Combination drug for treating malignant tumor

Abstract

The purpose of the present invention is to provide a novel drug having excellent anti-malignant tumor effect.　A drug characterized by comprising the combination of liposomes encapsulating an anti-malignant tumor agent with a bacterium.

Description

Combination medicines for treating malignant tumors

[0001] The present invention relates to a pharmaceutical for treating malignant tumors, which comprises a combination of a liposome encapsulating an anti-cancer agent and a bacterium.

[0002] Anaerobic bacteria such as Salmonella are known to selectively colonize and grow in tumor tissues when administered intravenously to tumor-bearing subjects. Based on the discovery that VNP20009, an attenuated strain of Salmonella Typhimurium, targets tumors and inhibits tumor growth in mice, clinical trials were conducted using VNP20009 administered intravenously to metastatic cancer patients. However, although safety of VNP20009 was confirmed, no antitumor effect was observed (Non-Patent Document 1). Various studies have been conducted on the use of attenuated Salmonella strains to treat malignant tumors. For example, reports have included the use of attenuated Salmonella strains in combination with tumor-penetrating agents (Patent Document 1), the use of recombinant attenuated Gram-negative bacterial strains (Patent Document 2), the use of attenuated Salmonella strains in combination with anti-cancer agents (Non-Patent Documents 2-7), and the use of attenuated Salmonella strains in combination with antibodies (Non-Patent Document 8). In addition, it is known that Listeria monocytogenes, a type of Listeria bacteria, Clostridium novyi, a type of Clostridium bacterium, and its mutant Clostridium novyi-NT are used in the treatment of malignant tumors (Non-Patent Document 9).

[0003] Anti-cancer agents in which active ingredients are encapsulated in liposomes have been developed as DDS formulations. Examples include a formulation in which doxorubicin hydrochloride is encapsulated in liposomes (trade name: Doxil), a formulation in which irinotecan hydrochloride hydrate is encapsulated in liposomes (trade name: Onivyde), a formulation in which vincristine sulfate is encapsulated in liposomes (trade name: Marqibo), and a formulation in which daunorubicin citrate is encapsulated in liposomes (trade name: Daunoxome). It is known that by using DDS formulations in which these active ingredients are encapsulated in liposomes, the pharmacokinetics of the active ingredient drug itself is altered, enhancing the main pharmacological effect and reducing side effects. Preparations that are not easily recognized as foreign substances by the endothelial system and that exert anti-tumor effects by extending blood circulation time and selectively exuding into tumor tissue are also known.

[0004] WO2015 / 002969WO2017 / 085233

[0005] J Clin Oncol. 2002 January 1; 20(1) 142-152Saltzman D., et al., Surgery, 163: 509-514 (2018)Lee CH, et al., Mol. Ther., 11: 707-716 (2015)Kawaguchi K., et al., Cell Cycle, 17: 2019-2026 (2018)Jia LJ, et al., Int. J. Cancer, 121: 666-674 (2007)Chen J., et al., Theranostics, 7: 2250-2260 (2017)Miyake K., et al., Tissue Cell, 54: 144-149 (2018) Hiroshima Y. et al., PloS One., 10(6): e0120358 (2015) Mai Thi-Quynh Duong, et al., Experimental & Molecular Medicine 51:152 (2019)

[0006] An object of the present invention is to provide a novel drug having excellent anti-malignant tumor effects.

[0007] The present inventors have conducted extensive research into the above-mentioned problems and have found that (1) the intratumor distribution of liposomes is improved by allowing bacteria to colonize tumor tissue, and (2) the effectiveness of liposomes encapsulating anti-cancer agents is significantly improved by using them in combination with bacteria such as attenuated strains of Salmonella. In other words, the combination of liposomes encapsulating anti-cancer agents and bacteria provides a more significant anti-cancer effect than when either is used alone. Based on these findings, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention is as follows:

[0008] [1] A pharmaceutical comprising a combination of a liposome encapsulating an anti-cancer agent and a bacterium. [2] The pharmaceutical according to the above-mentioned [1], wherein the liposome encapsulating an anti-cancer agent and the bacterium are separately formulated and used in combination. [3] The pharmaceutical according to the above-mentioned [1] or [2], wherein the bacterium is selected from the group consisting of Salmonella Typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT. [4] The pharmaceutical according to the above-mentioned [1] or [2], wherein the bacterium is Salmonella Typhimurium. [5] The medicine according to the above-mentioned [1] or [2], wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1. [6] The medicine according to the above-mentioned [1] or [2], wherein the bacterium is VNP20009. [7] The medicine according to any of the above-mentioned [1] to [6], wherein the antineoplastic agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof. [8] The medicine according to any of the above-mentioned [1] to [6], wherein the antineoplastic agent is doxorubicin or a pharmaceutically acceptable salt thereof. [9] The medicament according to any one of the above-mentioned [1] to [6], wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

[10] The medicament according to any one of the above-mentioned [1] to [9], for treating a malignant tumor.

[11] The medicament according to any one of the above-mentioned [1] to

[10] , for treating a solid cancer.

[12] The pharmaceutical composition of the above-mentioned

[11] , wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[0009]

[13] A bacterium for administration simultaneously with or separately from liposomes.

[14] The bacterium according to

[13] above for use in treating a malignant tumor.

[15] The bacterium according to

[14] above, wherein the malignant tumor is a solid cancer.

[16] The bacterium according to

[15] above, wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, myosin, and skin cancer.

[17] The bacterium according to any one of

[13] to

[16] above, which is a bacterium selected from the group consisting of Salmonella typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT.

[18] The bacterium according to any one of

[13] to

[16] above, which is Salmonella typhimurium.

[19] The bacterium according to any one of

[13] to

[16] above, which is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[20] The bacterium according to any one of

[13] to

[16] above, which is VNP20009.

[21] The bacterium according to any one of

[13] to

[20] above, wherein the liposome encapsulates an anti-cancer agent.

[22] The bacterium according to the above-mentioned

[21] , wherein the anti-cancer agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof.

[23] The bacterium according to the above-mentioned

[21] , wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[24] The bacterium according to the above-mentioned

[21] , wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

[0010]

[25] A kit comprising a liposome encapsulating an anti-cancer agent and a preparation containing bacteria.

[26] The kit according to

[25] above, wherein the bacteria is selected from the group consisting of Salmonella typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT.

[27] The kit according to

[25] above, wherein the bacteria is Salmonella typhimurium.

[28] The kit according to

[25] above, wherein the bacteria is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[29] The kit according to

[25] above, wherein the bacteria is VNP20009.

[30] The kit according to any one of

[25] to

[29] above, wherein the anti-cancer agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof.

[31] The kit according to any one of

[25] to

[29] above, wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[32] The kit according to any one of

[25] to

[29] above, wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

[33] The kit according to any one of

[25] to

[32] above, for treating a malignant tumor.

[34] The kit according to any one of

[25] to

[32] above, for treating a solid tumor.

[35] The kit according to

[34] above, wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[0011]

[36] A method for treating malignant tumors in a subject, comprising the step of administering to the subject an effective amount of a combination of liposomes encapsulating an anti-cancer agent and bacteria.

[37] The method according to

[36] above, wherein the step of administering to the subject comprises separately formulating the liposomes encapsulating an anti-cancer agent and the bacteria, and administering the combined products to the subject.

[38] The method according to

[36] or

[37] above, wherein the bacteria is selected from the group consisting of Salmonella Typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT.

[39] The method according to

[36] or

[37] above, wherein the bacteria is Salmonella Typhimurium.

[40] The method according to

[36] or

[37] above, wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[41] The method according to

[36] or

[37] above, wherein the bacterium is VNP20009.

[42] The method according to any of

[36] to

[41] above, wherein the antineoplastic agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof.

[43] The method according to any one of the above-mentioned

[36] to

[41] , wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[44] The method according to any one of the above-mentioned

[36] to

[41] , wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

[45] The method according to any one of the above-mentioned

[36] to

[44] , wherein the malignant tumor in the subject is a solid cancer.

[46] The method of

[45] above, wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[0012]

[47] A combination of a liposome encapsulating an anti-cancer agent and a bacterium for use as a medicine.

[48] A combination of a liposome encapsulating an anti-cancer agent and a bacterium for use in the treatment of malignant tumors.

[49] The combination according to

[47] or

[48] above, wherein the liposome encapsulating an anti-cancer agent and the bacterium are separately formulated and used in combination.

[50] The combination according to any of

[47] to

[49] above, wherein the bacterium is selected from the group consisting of Salmonella Typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT.

[51] The combination according to any of

[47] to

[49] above, wherein the bacterium is Salmonella Typhimurium.

[52] The combination according to any of the above-mentioned

[47] to

[49] , wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[53] The combination according to any of the above-mentioned

[47] to

[49] , wherein the bacterium is VNP20009.

[54] The combination according to any of the above-mentioned

[47] to

[53] , wherein the antineoplastic agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof.

[55] The combination according to any one of the above-mentioned

[47] to

[53] , wherein the antineoplastic agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[56] The combination according to any one of the above-mentioned

[47] to

[53] , wherein the antineoplastic agent is irinotecan or a pharmaceutically acceptable salt thereof.

[57] The combination according to any one of the above-mentioned

[47] to

[56] , for use in the treatment of solid cancer.

[58] The combination according to

[57] above, wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, stomach cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[0013]

[59] Use of a combination of liposomes encapsulating an anti-cancer agent and bacteria in the manufacture of a medicament for treating malignant tumors.

[60] The use according to

[59] above, wherein the medicament is a combination of liposomes encapsulating an anti-cancer agent and bacteria, each formulated separately.

[61] The use according to

[59] or

[60] above, wherein the bacteria is selected from the group consisting of Salmonella Typhimurium, Listeria monocytogenes, Clostridium novyi, and Clostridium novyi-NT.

[62] The use according to

[59] or

[60] above, wherein the bacteria is Salmonella Typhimurium.

[63] The use according to

[59] or

[60] above, wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[64] The use according to

[59] or

[60] above, wherein the bacterium is VNP20009.

[65] The use according to any of

[59] to

[64] above, wherein the antineoplastic agent is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, eribulin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and topotecan or a pharmaceutically acceptable salt thereof.

[66] The use according to any one of the above-mentioned

[59] to

[64] , wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[67] The use according to any one of the above-mentioned

[59] to

[64] , wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

[68] The use according to any one of the above-mentioned

[59] to

[67] , wherein the medicament is a medicament for treating solid cancer.

[69] The use of the above-mentioned

[68] , wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cavity cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[0014]

[70] A pharmaceutical for treating malignant tumors, comprising bacteria, for administration simultaneously or separately with liposomes encapsulating an anti-cancer agent.

[71] A pharmaceutical for treating malignant tumors, comprising liposomes encapsulating an anti-cancer agent, for administration simultaneously or separately with bacteria.

[72] An enhancer for the anti-cancer effect of bacteria, comprising liposomes encapsulating an anti-cancer agent as an active ingredient.

[73] An enhancer for the anti-cancer effect of liposomes encapsulating an anti-cancer agent, comprising bacteria as an active ingredient.

[74] A pharmaceutical composition comprising a combination of liposomes encapsulating an anti-cancer agent and bacteria.

[75] The pharmaceutical composition according to

[74] , in which liposomes encapsulating an anti-cancer agent and bacteria are each formulated separately and then combined.

[76] The pharmaceutical composition according to

[74] or

[75] , wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[77] The pharmaceutical composition according to

[74] or

[75] , wherein the bacterium is VNP20009.

[78] The pharmaceutical composition according to any of

[74] to

[77] , wherein the antineoplastic agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[79] The pharmaceutical composition according to any of

[74] to

[77] , wherein the antineoplastic agent is irinotecan or a pharmaceutically acceptable salt thereof.

[80] The pharmaceutical composition according to any of

[74] to

[79] , for treating a malignant tumor.

[81] The pharmaceutical composition according to any of

[74] to

[79] , for treating a solid tumor.

[82] The pharmaceutical composition according to

[81] , wherein the solid cancer is selected from the group consisting of lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumor, brain tumor, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumor, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, myosarcoma, and skin cancer.

[83] The pharmaceutical composition according to any of

[74] to

[82] , which is in the form of a kit.

[84] A combination of a liposome encapsulating an anti-cancer agent and bacteria.

[85] The combination according to

[84] , wherein the bacterium is an attenuated strain of Salmonella typhimurium selected from the group consisting of VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[86] The combination according to

[84] , wherein the bacterium is VNP20009.

[87] The combination according to any of

[84] to

[86] , wherein the antineoplastic agent is doxorubicin or a pharmaceutically acceptable salt thereof.

[88] The combination according to any of

[84] to

[86] , wherein the antineoplastic agent is irinotecan or a pharmaceutically acceptable salt thereof.

[0015] The pharmaceutical composition of the present invention, which is characterized by combining a liposome encapsulating an anti-cancer agent with bacteria, has an excellent anti-cancer effect.

[0016] FIG. 1-1 shows the results of measuring the amount of liposome transferred to a U87MG tumor in Test Example 1. FIG. 1-2 shows the results of observing the distribution of liposomes in a U87MG tumor (confocal micrographs of tumor tissue sections) in Test Example 1. FIG. 2-1 shows the results of measuring the amount of liposome transferred to an A549 tumor in Test Example 1. FIG. 2-2 shows the results of observing the distribution of liposomes in an A549 tumor in Test Example 1 (confocal micrographs of tumor tissue sections). FIG. 3-1 shows the results of measuring the amount of liposome transferred to a BxPC3 tumor in Test Example 1. FIG. 3-2 shows the results of observing the distribution of liposomes in a BxPC3 tumor in Test Example 1 (confocal micrographs of tumor tissue sections). FIG. 4 shows the results of evaluating the antitumor effects on A549 tumors and BxPC3 tumors in Test Example 2. Figure 5 shows the evaluation results of the antitumor effect on BxPC3 tumors (comparative test after three administrations) in Test Example 3. Figure 6 shows the evaluation results of the antitumor effect on BxPC3 tumors (effect after one administration) in Test Example 3. Figure 7 shows the evaluation results of the antitumor effect on BxPC3 tumors in Test Example 4.

[0017] The present invention is described in detail below. The term "medicine" as used herein is not particularly limited as long as it is intended to be used for the diagnosis, treatment, or prevention of a disease in a subject, and includes, for example, diagnostic agents, therapeutic agents, preventive agents, pharmaceutical compositions, kits, devices, and their uses, diagnostic methods, therapeutic methods, and preventive methods. The medicament of the present invention uses a combination of liposomes encapsulating an anti-cancer agent and bacteria. In the medicament of the present invention, the liposomes encapsulating an anti-cancer agent and the bacteria may be formulated simultaneously and contained in the same formulation, or the liposomes encapsulating an anti-cancer agent and the bacteria may be formulated separately and administered simultaneously or at different times to the same subject by the same or different routes. That is, the medicament of the present invention includes (1) a medicament containing liposomes encapsulating an anti-cancer agent and bacteria in a single formulation, and (2) a medicament in which the liposomes encapsulating an anti-cancer agent and the bacteria are separately formulated and used in combination.

[0018] The pharmaceutical of the present invention can be formulated by combining liposomes encapsulating an anti-cancer agent and bacteria in the same formulation, but it is preferable to formulate them separately and use them in combination. This pharmaceutical is used for the treatment of malignant tumors, particularly solid tumors. The bacteria of the present invention selectively colonize and grow in tumor tissue, improving the accumulation and distribution of administered liposomes in tumor tissue. That is, when liposomes encapsulating an anti-cancer agent and bacteria are used in combination in the present invention, the anti-cancer agent encapsulated in the liposomes can be distributed more widely within tumor tissue than when the liposomes are used alone, thereby improving the accumulation of the anti-cancer agent in tumor tissue (i.e., increasing the concentration of the anti-cancer agent in tumor tissue). The bacteria of the present invention are preferably used simultaneously or separately with liposomes encapsulating an anti-cancer agent, and are used for the treatment of malignant tumors, particularly solid tumors. The kit of the present invention is preferably one in which the liposomes encapsulating the anti-cancer agent and the bacteria are each formulated and ready for use, and is used for the treatment of malignant tumors, particularly solid cancers.

[0019] In the present invention, the term "bacteria" is not particularly limited, but examples thereof include Salmonella bacteria (e.g., Salmonella typhimurium), Listeria monocytogenes, Clostridium novyi, or its mutant Clostridium novyi-NT, and attenuated strains thereof. For the purposes of the present invention, the above-mentioned attenuated strains are preferred, and among these, attenuated strains of Salmonella bacteria (particularly Salmonella typhimurium) are more preferred. Specific examples of attenuated strains of Salmonella bacteria include attenuated strains of Salmonella typhimurium, such as VNP20009, A1-R, SHJ2037, SL3261, SL7207, BRD509, and YB1.

[0020] In the present invention, "Salmonella" refers to Gram-negative, facultative anaerobic bacilli belonging to the genus Salmonella of the family Enterobacteriaceae that primarily inhabit the digestive tracts of humans and animals. Among these, S. typhimurium is a type of Salmonella and is included in the Gram-negative, facultative anaerobic bacilli / non-typhoidal Salmonella. The Salmonella used in the present invention is preferably an attenuated strain of Salmonella, such as S. typhimurium DSLpNG, S. typhimurium A1-R, S. typhimurium VNP20009, S. typhimurium SHJ2037, or S. typhimurium SL3261. S. typhimurium VNP20009, an attenuated strain with purI and msbB mutations and an adenine-requiring / LPS mutation, is preferred. In the present invention, commercially available "Salmonella bacteria" can also be used. In this specification, Salmonella bacteria may be simply referred to as Salmonella.

[0021] In the present invention, the bacteria can be used alone or in a formulation prepared by a known method together with commonly used pharmaceutically acceptable additives (e.g., solvents, stabilizers, isotonicity agents, soothing agents, buffers, pH adjusters). For example, in the case of an aqueous solution formulation containing an isotonicity agent such as sugar or sodium chloride, the bacteria can be used in a concentration of about 10 to about 10 per ml. １０ cfu, preferably about 10 per ml ３ ~about 10 ９cfu, more preferably about 10 per ml ５ ~about 10 ７ The bacteria may be contained in an amount of 0.01 cfu. In the present invention, the bacteria are preferably used as an injection, more preferably as an intravenous injection. In this specification, the term "injection" also includes those administered by infusion, local perfusion, catheter, etc.

[0022] The "liposome" in the "liposome encapsulating an anti-cancer agent" used in the present invention is not particularly limited, and liposomes known in the field of pharmaceutical formulations can be used. As used herein, a liposome refers to a lipid bilayer vesicle with an aqueous interior. Examples of liposomes include multilamellar liposomes, in which multiple lipid bilayers are stacked in an onion-like pattern, and unilamellar liposomes. The lipid constituting the liposome is typically a phospholipid. Examples of phospholipids include acidic phospholipids such as phosphatidylcholines (e.g., lecithin and lysolecithin), phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid, as well as phospholipids in which the acyl groups of these phospholipids are substituted with lauroyl, myristoyl, or oleoyl groups, phosphatidylethanolamine, sphingophospholipids (e.g., sphingomyelin), glyceroglycolipids, and cationic lipids. Lipid derivatives in which a water-soluble polymer (e.g., polyethylene glycol) is bound to the lipid may also be used in combination. More specifically, examples of such phospholipids include hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Phospholipids obtained by binding these phospholipids with polyethylene glycol (PEG), such as N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, can also be used. Cholesterol and the like can also be added. Liposomes encapsulating anti-cancer agents can be produced, for example, by suspending a thin film of purified phospholipids in a solution containing the anti-cancer agent and subjecting the suspension to ultrasonic treatment or the like. Methods for producing liposome preparations encapsulating anti-cancer agents are well known in the art. For details of such production methods, see, for example, Annals of Oncology, vol. 15, pp. 517-525, 2004; Cancer Science, vol. 95, pp. 608-613, 2004, etc.

[0023] The "anti-cancer agent" in the "liposomes encapsulating an anti-cancer agent" used in the present invention is not particularly limited, and examples thereof include doxorubicin, irinotecan, eribulin, gemcitabine, topotecan, or pharmaceutically acceptable salts thereof, with doxorubicin or a pharmaceutically acceptable salt thereof and irinotecan or a pharmaceutically acceptable salt thereof being preferred. Nucleic acid pharmaceuticals such as antisense oligonucleotides, siRNA, miRNA, CpG oligos, and aptamers can also be used if they are intended for the treatment of malignant tumors. The "liposomes encapsulating an anti-cancer agent" used in the present invention are not particularly limited, as long as they are in a form in which an anti-cancer agent is encapsulated in liposomes as an active ingredient. For example, they can be produced by incorporating the above-mentioned anti-cancer agent into liposomes using the known methods listed above. Commercially available products such as Doxil (registered trademark), Onivyde (registered trademark), and Halaven (registered trademark) can also be used.

[0024] In the present invention, the liposome preparation and pharmaceutical composition encapsulating the anti-cancer agent may contain commonly used pharmaceutically acceptable additives (e.g., solvents, stabilizers, isotonicity agents, soothing agents, buffers, pH adjusters). In the present invention, the liposome preparation and pharmaceutical composition encapsulating the anti-cancer agent are preferably in the form of, for example, an injection. When the liposome preparation encapsulating the anti-cancer agent in the present invention is a liquid preparation such as an injection, it may be stored by freezing or lyophilization to remove water. When using a lyophilized preparation, it is redissolved in, for example, distilled water for injection.

[0025] In the pharmaceutical of the present invention, the method of administration of the liposomes encapsulating the anti-cancer agent and the bacteria is not limited as long as it is a method that can deliver the liposomes encapsulating the anti-cancer agent and the bacteria to the affected area or its surroundings. Examples include oral administration or parenteral administration using injections or drip infusions. Due to the nature of each formulation, parenteral administration is preferred, and specific examples include intravenous administration, intraarterial administration, intramucosal administration, intralymph node administration, and intra-affected tissue administration, with intravenous administration being preferred.

[0026] In the pharmaceutical of the present invention, the dosage of the liposomes encapsulating the anti-cancer agent and the bacteria can be appropriately determined depending on the route of administration, the severity of symptoms, the age of the patient, the degree of side effects, etc. In the pharmaceutical of the present invention, the dosage of the liposomes encapsulating the anti-cancer agent varies depending on the type of anti-cancer agent and the type of liposome used, the type of malignant tumor to be treated, the condition of the patient, etc., but the dosage of commercially available liposomes is usually used as a reference, and for example, for an adult (body weight 60 kg), it is 1 mg / m per day. ２ ~2000 mg / m ２ , preferably 5 mg / m ２ ~1000mg / m ２ , more preferably 10 mg / m ２ ~500 mg / m ２ The amount of bacteria used varies depending on the type of bacteria used, the type of malignant tumor to be treated, the condition of the patient, etc., but for an adult (weight 60 kg), for example, it is usually about 10 to 10 １０ cfu / kg, preferably about 10 ２ ~10 ８ cfu / kg, more preferably about 10 ３ ~10 ７ cfu / kg. These may be administered in one dose or in several doses.

[0027] The pharmaceutical agent of the present invention can be safely administered to humans and non-human mammals (for example, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, pigs, etc.).

[0028] The pharmaceutical of the present invention is useful as a pharmaceutical for treating malignant tumors. The pharmaceutical of the present invention is particularly effective against solid cancers, among other malignant tumors. More specifically, solid cancers include lung cancer, pancreatic cancer, glioblastoma, ovarian cancer, Kaposi's sarcoma, multiple myeloma, breast cancer, osteosarcoma, esophageal cancer, liver cancer, gastric cancer, pancreatic cancer, colorectal cancer, rectal cancer, colon cancer, ureteral tumors, brain tumors, gallbladder cancer, bile duct cancer, biliary tract cancer, kidney cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, testicular tumors, maxillary cancer, tongue cancer, lip cancer, oral cancer, pharyngeal cancer, laryngeal cancer, sarcoma, and skin cancer, as well as tumors that have recurred or worsened after any other treatment, such as chemotherapy or radiation therapy. The kit of the present invention may be used as long as the liposomes encapsulating the anti-cancer agent and the bacteria are each formulated and available. Examples of such pharmaceutical compositions include those prepared as separate packs containing a bacteria-containing formulation and liposomes encapsulating an anti-cancer agent; those prepared so that the bacteria-containing formulation and liposomes encapsulating an anti-cancer agent can be administered simultaneously or separately via the same or separate tubes; and those in which liposomes encapsulating an anti-cancer agent and bacteria are formulated in the same pack or the like. The pharmaceutical composition of the present invention is not particularly limited as long as it contains liposomes encapsulating an anti-cancer agent and bacteria. For example, the pharmaceutical composition of the present invention may be formulated or kitized to contain each of the two, or each may be formulated separately. Furthermore, the pharmaceutical composition of the present invention is used for medicinal purposes such as the treatment, prevention, and diagnosis of diseases in subjects. Specific examples include those listed above in the description of pharmaceuticals.

[0029] The present invention will be described in more detail below based on examples and test examples, but the present invention is not limited to these.

[0030] Test Example 1: Amount of liposome transferred to tumors / intratumoral distribution after administration of VNP20009 Improvements in the amount of liposome transferred to various tumors / intratumoral distribution after administration of VNP20009 were examined. Human glioblastoma-derived U87MG cells, human lung cancer-derived A549 cells, and human pancreatic cancer-derived BxPC3 cells were used as tumor cells.

[0031] 1. Bacterial Cultivation and Bacterial Count Bacterial cultivation was performed using the following bacteria and medium according to the following procedure, and the resulting bacteria were used as the administration sample for the following test. (Bacteria) Attenuated strain VNP20009 of Salmonella Typhimurium (mouse typhi bacteria) (obtained from ATCC) (Culture medium (common to liquid / agar)) Modified-LB (ingredients: 1% tryptone, 0.5% yeast extract, 0.002N CaCl2, 0.002N MgSO4; pH: 7.0) (Procedure) (1) 10 μL of bacteria stored frozen at -80°C in 20% glycerol was added to 4 mL of modified-LB medium. (2) The culture was incubated overnight at 37°C with shaking (130 rpm). (3) The culture medium was diluted 1 / 20 with modified LB medium and cultured again for 2-3 hours at 37°C with shaking (130 rpm). (4) When the optical density (OD600), which indicates turbidity as an indicator of bacterial mass, reached 0.5-0.7, 10 mL of culture medium was collected. (5) The culture medium was centrifuged (8000 g, 3 min), and the resulting bacterial pellet was resuspended in 10 mL of physiological saline. (6) The procedure in (5) was repeated. (7) The culture medium was centrifuged (8000 g, 3 min), and the resulting pellet was resuspended in 10 mL of physiological saline. (8) The sample was diluted with physiological saline as needed to prepare the administration sample.

[0032] (Counting of bacterial quantity) In the following tests, CFU (colony forming unit) was determined by counting the number of colonies formed after spreading 100 μL of bacterial suspension diluted as needed on a modified LB agar medium and incubating overnight at 37°C. (Number of live bacteria present in the spread suspension = number of colonies formed)

[0033] 2. Creation of a tumor-bearing mouse model. Human glioblastoma-derived U87MG cells, human lung cancer-derived A549 cells, and human pancreatic cancer-derived BxPC3 cells were used as tumor cells to create tumor-bearing mouse models. Immunodeficient mice (BALB / cSlc nu / nu) lacking thymic function were used. 4 x 10 ６80 μL of each tumor cell suspension was injected using a syringe with a 27G needle. The long and short diameters of the tumor were measured using a digital caliper, and the tumor volume was calculated using the formula: (long diameter (mm) x short diameter (mm) x short diameter (mm)) / 2. ３ Once this level was reached, it was used in the following tests.

[0034] 3. Preparation of Fluorescently Labeled Liposomes Liposomes (no anti-cancer drug encapsulated) (particle diameter: approximately 100 nm) in which the lipid membrane was labeled with the carbocyanine dye DiD were prepared using the ingredients listed below and by the preparation method listed below, and these were used as administration samples for the following tests. (Ingredients and Ratio) DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine (NOF Corporation) Cholesterol (Nacalai Tesque, Inc.) mPEG(2000)-DSPE: N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphorylethanolamine, sodium salt (Laysan Bio, Inc.) Ratio: DOPC / Cholesterol / mPEG(2000)-DSPE = 60 / 40 / 5 mol% DiD 5 mol% labeling (Preparation Method) 1.2 mL of 20 mM HEPES buffer was added to a 15 mL conical tube (Falcon / 352095) (Solution 1). An ethanol solution containing 20 mM DOPC, 20 mM cholesterol, and 10 mM mPEG(2000)-DSPE was added to a 5 mL tube at a molar ratio of 60 / 40 / 5. 250 μL of a 1 mM DiD ethanol solution was added, and the total volume was adjusted to 1 mL with isopropyl alcohol (Solution 2). While stirring Solution 1 with a vortex mixer, the entire volume of Solution 2 was added. After stirring for 30 seconds, 1 mL of PBS (-) was added dropwise. The entire volume was transferred to an AmiconUltra-15 (Millipore, MWCO 100,000) and subjected to ultrafiltration to remove the ethanol and isopropyl alcohol, exchange the buffer, and concentrate. Finally, the appropriate volume was adjusted to the appropriate volume with PBS (-). The particle size, polydispersity index, and electrical potential of the liposomes were measured using a Zetasizer nano ZSP.

[0035] 4. Administration Method Fluorescently labeled liposomes and VNP20009 were administered to the U87MG cell, A549 cell, and BxPC3 cell cancer model mice prepared above, respectively, using the administration method described below. (Administration groups) (i) A group administered with fluorescently labeled liposomes but not VNP20009 (control group, hereinafter referred to as the "VNP20009(-) group"). (ii) A group administered with VNP20009, followed immediately by fluorescently labeled liposomes (hereinafter referred to as the "VNP20009(+) DAY0 group"). (iii) A group administered with VNP20009, followed three days later by fluorescently labeled liposomes (hereinafter referred to as the "VNP20009(+) DAY3 group"). (Administration method, evaluation schedule) (i) VNP20009(-) group Fluorescently labeled liposomes (approximately 1 μmol / mouse in terms of lipid amount) were administered into the tail vein of cancer-bearing model mice under isoflurane anesthesia using a 29G syringe with a needle. One day after administration of the fluorescently labeled liposomes, tumor tissues were collected from the cancer-bearing model mice. (ii) VNP20009(+) DAY0 group: Under isoflurane anesthesia, tumor-bearing model mice were injected with VNP20009 (2-4x10) using a syringe with a 29G needle. ６ Immediately thereafter (within 1-2 minutes), fluorescently labeled liposomes (approximately 1 μmol / mouse as lipid amount) were administered into the tail vein using a syringe with a 29G needle. One day after administration of the fluorescently labeled liposomes, tumor tissues were collected from the cancer-bearing model mice. (iii) VNP20009(+) DAY3 group: Under isoflurane anesthesia, VNP20009 (2-4 x 10 CFU / mouse) was administered into the tail vein using a syringe with a 29G needle. ６ Three days later, fluorescently labeled liposomes (approximately 1 μmol / mouse as lipid amount) were administered into the tail vein using a syringe with a 29G needle. One day after administration of the fluorescently labeled liposomes, tumor tissues were collected from the cancer-bearing model mice.

[0036] 5. Evaluation Method 5-1. Measurement of the Amount of Fluorescently Labeled Liposome Translocated into Tumors (1) Tumor tissue collected from mice was diluted 10-fold with physiological saline and crushed to prepare a tumor tissue suspension. (2) 500 μL of the tumor tissue suspension was mixed with 500 μL of 2% SDS (sodium dodecyl sulfate) solution to dissolve the tissue. (3) After centrifugation (4°C, 10,000 g, 10 minutes), 600 μL of the supernatant was collected and used as the measurement sample. (4) A similar procedure was performed using tumor tissue collected from a cancer-bearing mouse model that had not been administered with fluorescently labeled liposomes and an arbitrary amount of fluorescently labeled liposomes to prepare a calibration curve sample for fluorescence intensity. (5) The fluorescence intensity of the calibration curve sample and the measurement sample was measured, and the amount of fluorescently labeled liposome in the tumor tissue was calculated. The measurement results of the amount of fluorescently labeled liposome transferred to U87MG tumors are shown in FIG. 1-1, the measurement results of the amount of fluorescently labeled liposome transferred to A549 tumors are shown in FIG. 2-1, and the measurement results of the amount of fluorescently labeled liposome transferred to BxPC3 tumors are shown in FIG. 3-1.

[0037] 5-2. Observation of Intratumoral Distribution of Fluorescently Labeled Liposomes (1) Tumor tissues collected from mice were placed in 4% PFA (paraformaldehyde) and immersed and fixed overnight at 4°C. (2) The fixed tumor tissues were sliced ​​to a thickness of 300 μm. (3) Cell nuclei were stained using Hoechst 33342 (shown in blue in the image). (4) Cell nuclei (shown in blue) and liposome fluorescence (shown in red) were observed using a confocal microscope. The results of the observation of the intratumoral distribution of fluorescently labeled liposomes in U87MG tumors are shown in Figure 1-2, the results of the intratumoral distribution in A549 tumors are shown in Figure 2-2, and the results of the intratumoral distribution in BxPC3 tumors are shown in Figure 3-2. Figures 1-2, 2-2, and 3-2 are originally color photographs, with light gray areas originally red and dark gray areas originally blue.

[0038] Observation of the intratumoral distribution of fluorescently labeled liposomes in U87MG tumors (Figure 1-2) showed that in the VNP20009(-) group (left photo in Figure 1-2), liposome fluorescence (shown in red) was distributed only to the outer edge of the tissue, whereas in the VNP20009(+) DAY 0 group (center photo in Figure 1-2) and the VNP20009(+) DAY 3 group (right photo in Figure 1-2), liposome fluorescence (shown in red) spread to the interior of the tissue, demonstrating an expansion of the intratumoral distribution of liposomes. Furthermore, the necrotic area (area surrounded by the dotted line) expanded in the VNP20009(+) DAY 3 group (right photo in Figure 1-2).

[0039] Measurement of the amount of fluorescently labeled liposomes transferred to A549 tumors (Figure 2-1) showed a significantly increased amount of liposomes transferred to tumors in the VNP20009(+) DAY 0 group compared to the VNP20009(-) group. Observation of the distribution of fluorescently labeled liposomes within A549 tumors (Figure 2-2) showed that in the VNP20009(-) group (left photo in Figure 2-2), liposome fluorescence (shown in red) was distributed only to the outer edge of the tissue, whereas in the VNP20009(+) DAY 0 group (center photo in Figure 2-2), liposome fluorescence (shown in red) spread throughout the tissue, indicating an expanded distribution area. Furthermore, scattered areas of necrosis (areas surrounded by dotted lines) were observed in the VNP20009(+) DAY 3 group (right photo in Figure 2-2).

[0040] Measurement of the amount of fluorescently labeled liposomes transferred to BxPC3 tumors (Figure 3-1) showed that the amount of liposomes transferred to tumors was significantly higher in the VNP20009(+) DAY 0 group compared to the VNP20009(-) group (*p<0.05, non-repeated ANOVA followed by SNK test). Observation of the distribution of fluorescently labeled liposomes within BxPC3 tumors (Figure 3-2) showed that in the VNP20009(-) group (left photo in Figure 3-2), there were scattered areas where the liposome fluorescence (shown in red) was poorly distributed within the tissue, whereas in the VNP20009(+) DAY 0 group (center photo in Figure 3-2) and the VNP20009(+) DAY 3 group (right photo in Figure 3-2), the liposome fluorescence (shown in red) was distributed throughout the entire tissue, indicating an expansion of the distribution area. In addition, in the VNP20009(+) DAY3 group (right photograph in FIG. 3-2), necrotic areas (areas surrounded by dotted lines) were observed.

[0041] The above results confirmed that administration of Salmonella (VNP20009) promotes the accumulation of liposomes in tumor tissue, that co-administration of Salmonella (VNP20009) and liposomes (particularly by administering liposomes immediately (substantially simultaneously) with Salmonella (VNP20009) administration) increases the amount of liposomes transferred to tumor tissue, and that administration of liposomes three days after Salmonella (VNP20009) administration can increase the distribution of liposomes in tumor tissue. Furthermore, the above results confirmed that co-administration of Salmonella (VNP20009) and liposomes (particularly by administering liposomes immediately after Salmonella (VNP20009) administration or by administering liposomes at a later time after Salmonella (VNP20009) administration) can expand the distribution area of ​​liposomes in tumor tissue.

[0042] Test Example 2 Antitumor Effect of Combined Use of Liposomes Encapsulating an Anticancer Agent and VNP20009 The antitumor effect of combined use of VNP20009 and liposomes encapsulating an anticancer agent was evaluated against A549 tumor and BxPC3 tumor, where the amount of liposomes transported into tumors was increased by combined administration with VNP20009 in Test Example 1.

[0043] 1. Cultivation of bacteria and counting of bacterial amount Bacteria (VNP20009 (obtained from ATCC)) were cultured in the same manner as in Test Example 1, and the obtained bacteria were used as the administration sample for the following tests.

[0044] 2. Preparation of Cancer-Bearing Model Mice Cancer-bearing model mice were prepared in the same manner as in Test Example 1, using human lung cancer-derived A549 cells and human pancreatic cancer-derived BxPC3 cells as tumor cells.

[0045] 3. Liposomes Encapsulating Antitumor Agents As liposomes encapsulating an antitumor agent, doxorubicin-encapsulated liposomes (Doxil (registered trademark) injection 20 mg) were used in the following tests.

[0046] 4. Administration Method The tumor-bearing mice using A549 cells and the tumor-bearing mice using BxPC3 cells prepared above were administered with the following administration methods: (a) a combination of doxorubicin-encapsulated liposomes and VNP20009 (combined administration group), (b) doxorubicin-encapsulated liposomes alone (liposome administration group), and (c) VNP20009 alone (VNP20009 administration group). (d) A control group was administered with no administration. (Administration Method) (a) Combination Administration Group Under isoflurane anesthesia, tumor-bearing model mice were administered VNP20009 (approximately 5 x 10 ５ (CFU / mouse) was administered into the tail vein, and immediately thereafter (within 1-2 minutes), doxorubicin-encapsulated liposomes (5 mg doxorubicin / kg body weight) were administered into the tail vein using a syringe with a 29G needle. 7 and 14 days after administration, VNP20009 and doxorubicin-encapsulated liposomes were administered in the same manner. (b) Liposome-administered group: Under isoflurane anesthesia, tumor-bearing model mice were administered doxorubicin-encapsulated liposomes (5 mg doxorubicin / kg body weight) into the tail vein using a syringe with a 29G needle. 7 and 14 days after administration, doxorubicin-encapsulated liposomes were administered in the same manner. (c) VNP20009 administration group. Under isoflurane anesthesia, tumor-bearing model mice were inoculated with VNP20009 (approximately 5 × 10５ CFU / mouse) was administered into the tail vein. Seven and 14 days after administration, VNP20009 was administered in the same manner.

[0047] 5. Evaluation Method From the day of the start of administration until day 18, the major and minor diameters of the tumor were measured once a day or once every two days using digital calipers, and the tumor volume was calculated using the formula: (major diameter (mm) x minor diameter (mm) x minor diameter (mm)) / 2. The results are shown in Figure 4.

[0048] The results in Figure 4 confirmed that combined administration of doxorubicin-encapsulated liposomes and VNP20009 ("VNP20009 + doxorubicin-encapsulated liposomes" in Figure 4) resulted in a stronger antitumor effect (effect of suppressing tumor volume increase) than either administration alone ("VNP20009" or "doxorubicin-encapsulated liposomes" in Figure 4). Furthermore, combined administration of doxorubicin-encapsulated liposomes and VNP20009 ("VNP20009 + doxorubicin-encapsulated liposomes" in Figure 4) was shown to exert an antitumor effect even at a dose at which VNP20009 alone was only ineffective. These effects of combined administration of doxorubicin-encapsulated liposomes and VNP20009 were not expected from the effects of either administration alone. Therefore, the combined use of liposomes encapsulating anti-cancer drugs and bacteria such as VNP20009 is expected to have a strong anti-tumor effect at a low dose, suggesting that it may be an effective treatment method even for cancer types that are generally difficult to treat.

[0049] Test Example 3 Antitumor Effect of a Combination of Liposomes Encapsulating an Antitumor Agent and VNP20009 (Comparative Test of a Combination of an Antitumor Agent and VNP20009) The antitumor effect of a combination of liposomes encapsulating an antitumor agent and VNP20009 was evaluated against BxPC3 tumors. For comparison, the antitumor effect of a combination of an antitumor agent and VNP20009 was examined.

[0050] 1. Cultivation of bacteria and counting of bacterial amount Bacteria (VNP20009 (obtained from ATCC)) were cultured in the same manner as in Test Example 1, and the obtained bacteria were used as the administration sample for the following tests.

[0051] 2. Preparation of Cancer-Bearing Model Mice Cancer-bearing model mice were prepared in the same manner as in Test Example 1, using BxPC3 cells derived from human pancreatic cancer as tumor cells.

[0052] 3. Liposomes Encapsulating Antitumor Agents, and Antitumor Agents As liposomes encapsulating antitumor agents, doxorubicin-encapsulated liposomes (Doxil (registered trademark) Injection 20 mg) were used in the following tests. As the antitumor agent, doxorubicin hydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.) was used in the following tests.

[0053] 4. Administration Method The tumor-bearing mice prepared above using BxPC3 cells were administered (a) a combination of doxorubicin-encapsulated liposomes and VNP20009 (liposome combination administration group), and (b) a combination of doxorubicin hydrochloride and VNP20009 (doxorubicin combination administration group) using the following administration methods. (c) A control group was administered nothing. (a) The combination administration of doxorubicin-encapsulated liposomes and VNP20009 was investigated not only three times (administration on days 0, 7, and 14) but also once (administration on day 0 only).

[0054] (Administration method) (1) Three administrations (administration on days 0, 7, and 14) (a) Liposome combination administration group VNP20009 (approximately 5 x 10 ５ CFU / mouse) was administered into the tail vein, and immediately thereafter (within 1 to 2 minutes), doxorubicin-encapsulated liposomes (doxorubicin amount 5 mg / kg body weight) were administered into the tail vein using a syringe with a 29G needle. 7 and 14 days after administration, VNP20009 and doxorubicin-encapsulated liposomes were administered in the same manner. (b) Doxorubicin combination administration group: Under isoflurane anesthesia, VNP20009 (approximately 5 x 10 ５CFU / mouse) was administered into the tail vein, and immediately thereafter (within 1 to 2 minutes), doxorubicin hydrochloride (5 mg / kg body weight) was administered into the tail vein using a syringe with a 29G needle. 7 and 14 days after administration, doxorubicin hydrochloride was administered in the same manner. (2) Single administration (administered only on day 0) (a) Liposome-coated group VNP20009 (approximately 5 x 10 CFU / mouse) was administered into the tail vein using a syringe with a 29G needle under isoflurane anesthesia. ５ CFU / mouse) was administered into the tail vein, and immediately thereafter (within 1 to 2 minutes), doxorubicin-encapsulated liposomes (doxorubicin amount: 5 mg / kg body weight) was administered into the tail vein using a syringe with a 29G needle.

[0055] 5. Evaluation Method From the day of administration start to the 18th day, the long and short diameters of the tumor were measured every two days using digital calipers, and the tumor volume was calculated using the formula: (long diameter (mm) x short diameter (mm) x short diameter (mm)) / 2. Figure 5 shows the results of three administrations (administered on days 0, 7, and 14): (a) the liposome-combined administration group ("VNP20009 + doxorubicin-encapsulated liposome" in Figure 5), (b) the doxorubicin-combined administration group ("VNP20009 + doxorubicin" in Figure 5), and (c) the control group ("Control" in Figure 5). Figure 6 shows the results of a single administration (administered only on day 0) of (a) the liposome-combined administration group ("VNP20009 + doxorubicin-encapsulated liposome x 1" in Figure 6). FIG. 6 also shows the results of three administrations (administrations on days 0, 7, and 14) shown in FIG. 5 for (a) the liposome-coadministered group (in FIG. 6, "VNP20009 + doxorubicin-encapsulated liposomes × 3") and (c) the control group (in FIG. 6, "Control").

[0056] The results in Figure 5 confirmed that combined administration of doxorubicin-encapsulated liposomes and VNP20009 provided a stronger antitumor effect (effect of suppressing an increase in tumor volume) than combined administration of doxorubicin hydrochloride and VNP20009. The results in Figure 6 confirmed that combined administration of doxorubicin-encapsulated liposomes and VNP20009, with a single administration (administration only on day 0), provided a stronger antitumor effect (effect of suppressing an increase in tumor volume) than combined administration of doxorubicin-encapsulated liposomes and VNP20009 with a single administration (administration on day 0, day 7, and day 14).

[0057] Test Example 4 Antitumor Effect of Combined Use of VNP20009 and Liposomes Encapsulating an Antitumor Agent The antitumor effect of combined use of VNP20009 and liposomes encapsulating an antitumor agent was evaluated against BxPC3 tumors.

[0058] 1. Cultivation of bacteria and counting of bacterial amount Bacteria (VNP20009 (obtained from ATCC)) were cultured in the same manner as in Test Example 1, and the obtained bacteria were used as the administration sample for the following tests.

[0059] 2. Preparation of a Cancer-Bearing Model Mouse A cancer-bearing model mouse was prepared using human pancreatic cancer-derived BxPC3 cells as tumor cells in the same manner as in Test Example 1. ３ Once this level was reached, it was used in the following tests.

[0060] 3. Liposomes Encapsulating Antitumor Agents Irinotecan-encapsulated liposomes (Onivyde (registered trademark) intravenous drip infusion 43 mg) were used as liposomes encapsulating antitumor agents in the following tests.

[0061] 4. Administration Method The tumor-bearing mice using BxPC3 cells prepared as described above were administered with (a) a combination of irinotecan-encapsulated liposomes and VNP20009 (combined administration group), and (b) irinotecan-encapsulated liposomes alone (liposome administration group) using the following administration methods. (Administration Method) (a) Combination Administration Group Under isoflurane anesthesia, tumor-bearing model mice were administered VNP20009 (2.8 x 10 ５Immediately thereafter (within 1-2 minutes), irinotecan-encapsulated liposomes (irinotecan dose: 20 mg / kg body weight) were administered into the tail vein using a syringe with a 29G needle (n=3). (b) Liposome administration group: Cancer-bearing model mice were anesthetized with isoflurane and administered irinotecan-encapsulated liposomes (irinotecan dose: 20 mg / kg body weight) into the tail vein using a syringe with a 29G needle (n=3).

[0062] 5. Evaluation Method The major and minor diameters of the tumor were measured using digital calipers on the day of administration (day 0), and on days 2, 6, 9, 12, and 15 after administration, and the tumor volume was calculated using the formula: (major diameter (mm) x minor diameter (mm) x minor diameter (mm)) / 2. The tumor growth rate (%) was calculated, assuming the value on the day of administration (day 0) as 100. Body weight was also measured. The results are shown in Figure 7.

[0063] The results in Figure 7 confirm that combined administration of irinotecan-encapsulated liposomes and VNP20009 ("VNP + Onivyde (trade name)" in Figure 7) resulted in a stronger antitumor effect than administration of irinotecan-encapsulated liposomes alone ("Onivyde (trade name)" in Figure 7) (left panel of Figure 7). No weight loss was observed in either group (right panel of Figure 7). Therefore, the combined use of liposomes encapsulating an anti-cancer agent and bacteria such as VNP20009 is expected to have a strong antitumor effect, suggesting that it may be an effective treatment method even for cancer types that are generally difficult to treat.

[0064] [Preparation Example 1] The following (1) and (2) are mixed to prepare an injection for anti-cancer treatment containing (1) and (2): (1) 10 mL (2 x 10 ６ CFU) (2) Doxorubicin-encapsulated liposome (Doxil (registered trademark) injection 20 mg) 10 mL

[0065] According to the present invention, a novel drug having excellent anti-malignant tumor effects can be provided.

[0066] This application is based on patent application No. 2022-047596 filed in Japan, the contents of which are incorporated in their entirety herein.

Claims

1. A pharmaceutical product characterized by combining liposomes that encapsulate an anti-cancer agent with VNP20009.

2. The pharmaceutical product according to claim 1, wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical product according to claim 1, wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical product according to any one of claims 1 to 3, for treating solid tumors.

5. A pharmaceutical composition containing a combination of liposomes encapsulating an anti-cancer agent and VNP20009.

6. The pharmaceutical composition according to claim 5, wherein the anti-cancer agent is doxorubicin or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition according to claim 5, wherein the anti-cancer agent is irinotecan or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition according to any one of claims 5 to 7 for treating solid tumors.