strain (NYC Health Department) and the Western Reserve strain, were purchased from the American Type Culture Collection (ATCC). For recombination, the TK region in the wild-type vaccinia virus was replaced with a shuttle plasmid vector containing the firefly luciferase reporter (p7.5 promoter) gene or the GFP gene.

[0104] Preparation Example 1.2. Production of Recombinant Vaccinia Virus

[0105] To obtain recombinant viruses, HeLa cells (ATCC) were seeded at 4×10 5 cells per well in a 6-well plate and then cultured in EMEM medium containing 10% fetal bovine serum. Subsequently, wild-type vaccinia virus was treated at 0.05 MOI. After 2 hours, the medium was replaced with EMEM medium containing 2% fetal bovine serum, and then the cells were transfected with 4 μg of the shuttle plasmid vector constructed and linearized in Preparation Example 1.1 using the Xfect TM Polymer (Clonetech 631317, USA). The cells were cultured for 4 hours. Subsequently, the medium was replaced with EMEM medium containing 2% fetal bovine serum and cultured for another 72 hours. Finally, the infected cells were collected and then frozen and thawed 3 times. Subsequently, the cells were lysed by sonication, and free recombinant vaccinia virus was obtained by the sucrose cushion method and named Wyeth VV tk- or WR VV tk- .

[0106] Preparation Example 2. Production of Recombinant Vaccinia Virus (OTS-412)

[0107] To produce a recombinant vaccinia virus lacking the thymidine kinase (TK) gene and expressing the mutant herpes simplex virus thymidine kinase (HSV-TK) gene, the pUC57amp+ plasmid (Genewiz, USA) in which the synthetic SEQ ID NO: 1 mutant type 1 HSV-TK gene (pSE / L promoter) and the firefly luciferase reporter (p7.5 promoter) gene were recombined was used as a shuttle vector to replace the TK region in the Wyeth strain wild-type vaccinia virus. The recombinant vaccinia virus was obtained in the same manner as in Preparation Example 1.2 using the shuttle vector constructed as above, and the virus was named OTS-412.

[0108] Preparation Example 3. Production of Recombinant Vaccinia Virus (WOTS-418)

[0109] To produce a recombinant vaccinia virus lacking the thymidine kinase (TK) gene and expressing the mutant herpes simplex virus thymidine kinase (HSV-TK) gene, the TK region in the wild-type vaccinia virus of the Western Reserve strain was replaced using the pUC57amp+ plasmid (Genewiz, USA) in which the mutant type 1 HSV-TK gene (pSE / L promoter) and the firefly luciferase reporter (p7.5 promoter) gene of SEQ ID NO: 2 were recombined as a shuttle vector. The recombinant vaccinia virus was obtained in the same manner as in Preparation Example 1.2 using the shuttle vector constructed as above, and this virus was named WOTS-418.

[0110] Preparation Example 4. Production of recombinant vaccinia virus (WR VV_DD)

[0111] To produce a recombinant vaccinia virus lacking the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene, the TK region in the wild-type vaccinia virus of the Western Reserve strain was replaced using a shuttle plasmid containing the enhanced green fluorescent protein (EGFP) gene, and the VGF gene region in the same virus was replaced using a shuttle plasmid containing the lacZ gene. The recombinant vaccinia virus was obtained in the same manner as in Preparation Example 1.2 using the shuttle plasmid containing the EGFP gene and the shuttle plasmid containing the lacZ gene, and this virus was named VV_DD.

[0112] Experimental Example 1. Confirmation of the cancer treatment effects of wild-type vaccinia virus (WR) and hydroxyurea on mice transplanted with mouse renal cancer cells: Renca (I)

[0113] Experimental Example 1.1. Generation of mice transplanted with mouse renal cancer cells and drug administration

[0114] Balb / c mice (female, 10 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimatized for 2 days and then subcutaneously transplanted with 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 50 mm 3 to 80 mm 3 and then the administration of wild-type vaccinia virus was started. On the other hand, the wild-type vaccinia virus of the Western Reserve strain (WR) has a stronger proliferative ability than the wild-type vaccinia virus of the Wyeth strain in an allograft model.

[0115] The mice transplanted with the generated mouse renal carcinoma cells were divided into 3 groups (n = 6). The group that received intraperitoneal administration of normal saline was set as the negative control group, and the group that received administration of wild-type vaccinia virus (WR, 1×10 5 pfu) was used as the positive control group. In addition, the group that received co-administration of wild-type vaccinia virus (WR, 1×10 5 pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The wild-type vaccinia virus was administered intratumorally once; and from 1 day before the administration of the wild-type vaccinia virus to the 14th day after the administration, except for the day of administration of the wild-type vaccinia virus, hydroxyurea was administered intraperitoneally 5 times a week.

[0116] Experimental Example 1.2. Examination of changes in tumor volume

[0117] The tumor volumes were measured on the 0th, 3rd, 7th, 10th, and 14th days after drug administration in the mice of each group in Experimental Example 1.1. The results confirmed that, compared with the negative control group, the tumor volume of the mice in the positive control group was inhibited, while the tumor volume of the mice in the experimental group was significantly inhibited ( Figure 1 ).

[0118] Experimental Example 1.3. Examination of changes in body weight

[0119] The body weights of the mice in the negative control group, positive control group, and experimental group in Experimental Example 1.1 were measured on the 3rd, 7th, 10th, and 14th days after each drug administration. As a result, there was no significant weight loss in all three groups ( Figure 2 ).

[0120] Experimental Example 2. Confirmation of the cancer treatment effect of recombinant vaccinia virus (WR VV tk- ) and hydroxyurea on mice transplanted with mouse renal carcinoma cells: Renca (II)

[0121] Experimental Example 2.1. Generation of mice transplanted with mouse renal carcinoma cells and drug administration

[0122] Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were allowed to undergo a one-week acclimation period, and then 5×10 6 cells were used for allogeneic transplantation of the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 100 mm 3 to 150 mm 3 , and then the administration of recombinant vaccinia virus was started. On the other hand, the recombinant vaccinia virus (WR VV tk- ) derived from the Western Reserve strain has stronger proliferative ability than the recombinant vaccinia virus derived from the Wyeth strain in the allogeneic transplantation model.

[0123] The mice transplanted with the generated mouse renal cancer cells were divided into 3 groups (n = 8). The group that received intraperitoneal administration of normal saline was set as the negative control group, and the group that received administration of recombinant vaccinia virus (WR VV tk- , 1×10 7 pfu) was set as the positive control group. In addition, the group that received co - administration of recombinant vaccinia virus and hydroxyurea (60 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally twice; and from 1 day before the administration of recombinant vaccinia virus to the 21st day after the administration, except on the days of recombinant vaccinia virus administration, hydroxyurea was administered intraperitoneally 6 times a week.

[0124] Experimental Example 2.2. Examination of changes in tumor volume

[0125] In Experimental Example 2.1, the tumor volumes of the mice in each group were measured on the 0th, 3rd, 7th, 10th, 14th, 17th, and 21st days after drug administration. It was confirmed that the tumor volume of the mice in the experimental group was significantly inhibited compared with that of the mice in the positive control group ( Figure 3 ).

[0126] Experimental Example 3. Confirmation of the cancer treatment effect of recombinant vaccinia virus (WR VV tk- ) and hydroxyurea on mice transplanted with mouse renal cancer cells: Renca(III)

[0127] Balb / c mice (female, 10 - week - old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days, and then 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank) were subcutaneously transplanted at the left thigh. The tumor volume was observed until it reached 50 mm 3 to 150 mm 3 , and then the administration of recombinant vaccinia virus was started.

[0128] The mice transplanted with the generated mouse renal cancer cells were divided into 3 groups (n = 6). The group that received intraperitoneal administration of normal saline was set as the negative control group, and the group that received administration of recombinant vaccinia virus (WR VV tk- , 1×10 5 pfu) was used as the positive control group. In addition, the group that received co - administration of recombinant vaccinia virus and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally once; and from 1 day before the administration of recombinant vaccinia virus to the 14th day after the administration, except on the days of recombinant vaccinia virus administration, hydroxyurea was administered intraperitoneally 6 times a week.

[0129] The tumor volumes of each group of mice were measured on days 0, 3, 7, 10, and 14 after drug administration. The results confirmed that the growth of the tumor volume of the experimental group mice was inhibited by approximately 25% compared to that of the positive control group mice ( Figure 4 ).

[0130] Experimental Example 4. Confirmation of the cancer treatment effect of recombinant vaccinia virus (WR VV_DD) and hydroxyurea on mice transplanted with murine melanoma: B16F10

[0131] C57BL / 6 mice (female, 7 weeks old) purchased from KOATECH (Korea) were subjected to a 2-day acclimation period, and then subcutaneously transplanted with murine melanoma cancer cell line (ATCC, B16F10) at 5×10 5 cells. The tumor volume was observed until it reached 50 mm 3 to 100 mm 3 , and then the administration of recombinant vaccinia virus (WR VV_DD) was started. Recombinant vaccinia virus (WR VV_DD) was obtained by double deletion of the thymidine kinase (TK) and vaccinia growth factor (VGF) regions in the Western Reserve strain of vaccinia virus and has limited proliferative ability in an allograft model.

[0132] The resulting mice transplanted with murine melanoma were divided into 4 groups (n = 6). The group receiving intraperitoneal administration of normal saline was set as the negative control group, and the groups receiving either hydroxyurea alone (30 mg / kg) or recombinant vaccinia virus (VV_DD, 1×10 6 pfu) were used as the positive control groups. Additionally, the group receiving the co-administration of recombinant vaccinia virus and hydroxyurea (30 mg / kg) was set as the experimental group. Recombinant vaccinia virus was intraperitoneally administered on days 0 and 5; and hydroxyurea was intraperitoneally administered 6 times a week from 1 day before the administration of recombinant vaccinia virus until day 15 after the administration, except on the days of recombinant vaccinia virus administration.

[0133] The tumor volumes of each group of mice were measured 1 day before drug administration and on days 4 and 7 after administration. The results confirmed that the tumor volume of the experimental group mice was significantly inhibited compared to that of the positive control group mice ( Figure 5 ). From these results, it was confirmed that a synergistic effect was observed when recombinant vaccinia virus (VV_DD) and hydroxyurea were co-administered.

[0134] Experimental Example 5. Confirmation of the cancer treatment effect of recombinant vaccinia virus (WOTS-418) and hydroxyurea on mice transplanted with human lung cancer cell line: NCI-H460

[0135] Female Balb / c nu / nu mice (7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then subcutaneously xenografted with 5×10 6 cells of the NCI-H460 human lung cancer cell line (Korea Cell LineBank). Tumor volume was observed until it reached 100 mm 3 to 150 mm 3 , and then administration of recombinant vaccinia virus (WOTS-418) was started. On the other hand, the recombinant vaccinia virus (WOTS-418) derived from the Western Reserve strain has the ability to proliferate in mice xenografted with the human lung cancer cell line (NCI-H460).

[0136] The mice transplanted with the generated human lung cancer cell line were divided into 2 groups (n = 4). The group that received intraperitoneal administration of physiological saline was set as the control group, and the group that received co-administration of recombinant vaccinia virus (WOTS-418, 1×10 7 pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intraperitoneally once; and from 1 day before the administration of the recombinant vaccinia virus to the 15th day after the administration, hydroxyurea was administered intraperitoneally 6 times a week except on the day of administration of the recombinant vaccinia virus.

[0137] On the -1st, 5th, 10th, 12th, and 15th days after drug administration in each group of mice, the tumor volume was measured. The results confirmed that the tumor volume of the experimental group mice was suppressed by about 40% compared with the tumor volume of the control group mice ( Figure 6 ).

[0138] Experimental Example 6. Analysis of the survival rate of recombinant vaccinia virus (WOTS-418) and hydroxyurea in mice transplanted with mouse colorectal cancer cells: CT-26

[0139] Female Balb / c mice (7 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days and then subcutaneously transplanted with 1×10 6 cells of the mouse colorectal cancer cell line (CT-26, Korea Cell Line Bank). After 7 days, administration of recombinant vaccinia virus (WOTS-418) and hydroxyurea was started. On the other hand, the recombinant vaccinia virus (WOTS-418) derived from the Western Reserve strain has stronger proliferative ability than the recombinant vaccinia virus derived from the Wyeth strain in the allograft model.

[0140] The mice transplanted with the generated mouse colorectal cancer cell line were divided into 2 groups (n = 12), that is, the group that received intraperitoneal administration of recombinant vaccinia virus (WOTS-418, 1×10 7The group that received recombinant vaccinia virus alone and the group that received co - administration of recombinant vaccinia virus and hydroxyurea (30 mg / kg). Recombinant vaccinia virus was administered intraperitoneally once; hydroxyurea was administered intraperitoneally 5 times continuously starting from 1 day after the administration of recombinant vaccinia virus.

[0141] The survival curves of mice in each group were analyzed. As a result, for the group that received only the administration of recombinant vaccinia virus, all mice died 25 days after the administration; however, 30% or more of the mice that received co - administration of hydroxyurea and recombinant vaccinia virus survived for 55 days or longer ( Figure 7 ). From these results, it was confirmed that the safety was improved in the case of co - administration of recombinant vaccinia virus and hydroxyurea compared with the case of administering only recombinant vaccinia virus.

[0142] Experimental Example 7. Recombinant vaccinia virus (Wyeth VV tk- ) and confirmation of the cancer treatment effect of hydroxyurea on mice transplanted with mouse renal cancer cells: Renca (IV)

[0143] Experimental Example 7.1. Generation of mice transplanted with mouse renal cancer cells and drug administration

[0144] Balb / c mice (female, 7 - week - old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days, and then allografted with 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 100 mm 3 to 150 mm 3 , and then the administration of recombinant vaccinia virus was started.

[0145] The generated mice transplanted with mouse renal cancer cells were divided into 4 groups (n = 4). The group that received intratumoral administration of normal saline was set as the negative control group, and the group that received administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) was set as the positive control group. In addition, the group that received co - administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) and recombinant human granulocyte - colony stimulating factor (rhG - CSF, 75 μg / kg), and the group that received recombinant virus (VV tk- , 1×10 7 pfu) and hydroxyurea (30 mg / kg) were set as experimental groups. Recombinant vaccinia virus was administered intratumorally, and rhG - CSF or hydroxyurea was administered intraperitoneally 5 times a week starting from 3 days before the administration of recombinant vaccinia virus until sacrifice.

[0146] Experimental Example 7.2. Examination of changes in tumor volume

[0147] On the 16th day after administration, the mice in each group of Experimental Example 7.1 were sacrificed, and the tumor volume was measured. As a result, compared with the initial tumor volume, the mice in the positive control group and the experimental group that received the co-administration of recombinant vaccinia virus and rhG-CSF showed an almost 10-fold increase. Compared with the initial tumor volume, the mice in the experimental group that received the co-administration of recombinant vaccinia virus and hydroxyurea showed an almost 8-fold increase, which was the most inhibited tumor volume observed( Figure 8 ).

[0148] Experimental Example 7.3. Confirmation of activation of antigen-specific cytotoxic T lymphocytes (CTL)

[0149] To confirm whether a tumor-specific anti-cancer effect was obtained in the case of co-administering recombinant vaccinia virus and hydroxyurea, the mice in each group of Experimental Example 7.1 were sacrificed on the 16th day, and then lymphocytes in the spleen were isolated from each group. Then, the isolated lymphocytes were respectively injected into new normal mice. Cancer transplantation was performed and the tumor volume was observed. Specifically, one week later, the mice were allografted with 5×10 6 cells of the Renca cancer cell line (Korea Cell LineBank), and the tumor volume was measured on the 19th day.

[0150] As a result, tumor growth was significantly inhibited in the mice injected with the splenocytes collected from the mice in the group that received the co-administration of recombinant vaccinia virus and hydroxyurea. On the other hand, tumor growth was not significantly inhibited in each of the mice injected with the splenocytes collected from the mice in the remaining groups( Figure 9 ). From these results, it was confirmed that the group that received the co-administration of recombinant vaccinia virus and hydroxyurea not only produced immune cells such as cytotoxic T cells, but also activated the adaptive immunity.

[0151] Experimental Example 8. Confirmation of the cancer treatment effect of recombinant vaccinia virus (Wyeth VV tk- ) and hydroxyurea on mice transplanted with renal cancer cells: Renca(V)

[0152] Experimental Example 8.1. Generation of mice transplanted with renal cancer cells and drug administration

[0153] Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were allowed to undergo a one-week acclimation period, and then were allografted with 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 50mm 3 to 100mm3 , and then the administration of recombinant vaccinia virus was started. On the other hand, the recombinant vaccinia virus derived from the Wyeth strain (Wyeth VV tk- ) hardly proliferated in the mouse model transplanted with mouse renal carcinoma cells.

[0154] The mice transplanted with the generated mouse renal carcinoma cells were divided into 4 groups (n = 4). The group that received intratumoral administration of normal saline was set as the negative control group, and the group that received administration of hydroxyurea alone and the group that received administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) alone were set as the positive control groups. In addition, the group that received co - administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) and hydroxyurea (30 mg / kg) was set as the experimental group. Recombinant vaccinia virus was administered intratumorally, and hydroxyurea was administered intraperitoneally 5 times a week starting 3 days before the administration of recombinant vaccinia virus until sacrifice.

[0155] Experimental Example 8.2. Examination of changes in tumor volume

[0156] In Experimental Example 8.1, the tumor volumes of the mice in each group were measured on the 0th, 4th, 10th, 15th, and 22nd days after drug administration. As a result, compared with the initial tumor volume, the tumor volume of the mice in the positive control group increased by about 11 to 13 times. On the other hand, compared with the initial tumor volume, the tumor volume of the mice in the experimental group increased by about 4 times ( Figure 10 ).

[0157] Experimental Example 8.3. Confirmation of activation of tumor - specific cytotoxic T lymphocytes (CTL)

[0158] To confirm whether a tumor - specific anticancer effect was obtained in the case of co - administration of recombinant vaccinia virus and hydroxyurea, the mice in each group in Experimental Example 8.1 were sacrificed on the 16th day, and then splenocytes and cytotoxic T lymphocytes (CD8 + T cells) were isolated from each group. Then, the isolated splenocytes and cytotoxic T lymphocytes were respectively injected into new normal mice. Cancer transplantation was performed and the tumor volume was observed. Specifically, one week later, the mice were allografted with 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank), and the tumor volumes were measured on the 7th, 10th, 14th, 18th, and 21st days.

[0159] As a result, tumor growth was significantly inhibited in the mice injected with splenocytes or T lymphocytes collected from the mice in the experimental group. On the other hand, tumor growth was not significantly inhibited in the mice injected with splenocytes or T lymphocytes collected from the mice in the remaining groups ( Figure 11)。It was confirmed from these results that in the group receiving the co - administration of recombinant vaccinia virus and hydroxyurea, adaptive immunity with anti - cancer efficacy was activated not only due to T lymphocytes but also due to other immune cells formed in the spleen( Figure 11 and Figure 12 )。

[0160] Experimental Example 9. Cancer treatment effect of recombinant vaccinia virus (Wyeth VV tk- ) and hydroxyurea on mice transplanted with mouse renal cancer cells: Renca(VI)

[0161] Experimental Example 9.1. Generation of mice transplanted with mouse renal cancer cells and drug administration

[0162] Balb / c mice (female, 8 - week - old) purchased from ORIENT BIO (Busan, Korea) were acclimated for one week and then allografted with 5×10 6 cells of the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 100mm 3 to 150mm 3 , and then the administration of recombinant vaccinia virus was started. On the other hand, the recombinant vaccinia virus derived from the Wyeth strain (Wyeth VV tk- ) hardly proliferated in the mouse model transplanted with mouse renal cancer cells.

[0163] The generated mice transplanted with mouse renal cancer cells were divided into 3 groups (n = 6). The group receiving intratumoral administration of physiological saline was set as the negative control group, and the group receiving the administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) was set as the positive control group. In addition, the group receiving the administration of recombinant vaccinia virus (Wyeth VV tk- , 1×10 7 pfu) and hydroxyurea (30mg / kg) was set as the experimental group. Recombinant vaccinia virus was administered intratumorally, and hydroxyurea was administered intraperitoneally 6 times a week starting from 1 day before the administration of recombinant vaccinia virus until sacrifice.

[0164] Experimental Example 9.2. Examination of changes in tumor volume

[0165] On the 22nd day after administration, the mice in each group of Experimental Example 9.1 were sacrificed and the tumor volume was measured. As a result, compared with the tumor volume of the mice in the negative control group, the tumor volume of the mice in the positive control group was inhibited by about 25%. In particular, compared with the tumor volume of the mice in the negative control group, the tumor volume of the mice in the experimental group was inhibited by about 37.5%, and compared with the tumor volume of the mice in the positive control group, the tumor volume of the mice in the experimental group was inhibited by about 15%( Figure 13 )。

[0166] Experimental Example 9.3. Confirmation of Spleen Tissue Microenvironment

[0167] When recombinant vaccinia virus and hydroxyurea were co-administered, the distribution of immune cells in the tumor microenvironment was analyzed. For the analysis, immunohistochemical staining was performed using 3,3'-diaminobenzidine (DAB). Specifically, spleens were collected from the mice in each group. The spleen tissues were cut into 0.4 μm and dried. Subsequently, the tissues were washed with PBS and then treated with bovine serum albumin (BSA). The tissues were treated with primary antibodies (anti-CD3 antibody (Abcam), anti-CD4 antibody (BD Biosciences), anti-CD8 antibody (BD Biosciences)) diluted at a ratio of 1:50, and the reaction was allowed to proceed overnight at 4 °C. The next day, the tissues were washed with PBS and then reacted with secondary antibody (Dako) for 30 minutes at room temperature. The tissues were washed with PBS again, reacted using an ABC kit (Dako), and then developed by adding H 2 O 2 to make it colored. Then, the tissues were dehydrated and then mounted.

[0168] The results confirmed that CD4+ T cells and CD8+ T cells were more abundantly distributed in the tumor tissues of the experimental group mice ( Figure 14 ). From these results, it was confirmed that in the case of co-administering recombinant vaccinia virus and hydroxyurea, compared with the case of administering only recombinant vaccinia virus, CD4+ T cells and CD8+ T cells in the spleen tissue were more differentiated and activated. That is, it was confirmed that in the case of co-administering recombinant vaccinia virus and hydroxyurea, compared with the case of administering only recombinant vaccinia virus, adaptive immunity was better activated.

[0169] Experimental Example 10. Confirmation of Antigen-Specific Cytotoxic T Lymphocyte (CTL) Activation Induced by Recombinant Vaccinia Virus (OTS-412) and Hydroxyurea in Mice with Transplanted Mouse Breast Cancer Cells: 4T1(I)

[0170] Balb / c mice (female, 7 weeks old) purchased from ORIENT BIO (Busan, Korea) were allowed to undergo a one-week acclimation period and then allografted with 4T1 cancer cell line (Korea Cell Line Bank) at 1 × 10 6 cells. The tumor volume was observed until it reached 100 mm 3 to 150 mm 3, and then the administration of recombinant vaccinia virus was initiated. On the other hand, the recombinant vaccinia virus (OTS-412) derived from the Wyeth strain hardly proliferated in a mouse model transplanted with mouse breast cancer cells. In addition, the mouse transplanted with a breast cancer cell line is an animal model that metastasizes systemically including the lung tissue, and metastasis is generally evaluated by the number of nodules on the tumor surface.

[0171] The mice transplanted with the generated mouse breast cancer cells were divided into 4 groups (n = 5). The group that received intratumoral administration of physiological saline was set as the negative control group, and the groups that received co-administration of recombinant vaccinia virus (OTS-412, 1×10 7 pfu) or hydroxyurea (30 mg / kg) were set as the positive control groups. The group that received co-administration of recombinant vaccinia virus and hydroxyurea was set as the experimental group. The recombinant vaccinia virus was administered intratumorally first, and then readministered on the 7th day after the first administration. Starting from 3 days before the administration of recombinant vaccinia virus until 3 days before euthanasia, hydroxyurea was administered intraperitoneally once a day except on the days of recombinant vaccinia virus administration.

[0172] On the 18th day after drug administration, the mice in each group were euthanized, and blood and spleens were collected from the mice. The distribution of immune cells in the blood and spleen cells was analyzed by flow cytometry. As a result, it was confirmed that the distribution of CD4+ T cells and CD8+ T cells that induce tumor immune responses was the highest in the mice of the experimental group in the blood and spleen. In addition, it was found that the number of myeloid-derived suppressor cells (MDSC) with immunosuppressive function was significantly reduced in the mice of the experimental group compared with the mice of the negative control group and the positive control group ( Figure 15 ).

[0173] Experimental Example 11. Confirmation of the effect of enhancing adaptive immunity of recombinant vaccinia virus (WR VV tk- ) and hydroxyurea on mice transplanted with mouse breast cancer cells: 4T1(II)

[0174] Balb / c mice (female, 10 weeks old) purchased from ORIENT BIO (Busan, Korea) were acclimated for 2 days, and then 4T1 cancer cell lines (Korea Cell Line Bank) were subcutaneously transplanted at 1×10 6 cells at the left thigh. Two days later, the same number of 4T1 cancer cell lines were subcutaneously transplanted into the right thigh of the mice. The tumor subcutaneously transplanted on the left thigh was observed until its volume reached 50 mm 3 to 200 mm 3 , and then the administration of recombinant vaccinia virus was initiated.

[0175] The mice transplanted with the generated mouse breast cancer cells were divided into 3 groups (n = 6). The group that received intratumoral administration of normal saline was set as the negative control group, and the group that received administration of recombinant vaccinia virus (WR VV tk- , 1×10 5 pfu) was set as the positive control group. In addition, the group that co-administered recombinant vaccinia virus and hydroxyurea (90 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered into the left tumor once, and from 1 day before the administration of recombinant vaccinia virus to the 14th day after the administration, except on the day of recombinant vaccinia virus administration, hydroxyurea was intraperitoneally administered 6 times a week.

[0176] On the 0th, 3rd, 7th, 10th, and 14th days after drug administration in each group of mice, the tumor volumes transplanted subcutaneously in both thighs were measured. The results confirmed that the growth of the left tumor volume in the experimental group of mice was inhibited by approximately 35% compared with the left tumor volume in the positive control group of mice ( Figure 16 ). In addition, the growth of the right tumor volume in the experimental group of mice was inhibited by approximately 45% compared with the right tumor volume in the positive control group of mice ( Figure 17 ). Based on these results, the effect of co-administering recombinant vaccinia virus and hydroxyurea on surrounding tumors was confirmed.

[0177] That is, it was confirmed that in the case of local treatment of tumors by co-administering vaccinia virus and hydroxyurea, an anticancer effect was also observed in tumors where the virus was not administered.

[0178] Experimental Example 12. Confirmation of the increased cancer selectivity in mice transplanted with renal cancer cells after co-administering recombinant vaccinia virus (WR VV tk- ) and hydroxyurea (I)

[0179] Balb / c mice (female, 8 weeks old) purchased from ORIENT BIO (Busan, Korea) were subjected to a one-week acclimation period, and then 5×10 6 cells were allografted with the Renca cancer cell line (Korea Cell Line Bank). The tumor volume was observed until it reached 100 mm 3 to 150 mm 3 , and then the administration of recombinant vaccinia virus was started. On the other hand, the recombinant vaccinia virus derived from the Western Reserve strain (WR VV tk- ) has stronger proliferative ability than the recombinant vaccinia virus derived from the Wyeth strain in the allograft model.

[0180] The mice transplanted with the generated mouse renal cancer cells were divided into 3 groups (n = 8). The group that received intraperitoneal administration of normal saline was set as the negative control group, and the group that received administration of recombinant vaccinia virus (WR VV tk-, 1×10 7 The group administered with the recombinant vaccinia virus (1×10

[0181] pfu) was set as the positive control group. Additionally, the group that received the co - administration of the recombinant vaccinia virus and hydroxyurea (60 mg / kg) was set as the experimental group. The recombinant vaccinia virus was administered intratumorally twice; and from 1 day before the administration of the recombinant vaccinia virus until day 21 after the administration, except on the days of the recombinant vaccinia virus administration, hydroxyurea was administered intraperitoneally 6 times a week. 2 O 2 to develop color. Then, the tissue was dehydrated and then mounted.

[0182] The results confirmed that the recombinant vaccinia virus was more abundantly distributed in the tumor tissues of the mice in the experimental group ( Figure 18 ). From these results, it was confirmed that in the case of co - administering hydroxyurea when the recombinant vaccinia virus was administered systemically, more effective tumor - specific proliferation of the recombinant vaccinia virus was observed.

[0183] Experimental Example 13. Confirmation of increased survival rate and cancer selectivity after co - administration of wild - type vaccinia virus (WR) and hydroxyurea in normal mice (II)

[0184] Balb / c nu / nu mice (female, 7 - week - old) purchased from ORIENTBIO (Busan, Korea) were subjected to a 2 - day acclimation period and then started to be administered with the wild - type Western Reserve strain vaccinia virus (WR). On the other hand, the wild - type Western Reserve strain vaccinia virus has limited proliferative ability in syngeneic mice.

[0185] The mice were divided into two groups (n = 12). The group that received the administration of the wild - type Western Reserve strain vaccinia virus (1×10 7The group that received only the pfu was set as the control group, and the group that received the co - administration of wild - type Western Reserve strain vaccinia virus and hydroxyurea (50 mg / kg) was set as the experimental group. The wild - type vaccinia virus was administered intranasally once; and starting from 1 day before the administration of the wild - type vaccinia virus, except on the day of wild - type vaccinia virus administration, hydroxyurea was administered intraperitoneally 5 times a week.

[0186] On the 8th day, the control group and experimental group mice were sacrificed, and kidney and liver tissues were isolated from the mice. Immunohistochemical staining was performed. Paraffin blocks were made, and each block was dewaxed using xylene and ethanol. Antigen retrieval was performed on the obtained blocks using a decloaking chamber. Then, the primary antibody (product number ABIN1606294, Antibodies - Online) was attached to the block, and the FITC - labeled secondary antibody (Alexa594, product number A21205, Invitrogen) was attached to it. Then, observation was carried out using a fluorescence microscope.

[0187] The results confirmed that, compared with the liver and kidney tissues of the control group mice, the virus was less distributed and proliferated in the liver and kidney tissues of the experimental group mice ( Figure 19 ).

Claims

1. A pharmaceutical composition for treating cancer, comprising the following as active ingredients: vaccinia virus; and hydroxyurea, wherein the vaccinia virus is a recombinant vaccinia virus with a deletion of the thymidine kinase gene and insertion of a gene encoding herpes simplex virus thymidine kinase, and the vaccinia virus is derived from a wild-type vaccinia virus.

2. The pharmaceutical composition according to claim 1, wherein, the vaccinia virus belongs to the Western Reserve (WR), New York Vaccinia Virus (NYVAC), Wyeth (New York City Board of Health (NYCBOH)), LC16m8, Lister, Copenhagen, Tian Tan, USSR, Tashkent, Evans, International Health Division-J (IHD-J) or International Health Division-White (IHD-W) vaccinia virus strain.

3. The pharmaceutical composition according to claim 1, wherein, the cancer is any one selected from the group consisting of: lung cancer, colorectal cancer, prostate cancer, thyroid cancer, breast cancer, brain cancer, head and neck cancer, esophageal cancer, skin cancer, thymic cancer, gastric cancer, colon cancer, liver cancer, ovarian cancer, uterine cancer, bladder cancer, rectal cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, kidney cancer and combinations thereof.

4. The pharmaceutical composition according to claim 1, wherein, the pharmaceutical composition for treating cancer is characterized by an increased cancer selectivity of the vaccinia virus.

5. A kit for preventing or treating cancer, comprising: a first composition containing vaccinia virus as an active ingredient; and a second composition containing hydroxyurea as an active ingredient, wherein the vaccinia virus is a recombinant vaccinia virus with a deletion of the thymidine kinase gene and insertion of a gene encoding herpes simplex virus thymidine kinase, and the vaccinia virus is derived from a wild-type vaccinia virus.

6. Use of a composition comprising vaccinia virus and hydroxyurea in the preparation of a drug for preventing or treating cancer, wherein the vaccinia virus is a recombinant vaccinia virus with a deletion of the thymidine kinase gene and insertion of a gene encoding herpes simplex virus thymidine kinase, and the vaccinia virus is derived from a wild-type vaccinia virus.

7. An anti-cancer adjuvant, comprising as an active ingredient: hydroxyurea, wherein the anti-cancer adjuvant is used as an anti-cancer adjuvant for an anti-cancer agent containing vaccinia virus as an active ingredient, wherein the vaccinia virus is a recombinant vaccinia virus with a deletion of the thymidine kinase gene and insertion of a gene encoding herpes simplex virus thymidine kinase, and the vaccinia virus is derived from a wild-type vaccinia virus.

8. The anti-cancer adjuvant according to claim 7, wherein, the hydroxyurea improves, enhances or increases the anti-cancer activity of the vaccinia virus.

9. The anti-cancer adjuvant according to claim 7, wherein, the anti-cancer adjuvant increases the cancer selectivity of the vaccinia virus.

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