Antitumor agent, side effect suppressant, and antitumor combination drug

Abstract

This antitumor agent is for use in combination with a cytokine inhibitor and contains purple non-sulfur bacteria and bacteria of a type different from that of the purple non-sulfur bacteria.

Description

Antitumor agents, side effect suppressants, and combination antitumor drugs

[0001] This invention relates to an antitumor agent, a side effect suppressant, and a combination antitumor drug.

[0002] Cancer-targeted therapy utilizing anaerobic microorganisms that selectively accumulate, grow, and proliferate within hypoxic tumor wounds is attracting attention. For example, Patent Documents 1 and 2 disclose antitumor agents containing bacteria of the genus Rhodopseudomonas and Proteus isolated from tumors. As shown in Patent Documents 1 and 2, these antitumor agents have extremely low toxicity and are useful for cancer treatment.

[0003] International Publication No. 2023 / 223869, International Publication No. 2023 / 223870

[0004] According to the above-mentioned Patent Documents 1 and 2, when the above-mentioned antitumor agent was administered to animals, a normal weight gain was observed, but a slight weight loss was seen the day after administration. Considering the possibility of high-dose use in clinical settings, it is important to examine even minor findings and take countermeasures.

[0005] This invention has been made in view of the above circumstances, and aims to provide an antitumor agent, a side effect suppressant, and an antitumor combination drug that can suppress side effects.

[0006] An antitumor agent according to the first aspect of the present invention comprises purple nonsulfur bacteria and bacteria of a different species from the purple nonsulfur bacteria, and is used in combination with a cytokine inhibitor.

[0007] The cytokine inhibitor may include at least one selected from the group consisting of interleukin-6 inhibitors, interleukin-6 receptor inhibitors, tumor necrosis factor-α inhibitors, interferon-γ inhibitors, and interleukin-1β inhibitors.

[0008] The cytokine inhibitor may include an interleukin-6 inhibitor, an interleukin-6 receptor inhibitor, a tumor necrosis factor-α inhibitor, and an interferon-γ inhibitor.

[0009] The antitumor agent according to the first aspect of the present invention described above may be used in combination with an anti-inflammatory agent.

[0010] The aforementioned anti-inflammatory agent may be a steroid.

[0011] The purple non-sulfur bacteria may be of the genus Rhodopseudomonas, and the bacteria of a different species from the purple non-sulfur bacteria may be of the genus Proteus.

[0012] The purple non-sulfur bacteria and bacteria of a different species from the purple non-sulfur bacteria may be a composite bacterium deposited under accession number: NITE BP-03627.

[0013] An antitumor agent for suppressing side effects of an antitumor agent, comprising purple nonsulfur bacteria and bacteria of a different type from the purple nonsulfur bacteria, according to a second aspect of the present invention, includes a cytokine inhibitor.

[0014] The side effect suppressant according to the second aspect of the present invention described above may be used in combination with an anti-inflammatory agent.

[0015] A combination antitumor drug according to a third aspect of the present invention comprises an antitumor agent containing purple nonsulfur bacteria and a different type of bacteria from the purple nonsulfur bacteria, and a cytokine inhibitor.

[0016] The antitumor combination drug according to the third aspect of the present invention may further comprise an anti-inflammatory agent.

[0017] According to the present invention, side effects can be suppressed.

[0018] This figure shows the difference in body weight of tumor-bearing mice before and after administration of the antitumor agent according to the present invention. This figure shows the concentration of interleukin (IL)-6 in the blood of tumor-bearing mice after administration of the antitumor agent according to the present invention. This figure shows the concentration of tumor necrosis factor (TNF)-α in the blood of tumor-bearing mice after administration of the antitumor agent according to the present invention. This figure shows the concentration of interferon (IFN)-γ in the blood of tumor-bearing mice after administration of the antitumor agent according to the present invention. This figure shows the change in tumor volume over time in tumor-bearing mice after administration of the antitumor agent according to the present invention.

[0019] Embodiments of the present invention will be described below with reference to the drawings. In each drawing, the same or equivalent parts are denoted by the same reference numerals. However, the present invention is not limited to the embodiments and drawings described below. In the embodiments described below, expressions such as “having,” “including,” or “containing” also include the meaning of “consisting of” or “composed of.”

[0020] The antitumor agent according to this embodiment includes purple nonsulfur bacteria and bacteria of a different species from the purple nonsulfur bacteria (hereinafter also referred to as "heterospecific bacteria"). Preferably, the purple nonsulfur bacteria and the heterospecific bacteria are viable bacteria. Preferably, the purple nonsulfur bacteria have the property of controlling the biological activity of the heterospecific bacteria in the presence of the heterospecific bacteria. When purple nonsulfur bacteria coexist with heterospecific bacteria, interactions such as biochemical reactions and interbacterial signaling may occur between the purple nonsulfur bacteria and the heterospecific bacteria. Preferably, the antitumor agent includes purple nonsulfur bacteria and heterospecific bacteria as a complex bacterium. The complex bacterium is a group of bacteria in which purple nonsulfur bacteria and heterospecific bacteria grow and multiply while interacting with each other.

[0021] Preferably, purple non-sulfur bacteria have iron-requiring properties, accumulate in tumors, and exhibit oncolytic activity. Furthermore, it is preferable that purple non-sulfur bacteria exert antitumor activity even without immunostimulatory activity. It is preferable that purple non-sulfur bacteria can proliferate even under low oxygen concentrations such as those found in tumor tissue (e.g., 0.5% or 1% or more, or 2% or 5% or less).

[0022] Preferably, the purple nonsulfur bacteria are nonpathogenic. Preferably, the purple nonsulfur bacteria are those that decrease in number as the tumor regresses. Preferably, the purple nonsulfur bacteria are purple nonsulfur bacteria isolated from the tumor, more preferably purple nonsulfur bacteria cultured after isolation, or purple nonsulfur bacteria cultured and subcultured after isolation. For methods of isolating and culturing purple nonsulfur bacteria, please refer to Patent Documents 1 and 2 mentioned above. For example, the purple nonsulfur bacteria are purple nonsulfur bacteria that are not normally present in the body and are inoculated into the tumor, and then isolated from the tumor. Even more preferably, the purple nonsulfur bacteria are purple nonsulfur bacteria that, after being inoculated into the tumor, form a community as a complex bacterium with heterogeneous bacteria normally present in the tumor.

[0023] Examples of purple non-sulfur bacteria in this embodiment include: bacteria of the genus Rhodopseudomonas such as Rhodopseudomonas Palustris and Rhodopseudomonas pseudopalustris; bacteria of the genus Blastochloris such as Blastochloris viridis and Blastochloris sulfoviridis; bacteria of the genus Afifella such as Afifella marina; bacteria of the genus Rhodobacter such as Rhodobacter blasticus, Rhodobacter capsulatus and Rhodobacter sphaeroides; bacteria of the genus Rubrivivax such as Rubrivivax gelatinosus; bacteria of the genus Pararhodospirillum such as Pararhodospirillum oryzae and Pararhodospirillum sulfurexigens; and Rhodocista Examples include bacteria of the genus Rhodocista such as centenaria; bacteria of the genus Marichromatium such as Marichromatium litoris; bacteria of the genus Phaeochromatium such as Phaeochromatium fluminis; bacteria of the genus Rhodoferax such as Rhodoferax fermentans; bacteria of the genus Rhodomicrobium such as Rhodomicrobium udaipurense and Rhodomicrobium vannielii; and bacteria of the genus Rhodovulum such as Rhodovulum sulfidophilum. The purple non-sulfur bacteria in this embodiment may be one of the bacteria from the genera listed above, or a combination of two or more.

[0024] Preferably, the purple non-sulfur bacteria are bacteria of the genus Rhodopseudomonas or Blastochloris, or both. For example, the purple non-sulfur bacteria may be one, two, three, or four species of Rhodopseudomonas Palustris, Rhodopseudomonas pseudopalustris, Blastochloris viridis, and Blastochloris sulfoviridis.

[0025] Particularly preferred are purple non-sulfur bacteria belonging to the genus Rhodopseudomonas. Rhodopseudomonas bacteria isolated from tumors can exhibit particularly excellent antitumor activity.

[0026] It is preferable that the heterologous bacteria have the characteristic of completely lacking pathogenic factors such as cilia and adhesins. When the heterologous bacteria are in a compound bacterial state with purple non-sulfur bacteria, it is preferable that they have one or more flagella, and more preferably that they have motility due to these flagella. It is preferable that the heterologous bacteria, when in a compound bacterial state with purple non-sulfur bacteria, have the ability to change shape, for example, by coming into contact with a tumor, and more preferably that they have the ability to change shape from a swimming cell (swimmer) with a short total length to an elongated fibrous cell (swimmer) with a total length several times longer.

[0027] Preferably, the heterologous bacteria have iron-requiring properties, accumulate in tumors, and exhibit oncolytic activity. Furthermore, it is preferable that the heterologous bacteria exert antitumor activity even without immunostimulatory activity. Preferably, the heterologous bacteria are bacteria that can grow even under low oxygen concentrations such as those found in tumor tissue (e.g., 0.5% or 1% or more, or 2% or 5% or less).

[0028] Preferably, the heterologous bacteria are non-pathogenic. The heterologous bacteria are those that decrease in number as the tumor regresses. The heterologous bacteria may originate from bacteria normally present at the tumor site, or they may be bacteria that form a community with purple non-sulfur bacteria that are not normally present in the body after intravenous inoculation.

[0029] For example, examples of heterogeneous bacteria include, but are not limited to, bacteria of the genera Proteus, Lactococcus, Enterococcus, Acinetobacter, Bacillus, and Cutibacterium.

[0030] Examples of bacteria belonging to the genus Proteus include Proteus mirabilis, Proteus vulgaris, and Proteus myxofaciens. The bacteria of the genus Proteus may be any one, two, or three of these species. Preferably, the bacteria of the genus Proteus is Proteus mirabilis. Proteus mirabilis isolated from tumors exhibits particularly excellent antitumor activity. This Proteus mirabilis is, for example, a bacterium (accession number: NITE BP-03626) that was internationally deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) on March 23, 2022, with a deposit date of March 23, 2022.

[0031] The shape of bacteria of the genus Lactococcus may be spherical or oval. These bacteria grow individually, in pairs, or in chains. Furthermore, bacteria of the genus Lactococcus may not form spores and may be non-motile. Examples of bacteria of the genus Lactococcus include Lactococcus formosensis, Lactococcus garvieae, and Lactococcus garvieae subsp. garvieae. Bacteria of the genus Lactococcus may consist of one, two, or three of these species. These bacteria of the genus Lactococcus were internationally deposited on August 2, 2022, with the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan) (Accession number: NITE BP-03694).

[0032] Examples of bacteria belonging to the genus Enterococcus include Enterococcus faecalis, Enterococcus faecium, Enterococcus alcedinis, Enterococcus bulliens, Enterococcus caccae, Enterococcus devriesei, Enterococcus eurekensis, Enterococcus rivorum, Enterococcus saccharolyticus, and Enterococcus termitis. The Enterococcus bacteria may be any one, two, or three of these species. Preferably, the Enterococcus bacteria is Enterococcus faecalis. Enterococcus faecalis isolated from tumors exhibits particularly excellent antitumor activity. This particular Enterococcus faecalis is a bacterium (accession number: NITE BP-03690) that was internationally deposited with the National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) on July 19, 2022, as the deposit date.

[0033] Examples of bacteria belonging to the genus Acinetobacter include Acinetobacter radioresistens, Acinetobacter albensis, and Acinetobacter baumannii. The bacteria of the genus Acinetobacter may be any one, two, or three of these species. Preferably, the bacteria of the genus Acinetobacter is Acinetobacter radioresistens. Acinetobacter radioresistens isolated from tumors exhibits particularly excellent antitumor activity.

[0034] Examples of bacteria belonging to the genus Bacillus include Bacillus thuringiensis, Bacillus agri, and Bacillus badius. The Bacillus bacteria may be any one, two, or three of these species. Preferably, the Bacillus bacteria is Bacillus thuringiensis.

[0035] Examples of bacteria belonging to the genus Cutibacterium include Cutibacterium acnes, Cutibacterium avidum, Cutibacterium granulosum, etc. The bacteria belonging to the genus Cutibacterium may be any one, two, or three of these. Preferably, the bacteria belonging to the genus Cutibacterium is Cutibacterium acnes. Cutibacterium acnes isolated from tumors exhibits particularly excellent antitumor activity.

[0036] As the heterologous bacteria, bacteria belonging to the genus Proteus are preferable, and more preferably, the heterologous bacteria is Proteus mirabilis. Preferably, in the antitumor agent according to the present embodiment, the purple non-sulfur bacteria are bacteria belonging to the genus Rhodopseudomonas, and the heterologous bacteria are bacteria belonging to the genus Proteus.

[0037] Particularly preferably, the purple non-sulfur bacteria and the heterologous bacteria are composite bacteria that were internationally deposited on March 23, 2022, at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD, Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture 292-0818) (Accession Number: NITE BP-03627).

[0038] The composition ratio of the purple non-sulfur bacteria and the heterologous bacteria in the antitumor agent according to the present embodiment may be, for example, 99:1 to 1:99, but preferably, the content ratio of the purple non-sulfur bacteria is higher in order to obtain excellent antitumor activity. The composition ratio of the purple non-sulfur bacteria and the heterologous bacteria in the antitumor agent is, for example, 99:1 to 50:50, and more preferably may be 99:1 to 55:45. The composition ratio may be 80:20 to 55:45, or may be 70:30 to 60:40. The composition ratio may be based on the CFU of each bacterium.

[0039] When the antitumor agent contains a composite bacterium composed of Rhodopseudomonas Palustris and Proteus mirabilis, the composition ratio based on CFU is preferably 95 to 99:5 to 1, more preferably 96 to 98:4 to 2, and particularly preferably 97:3.

[0040] The composition ratio based on CFU can be determined by inoculating the complex bacteria in the anti-tumor agent onto the culture medium in a petri dish and counting the number of colonies formed by culturing for a predetermined period. Alternatively, the composition ratio based on CFU may be determined by performing quantitative PCR specific to the gene sequences of each bacterium.

[0041] For example, Proteus mirabilis forms colonies 2 to 3 days after the start of culture, while Rhodopseudomonas Palustris forms colonies 7 to 10 days after the start of culture. Therefore, by counting the number of colonies formed 2 to 3 days after the start of culture and the number of colonies formed 7 to 10 days after the start of culture, the number of colonies of Proteus mirabilis and Rhodopseudomonas Palustris can be obtained respectively. The composition ratio may be determined based on these colony numbers.

[0042] In addition, when the periods for forming colonies are similar, alternatively, the number of colonies may be counted based on the optical properties of the purple non-sulfur bacteria and the heterologous bacteria. Purple non-sulfur bacteria have specific optical properties (for example, absorbance at 808 nm or 865 nm, fluorescence spectrum (fluorescence intensity at excitation wavelengths 805 nm and 888 nm)), while heterologous bacteria do not have such optical properties. Therefore, the colonies formed on the culture medium in the petri dish may be determined as to which bacteria they are based on the presence or absence of the optical properties, and the number of colonies of each bacterium may be counted.

[0043] The anti-tumor agent according to the present embodiment may contain purple non-sulfur bacteria and heterologous bacteria in a living state. Thereby, it is considered that the purple non-sulfur bacteria and the heterologous bacteria are more likely to reach the tumor site, and the anti-tumor activity by the purple non-sulfur bacteria and the heterologous bacteria is more effectively exerted. Note that the anti-tumor agent may contain the purple non-sulfur bacteria and the heterologous bacteria in a dead state.

[0044] An antitumor agent may mean a medicine used to treat tumors in animals. "Treatment of tumors" may mean, for example, reducing the size of the tumor, inhibiting tumor growth, killing or reducing tumor cells, or inhibiting the proliferation of tumor cells. An antitumor agent may mean a medicine used to treat or prevent tumors in animals. The animal is, for example, a mammal, and in particular a human, but may also be a non-human animal. A non-human animal may be, for example, an agricultural animal or a pet, and may include, for example, a cattle, horse, sheep, goat, pig, dog, cat, or rabbit.

[0045] The antitumor agent according to this embodiment may be used in particular to treat malignant tumors or benign tumors, and is particularly preferably used to treat malignant tumors. Malignant tumors are also called "cancer." Cancers are classified into solid tumors and hematological cancers. Solid tumors are further classified into carcinomas and sarcomas. The antitumor agent may be used to treat solid tumors or hematological cancers, and is particularly useful for treating solid tumors, for example, for treating carcinomas or sarcomas. The antitumor agent may also be an anticancer agent, a composition for treating cancer, or a composition for the treatment of cancer.

[0046] For example, the antitumor agent according to this embodiment is used to treat pharyngeal cancer, laryngeal cancer, tongue cancer, head and neck cancer, esophageal cancer, gastric cancer, duodenal cancer, colorectal cancer, colon cancer, rectal cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, anal cancer, kidney cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, etc. Also, for example, the antitumor agent is used to treat osteosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, etc.

[0047] The antitumor agent according to this embodiment may be in the form of a liquid. That is, the liquid may be a liquid (particularly a bacterial dispersion) containing purple non-sulfur bacteria and heterologous bacteria. The liquid component of the liquid, other than purple non-sulfur bacteria and heterologous bacteria, may be, for example, an injection solution, an intravenous solution, etc., used in the pharmaceutical field, and more specifically, an isotonic solution, a hypotonic solution, or a hypertonic solution. For example, the liquid component may be saline solution (physiological saline), a sugar solution, a buffer solution, etc. The liquid component may also be phosphate-buffered physiological saline. The liquid is particularly suitable for delivering the antitumor agent of the present invention to the tumor while maintaining its antitumor activity.

[0048] The antitumor agent according to this embodiment is preferably administered parenterally. For example, the antitumor agent may be administered intravenously, intraarterially, subcutaneously, intramuscularly, or intrathecally. Preferably, the antitumor agent is administered intravenously. Purple non-sulfur bacteria and heterologous bacteria have the characteristic of accumulating at tumor sites, so intravenous administration makes it easier for purple non-sulfur bacteria and heterologous bacteria to reach the tumor site.

[0049] The antitumor agent according to this embodiment may be administered directly to the tumor site or near the tumor site, for example, using a syringe, another tube, etc. Alternatively, depending on the location of the tumor, the antitumor agent may be administered orally.

[0050] The dosage of the antitumor agent according to this embodiment is appropriately adjusted to the amount that causes tumor regression, but for example, per administration, the total amount of purple non-sulfur bacteria and heterologous bacteria is 10 ４ CFU / kg body weight ~10 １１ CFU / kg body weight, preferably 10 ５ CFU / kg body weight ~10 １１ CFU / kg body weight, more preferably 10 ６ CFU / kg body weight ~10 １１ It may be administered in a manner that results in CFU / kg body weight.

[0051] If the antitumor agent is in liquid form, the antitumor agent contains purple nonsulfur bacteria and heterologous bacteria, for example, a total of 10 purple nonsulfur bacteria and heterologous bacteria. ５ CFU / ml ~10 １２CFU / ml, preferably 10 ６ CFU / ml to 10 １２ CFU / ml, more preferably 10 ７ CFU / ml to 10 １２ It may be contained at a concentration of CFU / ml.

[0052] The anti-tumor agent according to this embodiment may be administered only once, or may be administered two or more times. The anti-tumor agent may be administered once a day, or may be administered multiple times a day. When the anti-tumor agent is administered multiple times, the anti-tumor agent may be administered daily, or may be administered every other day or every two days. Further, the anti-tumor agent may be administered once a week, once every two weeks, once every three weeks, or once every four weeks.

[0053] The anti-tumor agent may contain other components used in the formulation of purple non-sulfur bacteria and heterologous bacteria in the pharmaceutical field. Therefore, the anti-tumor agent may be an anti-tumor composition or an anti-tumor pharmaceutical composition. The above other components are appropriately selected according to, for example, the administration method and administration site of the anti-tumor agent. The other components are, for example, additives, pH adjusters, colorants, and the like. Further, as long as the effect of the anti-tumor agent is not impaired, the anti-tumor agent may contain known or future-discovered pharmaceutical components for tumor treatment.

[0054] The formulation of the anti-tumor agent is appropriately carried out by a known method according to the dosage form. Purple non-sulfur bacteria and heterologous bacteria may be produced, for example, as follows.

[0055] (Tumor Collection Process) First, in the tumor collection process, a tumor is collected from an animal that has a tumor. Tumor collection may be performed, for example, by biopsy. The tumor may be, for example, a tumor formed from mammalian tumor cells, and in particular may be a tumor formed from human tumor cells. The animal that has a tumor may be, for example, a mammal, in particular a rodent, but may also be a primate, and in particular a human. The animal that has a tumor may be administered the above-mentioned purple non-sulfur bacteria before tumor collection. When purple non-sulfur bacteria are administered, such administration may be, for example, parenteral, and in particular intravenous. After such administration, the tumor is collected after a predetermined period, for example, 1 to 10 days later, and in particular 2 to 5 days later. It is thought that the administered purple non-sulfur bacteria reach the tumor after such a period of time has elapsed. If purple non-sulfur bacteria are not administered, bacteria that are originally present in the tumor may be obtained.

[0056] (Bacterial Isolation Process) In the bacterial isolation process, bacteria are isolated from the tumor collected in the tumor collection process. For example, the tumor is added to a liquid such as a buffer solution and homogenized. Homogenization yields a liquid in which tumor cells and bacteria are suspended. By shaking this liquid, for example at a predetermined speed, the tumor cells settle and the bacteria remain in the supernatant. Bacteria may be isolated from tumor cells in this way, but the method of isolation is not limited to this. They may also be isolated by other methods known in the art.

[0057] (Bacterial Culture Process) In the bacterial culture process, the bacteria isolated from the tumor in the bacterial isolation process are cultured. In the bacterial culture process, the supernatant may be added to a general-purpose agar medium, for example, in a petri dish, and culture is carried out. Colonies are formed on the medium. These colonies may be used as purple non-sulfur bacteria and heterologous bacteria. The general-purpose agar medium may be, for example, a peptone-containing medium or a peptone-free medium.

[0058] The peptone contained in the peptone-containing medium may be one or more of the following: casein peptone, meat peptone, gelatin peptone, and soybean peptone. The peptone-containing medium may be, for example, LB medium or polypeptone medium. In addition to peptone, the peptone-containing medium may further contain an extract. The extract may be, for example, yeast extract or meat extract or a combination thereof. The peptone-free medium may contain, for example, an extract. The extract may be, for example, yeast extract or meat extract or a combination thereof. The peptone-free medium may be, for example, ATCC543 medium.

[0059] Preferably, the bacteria in the colonies formed on the agar medium are further cultured in a liquid medium. The liquid medium may be a general-purpose liquid medium. The general-purpose liquid medium may be, for example, a peptone-containing medium or a peptone-free medium. The peptone and extract contained in the general-purpose liquid medium are the same as those contained in the general-purpose agar medium. The general-purpose liquid medium may be a liquid medium without added cysteine. The liquid medium may be, for example, a liquid medium without added cysteine, containing peptone and an extract, particularly a yeast extract. The liquid medium may be, for example, LB medium or polypeptone medium without added cysteine, or ATCC543 medium without added cysteine.

[0060] Bacteria cultured in a liquid medium may be further cultured on a general-purpose agar medium to form colonies. This general-purpose agar medium may be, for example, a general-purpose agar medium to which deoxycholic acid has been added. The ratio (%) of the amount of deoxycholic acid in the agar medium to the amount of agar medium (g) may be, for example, 0.01% to 1%, preferably 0.03% to 0.5%, and more preferably 0.05% to 0.3%. This general-purpose agar medium may be the same agar medium (fresh medium) as the general-purpose agar medium described at the beginning of this step, except that deoxycholic acid has been added. Colonies are formed on the medium by this culture. The colonies formed here may be further cultured in a liquid medium. This liquid medium may be the same medium (fresh medium) as the medium used in the previous liquid medium culture.

[0061] Furthermore, the above-mentioned general-purpose liquid culture medium may be a liquid culture medium to which cysteine ​​has been added. In this case, the description of the liquid culture medium without cysteine ​​can be referred to above, except for the fact that cysteine ​​has been added. The cysteine ​​content in the liquid culture medium with added cysteine, the ratio (%) of the amount of cysteine ​​(g) to the amount (g) of liquid culture medium, may be, for example, 0.1% to 10%, more preferably 1% to 5%, and even more preferably 2% to 4%. Bacteria cultured in the liquid culture medium with added cysteine ​​may be further cultured on a general-purpose agar medium to form colonies. The general-purpose agar medium may be, for example, a general-purpose agar medium to which deoxycholic acid and cysteine ​​have been added. The ratio (%) of the amount (g) of deoxycholic acid in the agar medium to the amount (g) of the agar medium may be, for example, 0.01% to 1%, more preferably 0.03% to 0.5%, and more preferably 0.05% to 0.3%. Furthermore, the ratio (%) of the cysteine ​​content (g) in the agar medium to the amount (g) of the agar medium may be, for example, 0.1% to 10%, more preferably 1% to 5%, and even more preferably 2% to 4%. The general-purpose agar medium may be the same agar medium (fresh medium) as the general-purpose agar medium described above, except that deoxycholic acid and cysteine ​​are added. Colonies are formed on the medium by this culture. The formed colonies may be further cultured in a liquid medium. The liquid medium may be the same medium (fresh medium) used in the previous liquid medium culture.

[0062] (Formulation Process) The bacteria cultured in the above bacterial culture process are used to formulate the antitumor agent. If the antitumor agent is a liquid formulation, it may be manufactured by mixing the bacteria with a predetermined liquid. The formulation method may be appropriately selected based on, for example, the dosage form, the components it contains, etc.

[0063] The antitumor agent according to this embodiment is used in combination with a cytokine inhibitor. A cytokine inhibitor is a substance that can inhibit, inactivate, or reduce the expression or activity level of a cytokine. Cytokine inhibitors are compounds that bind to cytokines and partially or completely block, reduce, prevent, delay, inactivate, desensitize, or downregulate their activity or expression, such as antagonists. Cytokine inhibitors are also compounds that partially or completely block, reduce, prevent, delay, inactivate, desensitize, or downregulate the activity of cytokines by binding to cytokine receptors or by inhibiting the binding of cytokines to receptors. Cytokine inhibitors include, but are not limited to, antigen-binding molecules, antibodies, antibody derivatives, antibody fragments, polypeptides such as soluble receptors, their derivatives, nucleic acids such as siRNA and antisense RNA, their derivatives, genetically modified forms of soluble factors, such as forms with altered activity, as well as soluble factor antagonists and small molecule compounds.

[0064] For example, cytokine inhibitors include IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IFN-α, IFN-γ, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemoattractant protein (MCP)-1, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte-macrophage colony-stimulating factor. It is an inhibitor of one or more cytokines selected from Ronnie's stimulating factor (G-CSF), C-X-C motif chemokine (CXCL) 9, CXCL 10, vascular endothelial growth factor (VEGF), RANTES, eotaxin, epidermal growth factor (EGF), hepatocyte growth factor (HGF), fibroblast growth factor (FGF)-β, CD30, CD30L, CD40, CD40L, ferritin, and RAGE. Cytokine inhibitors include one or more cytokines selected from IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IFN-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, TNF-α, GM-CSF, G-CSF, CXCL9, CXCL10, VEGF, RANTES, eotaxin, EGF, HGF, FGF-β, CD30, CD30L, CD40, CD40L, ferritin, RAGE, antibodies that bind to their receptors, antibody derivatives, or antibody fragments. Preferably, the cytokine inhibitor includes at least one selected from the group consisting of IL-6 inhibitors, IL-6 receptor (IL-6R) inhibitors, TNF-α inhibitors, interferon-γ inhibitors, and IL-1β inhibitors. More preferably, the cytokine inhibitor includes an IL-6 inhibitor, an IL-6R inhibitor, a TNF-α inhibitor, and an interferon-γ inhibitor.

[0065] To explain in detail using IL-6 inhibitors and IL-6R inhibitors as examples, IL-6 inhibitors and IL-6R inhibitors are inhibitors of IL-6 or IL-6 receptors, respectively. IL-6 inhibitors may be anti-IL-6 antigen-binding molecules, anti-IL-6 antibodies (including chimeric anti-IL-6 antibodies, humanized anti-IL-6 antibodies, and human anti-IL-6 antibodies), their antigen-binding fragments, or antibody derivatives. IL-6 receptor inhibitors may be anti-IL-6 receptor (IL-6R) antigen-binding molecules, anti-IL-6R antibodies (including chimeric anti-IL-6R antibodies, humanized anti-IL-6R antibodies, and human anti-IL-6R antibodies), their antigen-binding fragments, or antibody derivatives.

[0066] Cytokine inhibitors can be formulated according to conventional methods and may contain pharmacologically acceptable carriers or additives. Cytokine inhibitors may contain, for example, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc.

[0067] Antitumor agents are administered concurrently with, before, or after the administration of cytokine inhibitors. For example, antitumor agents are administered before the administration of cytokine inhibitors. The combination of antitumor agents and cytokine inhibitors includes not only administering a composition containing both agents, but also administering them separately and simultaneously, or separately and sequentially. The route of administration for cytokine inhibitors may be the same as that for antitumor agents, or a different route.

[0068] Cytokine inhibitors can be administered to subjects either orally or parenterally. Parenteral administration is preferred. Specific administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The dosage of cytokine inhibitors is not particularly limited, but for example, the dosage can be selected in the range of 0.0001 to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body per subject.

[0069] The antitumor agent according to this embodiment can suppress side effects caused by the antitumor agent when used in combination with a cytokine inhibitor. These side effects include, for example, severe thrombosis, thrombosis, weight loss, infections such as sepsis, fever, nausea, and vomiting.

[0070] The antitumor agent according to this embodiment may be used in combination with an anti-inflammatory agent. An anti-inflammatory agent is a substance that suppresses inflammation or an inflammatory response. The anti-inflammatory agent may be a steroid or a non-steroidal anti-inflammatory drug (NSAID), and examples include ε-aminocaproic acid, allantoin, berberine, glycyrrhizic acid, azulene sulfonic acid, zinc, tranexamic acid, lysozyme, and pranoprofen and their salts. Preferably, the anti-inflammatory agent is a steroid. The steroid is a glucocorticoid or a derivative thereof. Examples of steroids include dexamethasone, prednisolone, methylprednisolone, triamcinolone, betamethasone, and fluorometholone. Preferably, the anti-inflammatory agent is dexamethasone.

[0071] Anti-inflammatory agents can be formulated according to conventional methods and may contain pharmacologically acceptable carriers or additives. Anti-inflammatory agents may contain, for example, surfactants, excipients, colorants, flavorings, preservatives, stabilizers, buffers, suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc.

[0072] Antitumor agents are administered at the same time as, before, or after, the administration of anti-inflammatory agents. For example, antitumor agents are administered before anti-inflammatory agents. When anti-inflammatory agents are used in combination, this includes not only administering a composition containing antitumor agents, cytokine inhibitors, and anti-inflammatory agents to the target, but also administering the antitumor agents, cytokine inhibitors, and anti-inflammatory agents separately and simultaneously, or administering them separately and sequentially. The route of administration of the anti-inflammatory agent may be the same as the route of administration of the antitumor agent or cytokine inhibitor, or it may be a different route of administration.

[0073] Anti-inflammatory agents can be administered to the target subject either orally or parenterally. Parenteral administration is preferred. Specific methods of administration include injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection administration include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. The dosage of the anti-inflammatory agent is not particularly limited, but for example, the dosage can be selected in the range of 0.0001 to 1000 mg per kg of body weight per administration. Alternatively, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body per subject.

[0074] In another embodiment, an agent for suppressing the side effects of the antitumor agent described above is provided. This side effect suppressant includes the cytokine inhibitor described above. This side effect suppressant is used to suppress or reduce the side effects caused by the antitumor agent. This side effect suppressant may be used in combination with an anti-inflammatory agent.

[0075] In another embodiment, an antitumor combination drug is provided. This antitumor combination drug comprises the antitumor agent and the cytokine inhibitor. This antitumor combination drug may also be a combination agent administered in combination with the antitumor agent and the cytokine inhibitor.

[0076] The term "combination antitumor drug" encompasses providing the above-mentioned antitumor agent and cytokine inhibitor as a combination drug, as well as providing them as a kit containing each of these separately as a formulation. When the combination antitumor drug is a combination drug, the above-mentioned antitumor agent and cytokine inhibitor can be combined using the usual method of mixing multiple drugs to form a combination drug. The combination drug may further contain any components other than the above-mentioned antitumor agent and cytokine inhibitor. The optional components are other pharmacologically acceptable components that may be included in the above-mentioned antitumor agent or cytokine inhibitor. The form of the combination drug is arbitrary but preferably liquid. The mixing ratio of the above-mentioned antitumor agent and cytokine inhibitor should be an appropriate ratio that allows each to exert its respective effect.

[0077] If the combination antitumor drug is a kit comprising a first formulation containing the above-mentioned antitumor agent and a second formulation containing the above-mentioned cytokine inhibitor, the first and second formulations may be in the same form or in different forms. Furthermore, the first and second formulations may be the same formulation or different formulations in terms of administration route or method. For example, both the first and second formulations may be orally administered or parenterally administered formulations, the first formulation may be orally administered and the second formulation may be parenterally administered, or the first formulation may be parenterally administered and the second formulation may be orally administered. The first formulation may further contain any other components besides the above-mentioned antitumor agent. The second formulation may further contain any other components besides the above-mentioned cytokine inhibitor. The optional components are other pharmacologically acceptable components that may be included in the above-mentioned antitumor agent or cytokine inhibitor.

[0078] The antitumor combination drug may further comprise an anti-inflammatory agent. In this case, the antitumor combination drug may be provided as a combination preparation comprising the antitumor agent, the cytokine inhibitor, and the anti-inflammatory agent, or as a kit comprising each of these separately as a formulation. If the antitumor combination drug is a kit comprising the first and second formulations, plus the anti-inflammatory tumor agent as a third formulation, the third formulation may be the same form as the first or second formulation, or it may be a different form. Furthermore, the route or method of administration of the third formulation may be the same as or different from that of the first or second formulation. The third formulation may further comprise any other component besides the anti-inflammatory agent. The optional component is any other pharmacologically acceptable component that may be included in the antitumor agent or cytokine inhibitor described above.

[0079] Another embodiment provides a method for treating cancer, a method for tumor regression, or a method for suppressing tumor growth. The method for treating cancer, a method for tumor regression, or a method for suppressing tumor growth includes the steps of administering the antitumor agent to a subject having a tumor, and administering the cytokine inhibitor to the subject. The method for treating cancer, a method for tumor regression, or a method for suppressing tumor growth may further include the step of administering the anti-inflammatory agent to the subject. Another embodiment provides the use of the cytokine inhibitor for the manufacture of an antitumor agent for suppressing side effects of the antitumor agent. Another embodiment provides the use of the cytokine inhibitor for use in suppressing side effects of the antitumor agent. Another embodiment provides the use of the anti-inflammatory agent for the manufacture of an antitumor agent for suppressing side effects of the antitumor agent. Another embodiment provides the anti-inflammatory agent for use in suppressing side effects of the antitumor agent.

[0080] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

[0081] (Example 1) The complex bacterium AUN used in this example is a complex bacterium composed of Rhodopseudomonas Palustris and Proteus mirabilis (composition ratio 97:3 based on CFU) isolated from a tumor derived from mouse colon cancer (Colon 26, JCRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition). AUN was internationally deposited on March 23, 2022, at the Patent Microorganism Depository Center (NPMD, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan) (Accession number: NITE BP-03627). As shown in Patent Documents 1 and 2 above, AUN exhibits antitumor activity that reduces or eliminates tumors.

[0082] AUNs were cultured anaerobically at 26–30°C under a tungsten lamp in cysteine-free ATCC 543 liquid medium. ATCC 543 medium was prepared according to the cell bank's preparation method. Bacterial count and viability were confirmed by measurement using a bacterial counter (CASY Cell Counter & Analyzer; OMNI Life Science) and by colony assay.

[0083] Colon 26 was obtained from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition. Colon 26 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, gentamicin, and penicillin-streptomycin (100 IU / mL).

[0084] BALB / c nude mice with tumor transplanted with Colon26 (tumor size: approximately 200 mm) ３ AUN (1 x 10) is injected into the tail vein. ９ CFU / head was administered. One hour after AUN administration, various cytokine inhibitors (1 mg / head each) were administered by intravein injection. Twenty-four hours after cytokine inhibitor administration, the body weight of the mice was measured and compared to the body weight of the mice before AUN administration.

[0085] As cytokine inhibitors, anti-IL-6 receptor α chain (CD126) antibody (Anti-mouse IL-6R-InVivo, product code A2157, Clone NO. 15A7, manufactured by CEREC Biotechnology, referred to as "IL-6R" in Figure 1), anti-TNF-α antibody (Anti-mouse TNFα-InVivo, product code A2124, Clone NO. XT3.11, manufactured by CEREC Biotechnology, referred to as "TNF-α" in Figure 1), anti-interferon α / β receptor subunit 1 antibody (Anti-mouse IFNAR-1-InVivo, product code A2121, Clone Five types of antibodies were used: NO. MAR1-5A3, manufactured by CEREC Biotechnology, and labeled "IFN-γ" in Figure 1; anti-IL-6 antibody (Anti-mouse IL-6-InVivo, product code A2118, Clone NO. MP5-20F3, manufactured by CEREC Biotechnology, and labeled "IL-6" in Figure 1); and anti-IL-1β antibody (Anti-mouse / rat IL-1β-InVivo, product code A2142, Clone NO. B122, and labeled "IL-1β" in Figure 1).

[0086] As shown in Figure 1, the administration of cytokine inhibitors reduced weight loss caused by AUN administration. In particular, combining multiple types of cytokine inhibitors significantly increased the effect of reducing weight loss.

[0087] (Example 2) An anti-inflammatory agent dispersion was prepared by dispersing dexamethasone as an anti-inflammatory agent in an injection solution containing 10% dimethyl sulfoxide (DMSO). Colon26 transplanted tumor-bearing BALB / c mice (female, approximately 7 weeks old, n=5, average body weight=20g, average tumor size=200mm) ３、 400 μL of an anti-inflammatory agent dispersion (dexamethasone 0.1 mg / head) was administered subcutaneously to BALB / cCrSIC-nu / nu (manufactured by Nippon SLC Co., Ltd.).

[0088] 24 hours after administration of the anti-inflammatory agent dispersion, AUN (1 x 10) was administered in the same manner as in Example 1 above. ９CFU / head was administered by injection into the tail vein. One hour after administration of AUN, four cytokine inhibitors (0.2 mg / head each) were administered by injection into the tail vein (combination group). The cytokine inhibitors were the anti-IL-6 antibody, anti-IL-6 receptor α chain (CD126) antibody, anti-TNF-α antibody, and anti-interferon α / β receptor subunit 1 antibody used in Example 1 above.

[0089] Blood levels of IL-6, TNF-α, and IFN-γ were measured before cytokine inhibitor administration and 3, 6, and 24 hours after cytokine inhibitor administration using Bio-Plex Multiplex immunoassay system (Bio-Rad) and Bio-Plex Pro Mouse Th17 Panel 6-Plex (Bio-Rad). These levels were compared to an AUN-administered group (without concomitant anti-inflammatory agents or cytokine inhibitors) and a phosphate-buffered saline (PBS)-administered group (without anti-inflammatory agents, cytokine inhibitors, or AUN). L and W represent the longest and shortest diameters of the tumor, respectively, and the tumor volume V was defined as V = L × W. ２ Estimated by 2. Note that the tumor volume was 1500 mm³. ３ Mice exceeding a certain threshold were euthanized, as it was determined that they had reached the endpoint.

[0090] As shown in Figure 2, the increase in blood IL-6 concentration was suppressed in the combination therapy group that received anti-inflammatory drugs, cytokine inhibitors, and AUN. Similarly, as shown in Figures 3 and 4, the increase in blood TNF-α and IFN-γ concentrations was suppressed in the combination therapy group. As shown in Figure 5, tumor volume was significantly reduced in the combination therapy group.

[0091] The embodiments described above are for illustrative purposes only and do not limit the scope of the present invention. That is, the scope of the present invention is defined not by the embodiments, but by the claims. Various modifications made within the scope of the claims and equivalent inventive meaning are considered to be within the scope of the present invention.

[0092] This application is based on Japanese Patent Application No. 2025-26248, filed on 20 February 2025, and Japanese Patent Application No. 2025-127522, filed on 30 July 2025. The entire specifications, claims, and drawings of Japanese Patent Application No. 2025-26248 and Japanese Patent Application No. 2025-127522 are incorporated herein by reference.

[0093] This invention is useful for pharmaceuticals, particularly for the treatment of tumors.

Claims

1. An antitumor agent comprising purple non-sulfur bacteria and a different type of bacteria from the aforementioned purple non-sulfur bacteria, which is used in combination with a cytokine inhibitor.

2. The antitumor agent according to claim 1, wherein the cytokine inhibitor comprises at least one selected from the group consisting of an interleukin-6 inhibitor, an interleukin-6 receptor inhibitor, a tumor necrosis factor-α inhibitor, an interferon-γ inhibitor, and an interleukin-1β inhibitor.

3. The antitumor agent according to claim 2, wherein the cytokine inhibitor comprises an interleukin-6 inhibitor, an interleukin-6 receptor inhibitor, a tumor necrosis factor-α inhibitor, and an interferon-γ inhibitor.

4. The antitumor agent according to claim 1, which is used in combination with an anti-inflammatory agent.

5. The antitumor agent according to claim 4, wherein the anti-inflammatory agent is a steroid.

6. The antitumor agent according to any one of claims 1 to 5, wherein the purple nonsulfur bacteria are bacteria of the genus Rhodopseudomonas, and the bacteria of a different species from the purple nonsulfur bacteria are bacteria of the genus Proteus.

7. The antitumor agent according to claim 6, wherein the purple non-sulfur bacteria and bacteria of a different species from the purple non-sulfur bacteria are a complex bacterium deposited under accession number: NITE BP-03627.

8. An antitumor agent containing cytokine inhibitors, comprising purple non-sulfur bacteria and bacteria of a different species from the purple non-sulfur bacteria.

9. The side effect suppressant according to claim 8, which is used in combination with an anti-inflammatory agent.

10. An antitumor combination drug comprising an antitumor agent containing purple nonsulfur bacteria and a different type of bacteria from the purple nonsulfur bacteria, and a cytokine inhibitor.

11. The combination antitumor drug according to claim 10, further comprising an anti-inflammatory agent.

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