WO2021215405A1

WO2021215405A1 - Immunostimulant and composition for treatment or prevention

Info

Publication number: WO2021215405A1
Application number: PCT/JP2021/015904
Authority: WO
Inventors: 幸太岩堀
Original assignee: 国立大学法人大阪大学
Priority date: 2020-04-20
Filing date: 2021-04-19
Publication date: 2021-10-28

Abstract

Provided is a compound capable of treating or preventing a virus disease and tumors.　The compound is a compound for treatment or prevention, including at least one selected from the group consisting of a tetracycline compound and a pharmaceutically permissible salt thereof, wherein said compound for treatment or prevention is for administration for eight days or more, at a dose of from 1/10 to 1/2 of a defined dose for use in treatment of a bacterial infection, of the tetracycline compound or the pharmaceutically permissible salt thereof.

Description

Immunostimulators and therapeutic or prophylactic compositions

The present specification discloses an immunostimulatory agent and a therapeutic or prophylactic composition.

Since the end of 2019, the worldwide spread of coronavirus has caused human and economic losses in various countries, and the development of a treatment method for coronavirus disease 2019 (COVID-2019) has been carried out. It has become a global issue.

Non-Patent Document 1 describes that tetracycline (for example, tetracycline, doxycycline, minocycline) chelate a zinc compound in matrix metalloproteinase (MMP). And since the survival, cell infiltration, cell-cell adhesion, and replication of coronavirus are highly dependent on the host's MMP, it is hypothesized that the zinc chelating ability of tetracycline is effective in suppressing COVID-19 infection in humans. Is shown.

Tetracycline compounds can also be used to treat tumors. Non-Patent Documents 2 and 3 show that administration of chemically modified tetracycline to tumor cells suppresses the growth of tumor cells and causes cell damage. Patent Document 1 discloses a tumor immunostimulatory agent containing at least one selected from the group consisting of tetracycline compounds and pharmaceutically acceptable salts thereof.

International Publication No. 2019/177011

The inhibitory effect of tetracycline on COVID-19 described in Non-Patent Document 1 is only a hypothesis and has not been proved in practice.

Conventional antiviral drugs are drugs that target the virus itself and inhibit the production and proliferation of the virus, and can be a curative treatment for chronic viral infections such as hepatitis C virus and HIV. It is an adjunctive drug for acute viral infections such as influenza virus.

The present invention is different from conventional antiviral agents, acid-fast bacillosis agents, antifungal agents or antitumor agents, and presents an agent that exerts a therapeutic effect by enhancing the T-cell immune response of the patient itself. The challenge is to provide.

The present inventors have found that tetracycline compounds are effective for activating T-cell-mediated immunity from among existing drugs whose patent rights have expired. Further, it has been found that, conventionally, tetracycline compounds are used for the treatment of bacterial infectious diseases, but the volume used for the treatment of bacterial infectious diseases is lower than the volume used for the treatment of bacterial infectious diseases, and it is possible to activate T cell-mediated immunity.

The invention disclosed in the present specification has been completed based on the findings, and includes the following aspects.
Item 1. A therapeutic or prophylactic composition or an immunostimulatory agent comprising at least one selected from the group consisting of a tetracycline antibiotic represented by the following general formula (I) and a pharmaceutically acceptable salt thereof, and tetracycline. A therapeutic or prophylactic composition or a therapeutic or prophylactic composition for administering the tetracycline and its pharmaceutically acceptable salt at a dose of 1/10 to 1/2 of the prescribed dose used for the treatment of bacterial infections for 8 days or longer. Immunostimulator:

[During the ceremony,
R ¹ is a group represented by the following general formula (II) (R ⁸ is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms), or a lower alkyl group having 1 to 3 carbon atoms:

..
R ² is a group represented by the following general formula (III) (R ⁹ is a lower alkyl group having 1 to 3 carbon atoms or a hydrogen atom):

..
R ³ is a hydrogen atom, a hydroxyl group, or a lower alkyl group having 1 to 3 carbon atoms.
R ⁴ and R ⁵ are hydrogen atoms, hydroxyl groups or lower alkyl groups having 1 to 3 carbon atoms together or independently, or R ⁴ and R ⁵ are methylene groups in one.
R ⁶ is a hydrogen atom, a halogen or a group represented by the following general formula (IV) (R ¹⁰ is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms):

..
R ⁷ is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, -NH-CO-CH _2- NH-C (CH ₃ ) ₃ or -CH _2- NH-CH _2- C (CH ₃ ) ₃ . be. ]. Preferably, the therapeutic or prophylactic composition is used for the treatment or prevention of a viral infection or tumor.
Item 2. Item 2. The therapeutic or preventive composition according to Item 1, or an immunostimulatory agent, wherein the lower alkyl group having 1 to 3 carbon atoms is a methyl group.
Item 3. Item 2. The compound represented by the general formula (I) is at least one selected from the group consisting of demethylchlortetracycline, meclocycline, tetracycline, chlortetracycline, doxycycline, minocycline, and oxytetracycline. A therapeutic or prophylactic composition, or an immunostimulatory agent.
Item 4. A composition for treating a viral infection, which comprises the immunostimulatory agent according to any one of Items 1 to 3 and an antiviral agent.
Item 5. A composition for treating a tumor, which comprises the immunostimulatory agent according to any one of Items 1 to 3 and a tumor immunological agent.
Item 6. Item 5. The composition for treating a tumor according to Item 5, wherein the tumor immunological agent is at least one selected from an immune checkpoint inhibitor, a CAR-T cell drug, a bispecific molecular drug, and a cancer vaccine.
Item 7. A composition for treating an acid-fast bacillus infection, which comprises the immunostimulatory agent according to any one of Items 1 to 3 and an acid-fast bacillus agent.
Item 8. A composition for treating a fungal infection, which comprises the immunostimulatory agent according to any one of Items 1 to 3 and an antifungal agent.

The tetracycline compound can activate T cell-mediated immunity.

FIG. 1 shows an outline of a system for evaluating the antitumor activity of T cells in vitro. FIG. 2 shows the effect of the test drug on the antitumor activity of T cells in vitro. "BiTE" indicates the addition of BiTE alone, and "BiTE + DMC" indicates the addition of BiTE and demethylchlortetracycline (DMC) in combination. FIG. 3 shows the effect of the test drug on the tumor cytotoxic activity ^{of CD8 + T cells in vitro.} “BiTE” indicates the addition of BiTE alone, and “BiTE + DMC” indicates the addition of BiTE and DMC in combination. FIG. 4 shows the effect of the test drug on the abundance ratio of granzyme B-expressing CD8 ^{+ T cells in vitro.} “BiTE” indicates the addition of BiTE alone, and “BiTE + DMC” indicates the addition of BiTE and DMC in combination. FIG. 5 shows the effect of the test drug on ^{the proliferative capacity of CD8 + T cells in vitro.} “BiTE” indicates the addition of BiTE alone, and “BiTE + DMC” indicates the addition of BiTE and DMC in combination. FIG. 6 shows the effect of the test drug on the induction of CMV (cytomegalovirus) -specific cytotoxic T cells in vitro. A shows the scattergram of FACS analysis on the 7th and 14th days after CMV treatment. ^{CMV Tetramer +} cells are fractionated into the P7 fraction. B indicates the abundance ratio ^{of CMV Tetramer +} cells 14 days after CMV treatment. FIG. 7A shows the effect of the test drug on the cytotoxic activity of T cells in lung cancer tissue in vitro. FIG. 7B shows the effect of the test drug on ^{the abundance ratio of IFNγ-producing CD8 + T cells in vitro.} FIG. 7C shows a scattergram for FACS analysis. ^{IFNγ-producing CD8 +} T cells are fractionated into the P8 fraction. FIG. 8A shows the administration protocol of the test drug. FIG. 8B shows the change in tumor volume. In FIG. 8B, the broken line indicates the Vehicle group, and the solid line indicates the test drug administration group. The combined effect of demethylchlortetracycline and anti-PD-L1 antibody is shown. p indicates a significant difference. It is shown that the enhancing effect of the immune checkpoint inhibitor of demethylchlortetracycline on the antitumor effect ^{depends on CD8 + T cells.} p indicates a significant difference. The effect of demethylchlortetracycline on the abundance ratio of cancer antigen-specific CD8-positive T cells in the peripheral blood of mice is shown. p indicates a significant difference. We show that tetracycline compounds induce tumor cytotoxic activity and IFNγ production. A indicates the tumor cytotoxic activity when DMC is administered alone. B shows the tumor cytotoxic activity when entinostat alone is administered. C indicates tumor cytotoxic activity when olaparib alone is administered. D indicates the tumor cytotoxic activity when DMC was added when PBMC and BiTE were contacted with the U251 cell line. E indicates the tumor cytotoxic activity when entinostat was added when PBMC and BiTE were contacted with the U251 cell line. F indicates the tumor cytotoxic activity when olaparib was added when PBMC and BiTE were contacted with the U251 cell line. G indicates the IFNγ concentration in the culture supernatant when DMC was added when PBMC and BiTE were contacted with the U251 cell line. H indicates the IFNγ concentration in the culture supernatant when entinostat was added when PBMC and BiTE were contacted with the U251 cell line. I indicates the IFNγ concentration in the culture supernatant when olaparib was added when PBMC and BiTE were contacted with the U251 cell line. It shows the tumor growth inhibitory effect of the tetracycline compound and the antitumor effect enhancing effect of the tetracycline compound on the immune checkpoint inhibitor.

1. 1. Immunostimulators As used herein, immunostimulators include tetracycline compounds or pharmaceutically acceptable salts thereof as active ingredients.

In the present specification, "immunity" is not particularly limited, but is intended to be immunity that depends on T-cell-mediated immunity. More preferably, the immunity may include anti-viral immunity, anti-acid bacterium immunity, anti-fungal immunity, anti-tumor immunity and the like.

The virus is not particularly limited. It may be a DNA virus or an RNA virus. In addition, the genomic polynucleotide carried by the virus may be single-stranded or double-stranded. When the genomic polynucleotide carried by the virus is single-stranded, it may be positive or negative.

For example, as viruses, coronavirus family, adenovirus family, poxvirus family, polyomavirus family, papillomavirus family, parvovirus family, calicisvirus family, orthomixovirus family, paramixovirus family, lovedvirus family, phyllo It may include viruses of the family Virals, Arenaviruses, Togaviruses, Flaviviruses, Picornaviruses, Bunyaviruses, Leoviruses, Retroviruses, Hepedoviruses, Hepeviruses.

When a virus infects a human, it is preferable that the virus causes a disease in the human due to the infection. Here, causing a disease in humans does not mean that the incidence of the disease when infected with a virus is 100%. The incidence of disease when infected with a virus is greater than 0% and 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, It is 10% or less, or 5% or less.

Diseases can be classified into acute diseases, subacute diseases, and late-onset diseases according to the incubation period. Acute disease is intended to be a disease that develops with an incubation period of 1 day to less than 1 year after infection. Subacute disease is intended to be a disease that develops with an incubation period of 1 year or more and less than 8 years after infection. Late-onset disease is intended to be a disease that develops with an incubation period of 8 years or more after infection. The disease is preferably an acute disease or a subacute disease. The virus may also cause at least two diseases selected from acute, subacute, and late-onset diseases in a single infected person. In this case, at least one disease may include an acute disease or a subacute disease.

Examples of viruses that cause acute and subacute diseases in humans include Severe Acute Respiratory Syndrome (SARS) coronavirus 2 (SARS-CoV2), SARS coronavirus, and humans that cause COVID-19. Coronavirus 229E strain, human coronavirus NL63 strain, human coronavirus OC43 strain, human coronavirus HKU1 strain and other viruses belonging to the coronavirus family; cytomegalovirus, EB virus, simple herpesvirus, varicella-herpes zoster virus and other herpes Viruses belonging to the family of viruses; viruses belonging to the family Calisis virus such as norovirus; viruses belonging to the family of orthomixoviruses such as influenza virus; viruses belonging to the family paramixoviral family such as measles virus, mumps virus, parainfluenza virus, RS virus; Rabdovirus family virus that causes mad dog disease, bullous stomatitis, etc .; Phyllovirus family virus such as Ebola hemorrhagic fever virus; Arenavirus family virus that causes Lassa fever, etc .; Flavivirus that causes encephalitis, yellow fever, dengue fever, etc. Family viruses; Picornavirus family viruses such as poliovirus, enterovirus, coxsackie virus, echovirus; Bunyavirus family viruses such as huntervirus, orthobunyavirus, nylovirus, and frevovirus; Viruses; viruses of the Hepedovirus family such as hepatitis A virus; viruses of the Hepeviridae family such as hepatitis E virus can be mentioned.

Since this immunostimulatory agent is effective at a low dose, it can be administered for a long period of time. Therefore, it can be suitably used for the treatment or prevention of diseases caused by viruses of the Coronaviridae family.

Antiviral immunity (also called antiviral immunity) is considered to be a type of antigen-specific immune response of T cells. Therefore, it can be said that activating antiviral immunity means enhancing the proliferation of T cells specific to the viral antigen and / or the activity against the viral antigen.

The type of acid-fast bacillus is not particularly limited. Examples of mycobacteria include tubercle bacilli and nontuberculous mycobacteria. The tubercle bacillus group may include Mycobacterium tuberculosis (Human Mycobacterium tuberculosis), Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti. The non-tuberculous mycobacteria group may include Mycobacterium kansasii, Mycobacterium marimum, Mycobacterium avium (including Mycobacterium avium complex), Mycobacterium intracellulare, Mycobacterium xenopi, Mycobacterium abscessus.

Counter-acid bacterium immunity is considered to be a type of antigen-specific immune response of T cells. Therefore, it can be said that activating acid-fast bacillus immunity means enhancing the proliferation of T cells specific to the acid-fast bacillus antigen and / or the activity against the acid-fast bacillus antigen.

The type of fungus is not particularly limited. As a fungus, it is preferably a fungus that exhibits pathogenicity. Pathogenic fungi can include, for example, Aspergillus spp., Candida spp., Cryptococcus spp., Pneumocystis spp. Aspergillus spp. May include Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ochraceus, Aspergillus, Aspergillus versicolor. Candida spp. May include Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida lusitaniae. Cryptococcus neoformans may be included in the genus Cryptococcus. Pneumocystis genus may include Pneumocystis carinii and Pneumocystis jirovecii.

Antifungal immunity is considered to be a type of antigen-specific immune response of T cells. Therefore, it can be said that activating anti-fungal immunity means enhancing the proliferation of T cells specific to the fungal antigen and / or the activity against the fungal antigen.

The type of tumor is not particularly limited. It may be a benign tumor or a malignant tumor. It is preferably a malignant tumor. In addition, the tumor includes an epithelial tumor and a non-epithelial tumor, and is preferably an epithelial tumor. In the present invention, the most preferable tumor is an epithelial malignant tumor.

Examples of malignant tumors include respiratory malignant tumors originating from the trachea, bronchi, lungs, etc .; head and neck cancer; esophagus, stomach, duodenum, empty intestine, ileum, leukemia, leukemia, ascending colon, transverse colon, sigmoid colon, etc. Gastrointestinal malignant tumors originating from the rectal or anal region; liver cancer; pancreatic cancer; urinary system malignant tumors originating from the bladder, urinary tract or kidney; female reproductive system malignant tumors originating from the ovary, oviduct, uterus, etc. Leukemia: Prostatic cancer; Skin cancer; Endocrine malignant tumors such as hypothalamus, pituitary gland, thyroid gland, parathyroid gland, adrenal gland; Central nervous system malignant tumors; Solid tumors such as malignant tumors originating from bone and soft tissue, and bone marrow abnormalities Plastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid monocytic leukemia, chronic myeloid monocytic leukemia, acute monocytic leukemia, chronic monocytic leukemia, acute Whole myeloid leukemia, acute macronuclear leukemia, red leukemia, eosinophil leukemia, chronic eosinophil leukemia, chronic neutrophil leukemia, adult T cell leukemia, hairy cell leukemia, plasma cell leukemia, multiple leukemia Hematopoietic malignant tumors such as myeloma and malignant lymphoma; Hematopoietic tumors such as lymphoid malignant tumors can be mentioned. More preferably, respiratory epithelial malignant tumors such as lung cancer (flat epithelial cancer, small cell cancer, large cell cancer, adenocarcinoma), malignant pleural mesenteric tumor; head and neck cancer; esophageal cancer, gastric cancer, colon cancer (S) Gastrointestinal epithelial malignant tumors such as colon cancer, rectal cancer, etc.); liver cancer; pancreatic cancer; renal cell cancer; bladder cancer; malignant melanoma; classical Hodgkin lymphoma; ovarian cancer; breast cancer: prostate cancer; be able to. Most preferably, lung cancer.

Tumor immunity is considered to be a biological reaction to tumors based on the damaging activity of T cells against tumor cells. Therefore, activating tumor immunity can be said to increase the cytotoxic activity of T cells against tumor cells.

The tetracycline compound used in the present invention or a pharmaceutically acceptable salt thereof is intended to exert a growth inhibitory effect or a cytotoxic effect on direct tumor cells as described in Non-Patent Documents 2 and 3. It's not something to do. In addition, the tetracycline compounds used in the present invention or pharmaceutically acceptable salts thereof are not intended to directly damage virus-infected cells. As shown in Examples described later, the tetracycline compound used in the present invention or a pharmaceutically acceptable salt thereof has an action of enhancing the cytotoxic activity of T cells.

As the tetracycline compound, for example, a compound represented by the following general formula (I) can be exemplified.

..
R ⁷ is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, -NH-CO-CH _2- NH-C (CH ₃ ) ₃ or -CH _2- NH-CH _2- C (CH ₃ ) ₃ . be. ].

Examples of the lower alkyl group having 1 to 3 carbon atoms include a methyl group, an ethyl group, a propyl group, and an isopropyl group. It is preferably a methyl group or an ethyl group, and more preferably a methyl group.
Halogen is not particularly limited. For example, a chlorine atom, a fluorine atom, a bromine atom, an iodine atom and the like can be mentioned. It is preferably a chlorine atom.
The tetracycline compound is preferably at least one selected from the group consisting of the tetracycline compounds listed in Table 1 below.

The tetracycline-based compound is more preferably at least one selected from the group consisting of demethylchlortetracycline, mecrocycline, tetracycline, chlortetracycline, doxycycline, minocycline, and oxytetracycline.

Tetracycline compounds and salts thereof disclosed in the present specification are known. Therefore, methods for producing tetracycline compounds and salts thereof are also known.

The salt of the tetracycline compound is not limited as long as it is a pharmaceutically acceptable salt. For example, a salt can be produced by reacting a tetracycline-based compound acid salt containing an amine or other basic group with a suitable organic or inorganic acid to produce an anionic salt. Examples of anionic salts are acetate, benzenesulfonate, benzoate, bicarbonate, heavy tartrate, bromide, calcium edetate, cansilate, carbonate, chloride, citrate, dihydrochloride. , Edetate, edicylate, estlate, esilate, fumarate, gluceptate, gluconate, glutamate, glycolyllylarsanylate, hexylresorcin Acid salt, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, ISEthionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl sulfate, Mutate, napsylate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, basic acetate, Includes succinates, sulfates, tannates, tartrates, theocrates, tosylates, triethiodide salts and the like. The salt is preferably hydrochloride or dihydrochloride.

Tetracycline compounds can generate cationic salts by reacting with suitable bases. Cationic salts may be made of bases that give pharmaceutically acceptable cations, alkali metal salts (particularly sodium and potassium), alkaline earth metal salts (particularly calcium and magnesium), aluminum salts and ammonium. Salts, as well as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picolin, dicyclohexylamine, N, N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis- (2-hydroxyethyl) amine, tri- (2-hydroxyethyl) Contains salts such as amines, prokines, dibenzylpiperidin, dehydroabiethylamine, N, N'-bisdehydroabiethylamine, glucamine, N-methylglucamine, colidin, quinine, quinoline, and basic amino acids such as lysine and arginine. ..

The immunostimulatory agent contains a tetracycline compound or a pharmaceutically acceptable salt thereof. The immunostimulatory agent may be prepared by combining a tetracycline compound or a pharmaceutically acceptable salt thereof with a suitable carrier or additive. As the carrier and the additive used for the preparation of the immunostimulatory agent, various substances commonly used in ordinary drugs depending on the dosage form of the immunostimulatory agent, such as excipients, binders, disintegrants, and slippers, are used. Examples thereof include swamps, colorants, flavoring agents, odorants, and surfactants.

When the above immunostimulatory agent is orally administered, the dosage form is not particularly limited, but tablets, powders, granules, capsules (including hard capsules and soft capsules), liquids, pills, etc. Suspension agents, emulsions and the like can be exemplified. When the immunostimulatory agent is administered parenterally, examples thereof include injections, infusions, suppositories, nasal drops, and transpulmonary administrations.

When the immunostimulatory agent is an oral solid composition such as tablets, powders, granules, pills, capsules, etc., as a carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, etc. Excipients such as calcium carbonate, kaolin, crystalline cellulose, silicic acid, methyl cellulose, glycerin, sodium alginate, rubber arabic; simple syrup, pudo sugar solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, carboxymethyl cellulose , Celac, methylcellulose, ethylcellulose, water, ethanol, potassium phosphate, etc .; dried starch, sodium alginate, canten powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, Disintegrants such as stearic acid monoglyceride, starch, lactose; disintegrators such as sucrose, stearic acid, cacao butter, hydrogenated oil; absorption promoters such as sodium lauryl sulfate; moisturizers such as glycerin, starch; starch, lactose, Excipients such as kaolin, bentonite and colloidal silicic acid; and lubricants such as purified talc, stearate, boric acid powder and polyethylene glycol can be used. Further, the tablet may be a tablet coated with a normal skin, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, a multi-layer tablet, or the like.

When the above immunostimulatory agent is an oral solid composition of pills, as a carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, talc; gum arabic. Binders such as powder, tragant powder, gelatin; disintegrants such as laminarin and canten can be used.

When the immunostimulatory agent is an oral solid composition of a capsule, the capsule is prepared by mixing the active ingredient with various carriers exemplified above and filling the capsule into a hard capsule, a soft capsule, or the like. Is prepared.
When the above-mentioned preparation is a liquid preparation, it may be an aqueous or oily suspension, a solution, a syrup, or an elixir preparation, and is prepared according to a conventional method using ordinary additives.

When the immunostimulatory agent is an injection, the carrier may be, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, or the like. Diluting agent; pH adjuster such as sodium citrate, sodium acetate, sodium phosphate; buffer such as dipotassium phosphate, trisodium phosphate, sodium hydrogen phosphate, sodium citrate; sodium pyrosulfate, EDTA, thioglycol Stabilizers such as acids and thiolactic acids; saccharides such as mannitol, inositol, maltose, sucrose and lactose can be used as molding agents when freeze-dried. In this case, a sufficient amount of glucose or glycerin to prepare an isotonic solution may be contained in the agent, or a usual solubilizing agent, pain-relieving agent, local anesthetic, etc. may be added. good. These carriers can be added to produce subcutaneous, intramuscular, and intravenous injections by conventional methods.

When the above-mentioned preparation is an intravenous drip, it can be prepared by dissolving the administered compound in an isotonic electrolyte infusion preparation based on physiological saline, Ringer's solution, or the like.

The immunostimulatory agent in the present invention may be either an oral composition or a parenteral composition, but is preferably an oral composition.

The tetracycline compound or a pharmaceutically acceptable salt thereof contained in the immunostimulatory agent may be one kind or a plurality of kinds. That is, the immunostimulatory agent may contain at least one selected from the group consisting of tetracycline compounds and pharmaceutically acceptable salts thereof.

The dose of the tetracycline compound or its pharmaceutically acceptable salt used as the immunostimulatory agent is preferably less than the prescribed dose used for the treatment of bacterial infectious diseases. For example, the dose of the tetracycline compound or its pharmaceutically acceptable salt used in the present embodiment is converted into a daily dose from 1/10 of the prescribed dose used for the treatment of bacterial infectious diseases. It is about 1/2. Preferably, the lower limit of the dose in this embodiment can be selected from 1/10, 1/8, and 1/6 of the specified dose. The upper limit of the dose in this embodiment can be selected from 1/2, 1/3, and 1/4 of the specified dose. For example, examples of prescribed doses of tetracycline compounds or pharmaceutically acceptable salts thereof used in the treatment of bacterial infections are as follows. The prescribed dose of tetracycline hydrochloride is 1 g per day for adults and 30 mg / kg for children, which are taken in 4 divided doses. The prescribed dose of demethylchlortetracycline hydrochloride is 450 mg to 600 mg per day for adults, and these are taken in 2 to 4 divided doses. The prescribed dose for the initial dose of doxycycline hydrochloride hydrate is 200 mg per day for adults, and it may be taken in a single dose or in two divided doses. The prescribed dose for the second and subsequent doses of doxycycline hydrochloride hydrate is 100 mg per day for adults. The prescribed dose of the initial dose of doxycycline hydrochloride hydrate is 4.4 mg / kg per day for children weighing 45 kg or less, and is taken in two divided doses. The prescribed dose for the initial dose of doxycycline hydrochloride hydrate is 200 mg / kg per day for children weighing over 45 kg and is taken in two divided doses. The prescribed dose for the second and subsequent doses of doxycycline hydrochloride hydrate is 2.2 mg / kg to 4.4 mg / kg per day for children weighing 45 kg or less and should be taken once daily or 2 Take in divided doses. The prescribed dose for the second and subsequent doses of doxycycline hydrochloride hydrate is 100 mg to 200 mg per day for children weighing over 45 kg and may be taken once daily or in two divided doses. The prescribed dose for the initial dose of minocycline hydrochloride is 100 mg to 200 mg per day for adults, which is taken orally or intravenously infused. The prescribed dose for the second and subsequent doses of minocycline hydrochloride is 100 mg every 12 or 24 hours for adults and is taken orally or intravenously infused. The prescribed dose of administration of minocycline hydrochloride is 2 mg / kg to 4 mg / kg per day for children and is taken orally. The prescribed dose for the first dose of tigecycline is 100 mg for adults, which is given by intravenous drip infusion over 30 to 60 minutes. The prescribed dose for the second dose of tigecycline is 50 mg, which is given by intravenous drip infusion over 30 to 60 minutes every 12 hours. The dose can be appropriately adjusted according to the age, symptoms, tumor size, administration status of other drugs, and the like. When two or more tetracycline compounds and pharmaceutically acceptable salts thereof are administered in combination, the total amount of the tetracycline compounds or pharmaceutically acceptable salts thereof is used for the treatment of the above-mentioned bacterial infectious disease. It is preferable not to exceed 1/2 of the total prescribed dose. For tetracycline antibiotics, side effects such as abdominal pain, nausea, loss of appetite, gastrointestinal disorders and other gastrointestinal symptoms, or dizziness have been reported when administered at a prescribed dose used for the treatment of bacterial infections. In addition, administration of a prescribed dose used for the treatment of bacterial infections may cause a bacterial alternation phenomenon. However, according to the present embodiment, since the effect is exhibited at a dose smaller than the specified dose, long-term administration is possible without causing side effects or bacterial change phenomenon.

In addition, the administration period of the immunostimulatory agent can be from the time when the immunostimulatory agent is prescribed until the symptoms associated with the disease are relieved or cured. It is preferable to continue for 3 days or more, 5 days or more, 8 days or more, 10 days or more, 14 days or more, 21 days or more, 30 days or more, and 60 days or more. The upper limit of the administration period varies depending on the virus, acid-fast bacillus, and fungus, and is, for example, 90 days, 60 days, 48 days, 40 days, 36 days, 30 days, 28 days, 21 days, or 14 days. For example, it is preferably administered for 14 to 28 days for patients suffering from a coronaviridae infection, especially COVID-19. The immunostimulatory agent can be administered every day during the administration period. For example, when it is administered for 14 days or more, 21 days or more, 1 month or more and 2 months or more, it is administered every 2 days or 3 days. be able to. As another administration form, the immunostimulatory agent is administered daily for the first 8 days, 10 days, or 14 days after the administration of the immunostimulatory agent, and then every 2 days and every 3 days. You may change it. Further, it may be administered every 11 to 12 hours, for example, within the range not exceeding the daily dose.

The subjects to whom the immunostimulatory agent is administered for viral infections are those who have developed viral infections, those who are suspected of having close contact with patients who have developed viral infections, and those who have no symptoms of viral infections. A person who has a positive reaction in the virus test may be the subject of administration. Examples of the virus test include a virus antigen test, a virus antibody test, a PCR test, and a sequencing test using a next-generation sequencer.

The subjects to whom the immunostimulatory agent for acid-fast bacillus infection is administered are those who have developed acid-fast bacillus infection, those who are suspected of having close contact with patients who have developed acid-fast bacillus infection, and acid-fast bacillus infection. A person who has no symptoms of the disease but who shows a positive reaction in various acid-fast bacillus tests may be the subject of administration. Examples of the acid-fast bacillus test include a tuberculin test, an X-ray test, a Tyrnersen staining test of a sample collected from a tissue suspected of being infected or a related site thereof, a PCR test, and a sequencing test using a next-generation sequencer.

The recipients of the immunostimulatory agent for fungal infections may be patients who have developed fungal infections or those who have no symptoms of fungal infections but who have a positive reaction in various fungal tests. .. Examples of the fungal test include an X-ray test, a histopathological test of a sample collected from a tissue suspected of being infected or a related site thereof, a cytological test, a PCR test, and a sequencing test using a next-generation sequencer.

Tetracycline compounds or pharmaceutically acceptable salts thereof may be used in combination with other drugs. Other agents that can be used in combination are not particularly limited, but are preferably antiviral agents, antimycobacterial agents, antifungal agents, or antitumor agents.

Antiviral drugs include, for example, antiviral drugs (oseltamivir, baroxavir, zanamivir, peramivir, raninamidir, fabipiravir), anticytomegalovirus drugs (gancyclovir, balgancyclovir, hoscalnet), antiherpesvirus drugs (acyclovir, baracyclovir, femme). Cyclovir, Amenamevir), antiviral drugs (didobudin, ramibdin, abacavir, tenofovir, emtricitabine, nevirapin, efavirentz, etrabirin, lylpivirin, nerfinavir, ritonavir, lopinavir, atazanavir, hosamprenavir, daclatasvir, daclatasvir, daclatasvir, daclatasvir, daclatasvir, daclatasvir Anti-hepatitis virus drugs (teraprevir, simeprevir, vaniprevir, daclatasvir, asnaprevir, sophosbuvir, ribavirin, regipasvir, ombitasvir, paritaprevir, ritonavir, elvasville, glazoprevir, daclatasvir, asunaprevir, beclabvir, grecaprevir, pibrentasvir Examples thereof include hepatitis virus drugs (ramibdin, adehovir, entecavir, tenofovir), antievolavirus drugs (remdecibir) and the like.

The antiviral drug can be administered according to a known method.

The mode of combination (combination) of the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof with an antiviral drug is not particularly limited as long as it is a mode of use that exerts the effect of the present invention. For example, a tetracycline compound or a pharmaceutically acceptable salt thereof may be administered in parallel with the administration of the antiviral drug, and prior to the administration of the antiviral drug or after the administration of the antiviral drug is started. Tetracycline compounds or pharmaceutically acceptable salts thereof may be administered. In this case, the tetracycline compound or a pharmaceutically acceptable salt thereof and the antiviral drug may be alternately administered. Further, after the start of administration of the antiviral drug, a tetracycline compound or a pharmaceutically acceptable salt thereof may be co-administered during the administration of the antiviral drug, depending on the degree of improvement of symptoms and the like, and vice versa. , A tetracycline compound or a pharmaceutically acceptable salt thereof may be administered, and then an antiviral drug may be co-administered in the middle of the administration.

Further, the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof may be administered once, continuously, or intermittently as long as it is used in combination with an antiviral drug. For example, in the case of administering the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof prior to the administration of the antiviral drug, from 7 days or 3 days before the start of administration of the antiviral drug. An example can be given of a method of administering once a day every day for about 14 to 21 days. In addition, a tetracycline compound or a pharmaceutically acceptable salt may be administered every day during the administration of the antiviral drug.

The tetracycline compound of the present invention or a pharmaceutically acceptable salt is prepared as a pharmaceutical composition in various forms (dosage forms) according to its administration route (administration method), and is a subject in combination with an antiviral drug. It is administered to patients with viral infections.

As anti-antioxidant agents, for example, rifampicin, isoniazid (hydrazide), streptomycin, etambutol, pyrazinamide, which can be used for infection of tuberculosis flora, and combinations thereof; , Rifampicin, streptomycin, canamycin, and combinations of these; isoniazid, rifampicin, etambitol, which can be used for Mycobacterium kansasii infection, and combinations thereof; , And combinations thereof.

Administration of the antimycobacterial drug can be performed according to a known method.

The mode of combination (combination) of the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof with an acid-fast bacillus drug is not particularly limited as long as it is a mode of use that exerts the effect of the present invention. For example, a tetracycline compound or a pharmaceutically acceptable salt thereof may be administered in parallel with the administration of the acid-fast bacillus drug, or prior to the administration of the acid-fast bacillus drug, or the anti-acid-fast acid. A tetracycline compound or a pharmaceutically acceptable salt thereof may be administered after the start of administration of the mycobacterial drug. In this case, the tetracycline compound or a pharmaceutically acceptable salt thereof and the antimycobacterial drug may be administered alternately. Further, after the start of administration of the acid-fast bacillus drug, a tetracycline compound or a pharmaceutically acceptable salt thereof may be co-administered from the middle of the administration of the acid-fast bacillus drug according to the degree of improvement of symptoms and the like. On the contrary, after the tetracycline compound or a pharmaceutically acceptable salt thereof is administered, an antimycobacterial drug may be administered in combination in the middle of the administration.

In addition, the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof may be administered once, continuously, or intermittently as long as it is used in combination with an antimycobacterial drug. .. For example, in the case of administering the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof prior to the administration of the antimycobacterial drug, for example, from 7 days before the start of administration of the antimycobacterial drug. Alternatively, a method of administering the drug once a day from 3 days before for 14 to 21 days can be exemplified. In addition, a tetracycline compound or a pharmaceutically acceptable salt may be administered every day during the administration of the antimycobacterial drug.

The tetracycline compound of the present invention or a pharmaceutically acceptable salt is prepared as a pharmaceutical composition in various forms (dosage forms) according to the route of administration (administration method) thereof, and is combined with an acid-fast bacillus drug. , Administered to target mycobacterial infection patients.

Examples of antifungal agents include itraconazole, diflucan, prodif, buifend, neyrin, krenafin, nizoral, astat, lamicile, and luricon.

The antifungal drug can be administered according to a known method.

The mode of combination (combination) of the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof with an antifungal agent is not particularly limited as long as it is a mode of use that exerts the effect of the present invention. For example, a tetracycline compound or a pharmaceutically acceptable salt thereof may be administered in parallel with the administration of the antifungal drug, and prior to the administration of the antifungal drug or after the administration of the antifungal drug is started. Tetracycline compounds or pharmaceutically acceptable salts thereof may be administered. In this case, the tetracycline compound or a pharmaceutically acceptable salt thereof and the antifungal drug may be alternately administered. Further, after the start of administration of the antifungal drug, a tetracycline compound or a pharmaceutically acceptable salt thereof may be co-administered during the administration of the antifungal drug, or vice versa, depending on the degree of improvement of symptoms and the like. , A tetracycline compound or a pharmaceutically acceptable salt thereof may be administered, and then an antifungal drug may be administered in combination.

Further, the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof may be administered once, continuously, or intermittently as long as it is used in combination with an antifungal drug. For example, in the case where the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof is administered prior to the administration of the antifungal drug, for example, from 7 days or 3 days before the start of administration of the antifungal drug. An example can be exemplified of a method of administering once a day every day for about 14 to 21 days. In addition, a tetracycline compound or a pharmaceutically acceptable salt may be administered every day during the administration of the antifungal drug.

The tetracycline antibiotics or pharmaceutically acceptable salts of the present invention are prepared as pharmaceutical compositions in various forms (dosage forms) according to the route of administration (administration method), and are subject to combination with antifungal agents. It is administered to patients with fungal infections.

Examples of antitumor agents include those generally used as anticancer agents, such as alkylating agents, metabolic antagonists, antitumor antibiotics, microvascular inhibitors, hormones or hormone-like agents, platinum preparations, etc. Topoisomerase inhibitors, cytokines, antibody drugs, tumor immunological drugs (radioimmunotherapy drugs, non-specific immunoactive drugs, immune checkpoint inhibitors, CAR-T cell drugs, BiTE drugs, cancer vaccines, etc.), molecular target drugs, And other anti-tumor agents can be appropriately selected according to the target tumor.

Although not limited here, examples of alkylating agents include cyclophosphamide, iphosphamide, busulfan, melfaran, bendamstin hydrochloride, nimustin hydrochloride, lanimustin, dagalvazine, procarbazine hydrochloride temozolomid, etc .; as metabolic antagonists. , Metotrexate, Pemetrexed Sodium, Fluorouracil, Doxyflulysin, Capecitabin, Tegafur, Citarabin, Citarabin ocphosphate hydrate, Enocitabin, Gemcitabine hydrochloride, Mercaptoprin hydrate, Fludarabin phosphate ester, Nerarabin, Pentostatin , Holinate calcium, hydroxycarbamide, L-asparaginase, azacitidine, etc .; Examples of antitumor antibiotics include doxorubicin hydrochloride, daunorbisin hydrochloride, pirarubicin, epirubicin hydrochloride, idalbisin hydrochloride, acralbisin hydrochloride, amurubicin hydrochloride, mitoxane. Tron hydrochloride, mitomycin C, actinomycin D, bleomycin, pepromycin sulfate, dinostatin styramer, etc .; Examples of microvascular inhibitors include vincrystin sulfate, vinblastin sulfate, bindesin sulfate, binorelbin tartrate, paclitaxel, docetaxel water. Japanese products, elibrin mesylate, etc .; as hormones or hormone-like drugs (hormone agents), anastrozole, exemethan, retrozol, tamoxyphen citrate, tremiphen citrate, flubestland, flutamide, bicartamide, medroxy Progesterone acetate, estramustin phosphate sodium hydrate, goselelin acetate, leuprorelin acetate, etc .; as platinum preparations, cisplatin, milliplatin hydrate, carboplatin, nedaplatin, oxaliplatin, etc.; as topoisomerase inhibitors Is a topoisomerase I inhibitor such as irinotecan hydrochloride hydrate and nogitecan hydrochloride, and a topoisomerase II inhibitor such as etoposide and sobzoxane; , Trustuzumab, rituximab, gemtuzumab ozogamicin, bebashizumab, setuximab, panitumumab, alemtuzumab, etc .; Tinib, imatinib mesylate, voltezomib, erlotinib hydrochloride, sorafenib tosylate, sunitinib malate, salidamide, nivolumab hydrochloride hydrate, dasatinib hydrate, lapatinib tosilate hydrate, everolimus, renaridamide hydrate, Dexametazone, temsirolimus, bolinostat, tretinoin, tamibarotene, etc .; non-specific immunoactive agents include OK-432, dried BCG, Kawatake polysaccharides, lentinan, ubenimex, etc.; immune checkpoint inhibitors include atezolizumab, nivolumab. , Pembrolizumab, ipilimumab, durvalumab, avelumab, tretinolimus, etc .; as CAR-T cell drugs, Sisagenlecleucel, etc .; as bispecific molecular drugs, BiTE ™ drugs such as brinatsumomab; Etc .; In addition, acegraton, porphimer sodium, taraporfin sodium, ethanol, arsenic trioxide and the like can be exemplified. Here, the bispecific molecule is an antigen-binding region that binds to at least one surface antigen present in tumor cells and an antigen-binding region that binds to at least one surface antigen present in T cells within one molecule. Intended for molecules with and. The number of antigen-binding regions that bind to at least one of the surface antigens may be one or two or more for one antigen. For example, the antigen-binding region that binds to at least one of the surface antigens is a combination of the same type of Fab region, a combination of the same type of variable region, a combination of the same type of sc-Fv, and a heavy chain Fab region derived from one type of antibody. Combination of Chain Fab Regions A combination of a heavy chain variable region and a light chain variable region derived from one type of antibody may be used, and a combination of heavy chain sc-Fv derived from one type of antibody may be used. The surface antigen of a T cell is not limited as long as it exists on the surface of the T cell and at least one antigen-binding region contained in the bispecific molecule or trispecific molecule can bind to it. As the surface antigen of T cells, preferably, the surface antigen of cytotoxic T cells can be mentioned. For example, T cell surface antigens include CD3, CD8, TCR, CTLA-4, PD1, Tim3, CD27, CD28, CD40, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS). .. It is preferably CD3. The surface antigen of a tumor cell is present on the surface of the tumor cell and is not limited as long as at least one antigen-binding region contained in the bispecific molecule can bind. As surface antigens of tumor cells, EphA1 (ephrin type-A receptor 1), EphA2, FolR1 (forate receptor 1), EpCAM (Epidermal growth factor receptor 1), CD19, Erbb2 : ErbB1), Her2, CD20, EGFR (Epidermal Growth Factor Receptor), CCR4 (CC chemokine receptor type 4), CEA (Carcinomebryonic, G2G3G3G3DIG3, (Mucin1), PSCA (Prostate stem cell antigen), PSMA (Prostate-specific memory antigen), HLA-A1 + NY-ESO1 (HLA-A1 restricted NY-ESO1), HLA-A2 , HLA-A3 + NY-ESO1 (HLA-A3 antigened NY-ESO1) and the like. Bispecific molecules include BiTE ™, bispecific antibodies and the like. Examples of BiTE ™ include bispecific molecules targeting CD19 and CD3, and bispecific molecules targeting EphA2 and CD3. Examples of bispecific antibodies include antibodies that target EpCAM and CD3.

From the viewpoint of the action and effect of tetracycline compounds, tumor immunotherapeutic agents can be preferably mentioned as antitumor agents, and more preferably immune checkpoint inhibitors, CAR-T cell agents, bispecific molecular agents, and the like. At least one selected from the cancer vaccine can be mentioned. The most preferred of the tumor immunological agents are immune checkpoint inhibitors (atezolizumab, nivolumab, pembrolizumab, ipilimumab, durvalumab, avelumab, tremelimumab, etc.).

Administration of antitumor drug can be performed according to a known method. In addition, in determining the dose of the antitumor drug, Example 1. The intensity of tumor cell injury activity of T cells of peripheral blood mononuclear cells was evaluated for each patient according to the method for measuring tumor cell injury activity using peripheral blood mononuclear cells, and the dose was adjusted according to this intensity. May be determined.

The mode of combination (combination) of the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof with an antitumor drug is not particularly limited as long as it is a mode of use that exerts the effect of the present invention. For example, a tetracycline compound or a pharmaceutically acceptable salt thereof may be administered in parallel with the administration of the antitumor drug, or tetracycline may be administered prior to the administration of the antitumor drug or after the administration of the antitumor drug. Tetracyclines or pharmaceutically acceptable salts thereof may be administered. In this case, the tetracycline compound or a pharmaceutically acceptable salt thereof and the antitumor drug may be alternately administered. Further, after the administration of the antitumor drug, a tetracycline compound or a pharmaceutically acceptable salt thereof may be co-administered from the middle of the administration of the antitumor drug according to the degree of shrinkage of the tumor tissue, and vice versa. After administration of a tetracycline compound or a pharmaceutically acceptable salt thereof, an antitumor drug may be co-administered in the middle of the administration.

Further, the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof may be administered once, continuously, or intermittently as long as it is used in combination with an antitumor drug. For example, in the case where the tetracycline compound of the present invention or a pharmaceutically acceptable salt thereof is administered prior to the administration of the antitumor drug, for example, from 7 days or 3 days before the start of administration of the antitumor drug. An example can be exemplified of a method of administering once a day every day for about 14 to 21 days. In addition, a tetracycline compound or a pharmaceutically acceptable salt may be administered every day during the administration of the antitumor drug.

The tetracycline compound of the present invention or a pharmaceutically acceptable salt is prepared as a pharmaceutical composition in various forms (dosage forms) according to its administration route (administration method), and is a subject in combination with an antitumor drug. It is administered to tumor patients.

Patients to whom the immunostimulatory agent is administered in the treatment of tumors are not limited as long as the tumor immunotherapy can be applied. For example, as a patient, a patient who has any of the above-mentioned tumors and has not been treated, a patient who has already received some tumor treatment, a patient who is being treated for the tumor, or a tumor that has recurred or metastasized. Patients can be mentioned. The treatment of the tumor preferably includes surgical tumor resection, chemotherapy, radiotherapy and the like.

2. Therapeutic or preventive composition 1. The tetracycline compounds or pharmaceutically acceptable salts thereof described in the above, or immunostimulatory agents can be used as therapeutic or prophylactic compositions. Above 1. The description of the tetracycline compounds or pharmaceutically acceptable salts thereof described in the above is incorporated herein by reference. In addition, the composition as a composition for treatment or prevention, the dose, the administration period, the administration method, and the administration subject are as described in 1. above. Since it is the same as the immunostimulatory agent described in 1. above. The explanation of is used here. Therapeutic or prophylactic compositions can be used for the treatment and / or prevention of viral infections, or tumors. Treatment may include ameliorating or curing the disease. Prevention may include preventing the onset or recurrence of the disease and suppressing the exacerbation of the disease.

3. 3. Composition for Infectious Disease Treatment The composition for infectious disease treatment is described in 1. above. Includes the immunostimulatory agents described in 1 and antiviral agents, antimycobacterial agents, or antifungal agents. The antiviral drug, antimycobacterial drug, and antifungal drug are described in 2. above. The explanation of is used here. Hereinafter, antiviral drugs, acid-fast bacillus drugs, and antifungal drugs are collectively referred to as "anti-infectious disease drugs".
Infectious disease therapeutic compositions comprising an immunostimulatory agent and an anti-infective agent include:
(I) When the immunostimulatory agent and the anti-infectious disease drug are contained in the same preparation in a mixed manner (combination drug).
(Ii) An immunostimulatory agent alone or a preparation containing an immunostimulatory agent and an anti-infectious disease drug alone or a preparation containing an anti-infectious disease drug are packaged as separate preparations, and both are combined. When sold as (kit)
(Iii) A pharmaceutical product containing an immunostimulatory agent alone or an immunostimulatory agent and a pharmaceutical product containing an anti-infectious disease drug alone or an anti-infectious disease drug are individual pharmaceutical products, and these are combined into one package. Or (iv) the immunostimulatory agent alone or the preparation containing the immunostimulatory agent and the anti-infectious disease drug alone or the preparation containing the anti-infectious disease drug are packaged as separate preparations. If both exist in the market through separate distribution channels and are used in combination at the time of use.
Infectious disease therapeutic compositions containing antiviral agents are used as viral infectious disease therapeutic compositions. A composition for treating an infectious disease containing an acid-fast bacillus drug is used as a composition for treating an acid-fast bacillus infection. Infectious disease therapeutic compositions containing antifungal agents are used as fungal infectious disease therapeutic compositions.

4. Treatment method for viral infections This disclosure describes the above 1. The immunostimulatory agent described above, 2. The composition described and the above 3. Includes methods of treating infectious diseases using the compositions for treating infectious diseases described in. The administration method and administration target of the immunostimulatory agent and the composition for treating infectious diseases are as described in 1. above. The explanation described in is incorporated here.

5. Tumor therapeutic composition The tumor therapeutic composition is described in 1. above. Includes the immunostimulatory agent described in 1 and an antitumor agent. Preferably, the antitumor agent is a tumor immunology agent, and more preferably, the tumor immunology agent is at least one selected from an immune checkpoint inhibitor, a CAR-T cell drug, a bispecific molecular drug, and a cancer vaccine. be. The dose is as described in 1. above. The explanation described in is incorporated here.

Tumor therapeutic compositions comprising an immunostimulatory agent and an antitumor agent include:
(I) When the immunostimulatory agent and the antitumor drug are contained in the same preparation in a mixed manner (combination drug).
(Ii) An immunostimulatory agent alone or a preparation containing an immunostimulatory agent and an antitumor drug alone or a preparation containing an antitumor drug are packaged as separate preparations, and both are combined (kit). ) When sold as
(Iii) The individual product of the immunostimulatory agent or the preparation containing the immunostimulatory agent and the individual product of the antitumor drug or the preparation containing the antitumor drug are individual preparations, and these are combined and sold as one package. When, or (iv) the immunostimulatory agent alone or the preparation containing the immunostimulatory agent and the antitumor drug alone or the preparation containing the antitumor drug are packaged as separate preparations. When both exist in the market through separate distribution channels and are used in combination at the time of use.

6. Tumor Treatment Methods The present disclosure includes tumor treatment methods using the immunostimulatory agents described above and tumor therapeutic compositions. The administration method and administration target of the immunostimulatory agent and the composition for treating tumors are as described in 1. above. The explanation described in is incorporated here.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not construed as being limited to Examples.

I. Each experimental protocol 1. Measurement of tumor cell injury activity using peripheral blood mononuclear cells and evaluation of each test drug As shown in Fig. 1, in vitro, peripheral blood mononuclear cells (PBMC) and tumor cells By measuring how much tumor cells were damaged by PBMC (tumor cell-damaging activity) after contacting and culturing through an engager such as BiTE®, antitumor of T cells in PBMC The activity was evaluated.

(1) The U251 cell line was cultured in RPMI1640 medium supplemented with 10% FBS (RPMI1640 medium supplemented with 10% FBS), then peeled off with trypsin, and the RPMI1640 medium supplemented with 10% FBS so as to be ^{1 × 10 5 cells / mL.} A cell suspension was prepared by suspending in. The cell suspension was seeded on a 96-well plate to 100 μL / well and incubated at 37 ° C. for 18 hours in a wet incubator in the presence of 5% carbon dioxide.

(2) Peripheral blood (heparin blood sampling) was collected from healthy subjects ^{, and PBMC was recovered using Lymphoprep TM} (Alere Technologies AS) according to the attached protocol. ^{Specifically, the peripheral blood collected in Lymphoprep TM} placed in a tube was slowly layered, centrifuged at 400 g for 50 minutes, and then the lymphocyte layer was collected. RPMI1640 medium containing 10% FBS was added to the recovered solution and centrifuged, and then the supernatant was removed. Cell pellets were suspended in RPMI 1640 medium supplemented with 10% FBS to adjust to ^{1 × 10 6 / mL.} The recovery of PBMC was performed immediately before the step (3) described later.

(3) EphA2-CD3 BiTE®, which has an antigen-binding site for EphA2 and an antigen-binding site for CD3 in one molecule, was prepared at 400 ng / mL in RPMI1640 medium supplemented with 10% FBS (“BiTE solution”). "). In addition, each test drug was adjusted to 10 μM in the BiTE solution. To the 96-well plate after incubation in (1), 50 μL of PBMC cell suspension and 50 μL of BiTE solution containing each test drug were added per well. The final concentration of BiTE was 100 ng / mL, and the final concentration of each test drug was 2.5 μM. As controls, 50 μL of PBMC cell suspension and 50 μL of BiTE solution containing no test drug are added into a 96-well plate that has been incubated in (1), and a 96-well plate that has been incubated in (1). A well was prepared to which 50 μL of PBMC cell suspension and 50 μL of RPMI1640 medium supplemented with 10% FBS were added. The amount of RPMI medium with 10% FBS per well was finally adjusted to a total volume of 200 μL. A 96-well plate after the addition of PBMC and EphA2-CD3 BiTE® or after the addition of PBMC and EphA2-CD3 BiTE® and the study drug was incubated at 37 ° C. for 48 hours.

(4) After completion of the incubation, the medium of each well was collected together with PBMC. Further, 200 μL of RPMI medium containing 10% FBS was added to each well to wash the inside of the well, and the medium used for washing was collected. This operation was repeated 3 times and the inside of the well was washed 3 times. At this time, the medium collected first and the medium collected at the time of washing were put together in one tube for each well. After washing, 100 μL of 10% FBS-added RPMI medium and 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Reagent (MTS reagent) were added to each well from which the medium and PBMC had been removed. Then, the 96-well microplate was incubated at 37 ° C. for about 30 minutes to 1 hour in a wet incubator in which 5% carbon dioxide gas was present.

(5) For the 96-well microplates that had been incubated in (4), the absorbance of each well at 492 nm was measured using a microplate reader. Subsequently, the tumor cytotoxic activity was calculated according to the following formula.

Absorbance of wells containing only U251 cell line and PBMC = A
Absorbance of wells containing U251 cell line, PBMC and EphA2-CD3 BiTE®, or well containing U251 cell line, PBMC, EphA2-CD3 BiTE® and test drug = B tumor cytotoxic activity ( %) = [(AB) / A] x 100

2. Granzyme B staining Above 1. The medium collected in (4) was centrifuged at 400 g for 5 minutes, the supernatant was removed, and PBMC pellets were left. Each antibody (CD3, CD4, CD8, CD45RA) is diluted 100-fold with 2% FBS and 10 mM HEPES-added HBSS medium (hereinafter referred to as "HBSS + 2% FBS + 10 mM HEPES" medium), and these antibody diluents are used. Was added to the pellet from which the supernatant had been removed at 30 μL / tube each, and incubated at 4 ° C. for 30 minutes. HBSS + 2% FBS + 10 mM HEPES medium was added at 200 μL / tube each and centrifuged at 400 g for 5 minutes, and then the supernatant was removed to leave pellets. After adding 100 μL / tube of Fix buffer to suspend the remaining PBMC, it was incubated at 4 ° C. for 30 minutes.

The tube after incubation was centrifuged at 400 g for 5 minutes to remove the supernatant, leaving PBMC pellets. After adding 200 μL / tube of Wash buffer to suspend the pellets, the pellets were centrifuged at 400 g for 5 minutes to remove the supernatant, leaving the pellets of PBMC. The anti-Granzyme B antibody was diluted 100-fold with Wash buffer, and the remaining PBMC was suspended in 30 μL / tube of this antibody diluent and incubated at 4 ° C. for 30 minutes. 200 μL / tube of Wash buffer was added, and the mixture was centrifuged at 400 g for 5 minutes to remove the supernatant, leaving pellets of PBMC. The remaining PBMC was suspended in HBSS + 2% FBS + 10 mM HEPES medium, and the suspension was subjected to FACS analysis.

3. 3. CytoTell Assay Above 1. PBMC was collected from the peripheral blood of a healthy person according to the method (1). A solution of CytoTell red mixed with HBSS + 2% FBS + 10 mM HEPES medium was added to the recovered PBMC pellets, and the mixture was incubated at 37 ° C. for 30 minutes. After the incubation was completed, the mixture was centrifuged at 400 g for 5 minutes, the supernatant was removed, and pellets of PBMC were left. RPMI medium supplemented with 10% FBS was added to suspend the remaining PBMC, and a 1 × 10 ⁶ / mL PBMC cell suspension was prepared.

Above 1. In the same manner as in (3), the U251 cell line, PBMC and BiTE, or the U251 cell line, PBMC, BiTE and the test drug were brought into contact with each other and cultured at 37 ° C. for 96 hours.

After the culture was completed, the culture supernatant was collected, and the medium was further washed with RPMI1640 medium containing 10% FBS three times, and the medium was also collected. At this time, the medium collected first and the medium collected at the time of washing were put together in one tube for each well.

The collected medium was centrifuged at 400 g for 5 minutes to remove the supernatant, leaving PBMC pellets. Each antibody (CD3, CD4, CD8, CD45RA) was diluted 100-fold in HBSS + 2% FBS + 10 mM HEPES medium, these antibody solutions were added to pellets at 30 μL / tube, and the remaining PBMC was suspended at 4 ° C. Incubated for 30 minutes.

HBSS + 2% FBS + 10 mM HEPES medium was added to each tube after incubation at 200 μL / tube and centrifuged at 400 g for 5 minutes, the supernatant was removed, and PBMC pellets were left. The remaining PBMC was suspended in HBSS + 2% FBS + 10 mM HEPES medium, and the suspension was subjected to FACS analysis.

4. CMV-specific CTL induction (Mixed CMV peptide / Lymphocytes culture)
CMV peptide adjusted to 10 μg / mL in RPMI medium with 10% FBS was added to PBMC of healthy subjects with HLA type of 24:02, and cultured on a 96-well plate for 3 days. The medium was replaced with RPMI medium containing 10 μg / mL CMV peptide + 20 U / mL IL-2 ± 2.5 μM demethylchlortetracycline (DMC) and cultured for another 4 days (7-day culture). The medium of each well containing PBMC was transferred to a 24-well plate and scaled up, and then cultured in RPMI medium containing 10 μg / mL peptide + 20 U / mL IL-2 ± 2.5 μM DMC for another 3 days. The medium was replaced with RPMI medium containing 10 μg / mL peptide + 20 U / mL IL-2 ± 2.5 μM DMC and cultured for another 4 days (14-day culture).

After completion of the culture, centrifuge at 400 g for 5 minutes to remove the supernatant to leave PBMC pellets, and dilute each antibody (CD3, CD4, CD8) 100-fold in HBSS + 2% FBS + 10 mM HEPES medium and CMV Tetramer. Was diluted 10-fold) and added to PBMC pellets at 30 μL / tube to suspend PBMC and incubated at 4 ° C for 30 minutes. HBSS + 2% FBS + 10 mM HEPES medium was added at a rate of 200 μL / tube and centrifuged at 400 g for 5 minutes, and the supernatant was removed to leave PBMC pellets.

The remaining PBMC was suspended in HBSS + 2% FBS + 10 mM HEPES medium, and the suspension was subjected to FACS analysis.

5. Collection of T cells in lung cancer tissue For the tumor cytotoxic activity of T cells in lung cancer tissue, cells were recovered from lung cancer tissue according to the following method. Tumor cytotoxic activity was measured as the activity of the cell population.

After transferring the cancer tissue to a 6 cm Dish and shredding it, put it in a tissue stirring solution (HBSS + 2% FBS + 10 mM HEPES with Tumor Dissociation Kit (Miltenyi Biotec) added) and ^{stir with gentleMACS TM} Dissociator (Miltenyi Biotec). Incubated with rotation for 30 minutes in an incubator maintained at 37 ° C. Then, the tissue residue was removed by passing a stirring solution containing cells through a 70 μm mesh, and the filtrate was centrifuged at 600 xg for 10 minutes to collect the precipitate. BD Pharm lyse (BD Biosciences) was added to the cell-containing precipitate and allowed to stand for 2 minutes. HBSS Buffer was added to the lysate containing the cells after standing, and the mixture was centrifuged at 600 xg for 10 minutes, and the precipitate was collected again. A 30% Percoll solution was added to the recovered precipitate, and the mixture was centrifuged at 12000 xg for 30 seconds, and the precipitate was recovered again. The recovered precipitate was washed with HBSS Buffer and centrifuged at 12000 xg for 30 seconds to collect cells in the tissue. The tumor cytotoxic activity of the recovered cells is described in the above-mentioned I. 1. 1. The measurement was carried out in the same manner as in (1) to (5). Since a BiTE that binds to CD3 is used, the tumor cytotoxic activity of the cells recovered from the lung cancer tissue is considered to be the tumor cytotoxic activity of T cells in the lung cancer tissue.

6. IFNγ production assay Above 5. In the above 1. The IFNγ-producing ability of all the cells in the medium collected according to (4) was measured using the IFN-γ Secretation Assay Kit (Miltenyi Biotec).

Above 1. The medium collected in (4) was centrifuged at 400 g for 5 minutes to remove the supernatant. To the remaining PBMC pellets, IFNγ Catch Reagent diluted 100-fold with 10% human serum-added AIM medium was added at 100 μL / tube each, the PBMC was suspended, and then incubated at 4 ° C. for 10 minutes.

1 mL / tube of 10% human serum-added AIM medium warmed to 37 ° C was dispensed into the tube after incubation, and the tube was incubated at 37 ° C for 45 minutes while stirring. After completion of the incubation, the cells were cooled again on ice and then centrifuged at 400 × g for 5 minutes to remove the supernatant, leaving pellets of PBMC.

Suspend the remaining PBMC in HBSS + 2% FBS + 10 mM HEPES medium, divide each sample into two (one for isotype staining), and centrifuge again at 400 g for 5 minutes to remove the supernatant and leave pellets. rice field.

Each antibody (CD3, CD4, CD8, CD45RA) was diluted 100-fold and anti-i-IFNγ antibody was diluted 10-fold in HBSS + 2% FBS + 10 mM HEPES medium, and 30 μL / L of these antibody dilutions was added to the remaining PBMC. PBMC was suspended by adding tubes at a time and incubated at 4 ° C. for 30 minutes.

Add 200 μL / tube of HBSS + 2% FBS + 10 mM HEPES medium to the tube after incubation, centrifuge at 400 g for 5 minutes, remove the supernatant, and remove the supernatant again, and PBMC remaining in HBSS + 2% FBS + 10 mM HEPES medium. Was suspended and the suspension was subjected to FACS analysis.

7. Verification of tumor cell proliferation inhibitory effect in vivo CT26WT cells were thawed 5 days before inoculation and cultured in DMEM + 10% FBS + 1% penicillin / streptomycin medium. CT26WT cells were trypsinized and passaged 2 days prior to tumor cell inoculation.

After trimming the hair on the right dorsal side of BALB / c mice, CT26WT cells were intradermally inoculated at ^{2 × 10 5 cells / animal.} On the 6th day after inoculation of tumor cells, the body weight and tumor diameter of the mice were measured, and the mice were divided into a test drug-administered group and a Vehicle group (n = 9 each) so as to be uniform. In the test drug administration group, the test drug was dissolved in physiological saline to adjust the dose to 3.0 mg / mL, and 200 μL / animal was intraperitoneally administered at 30 mg / kg / day. In the Vehicle group, 200 μL / animal of saline alone was intraperitoneally administered. Administration of the test drug or saline was performed daily for 10 days (Fig. 8A). Tumor diameter was measured 10 days, 13 days, and 16 days after administration of tumor cells.

II. Example 1: Verification of the activating effect of the tetracycline compound on T cell antitumor activity. 1. 1. T cell antitumor activity using demethylchlortetracycline (DMC), meclocycline (MC), tetracycline (TC), chlortetracycline (CTC), minocycline (MINO) as test agents according to the method described in The activation effect of The difference between the BiTE single addition group and the BiTE and the test drug combination group was calculated by t-test, where n was 3 (independent healthy person). The result is shown in FIG.

In the BiTE and test drug combination group, the antitumor activity of T cells was enhanced in all of DMC, MC, TC, CTC and MINO compared with the case of adding BiTE alone (DMC, MC and CTC: p). <0.01, TC and MINO: p <0.05). From this, it was clarified that the tetracycline compound has an action of activating T cell antitumor activity.

III. Example 2: Verification of the effect of a tetracycline compound on enhancing the tumor cytotoxic activity of ^{CD8 + T cells.} 1. 1. ^{Tumors of CD8-positive T cells (CD8 +} T cells) in each PBMC collected from 3 healthy subjects using demeclocycline (DMC: final concentration 2.5 μM) as the test drug according to the method described in The effect of tetracycline compounds on cytotoxic activity was examined. CD8 ⁺ T cells were ^{negatively selected with the CD8 +} T cell Isolation Kit (Miltenyi Biotec). As shown in Fig. 3, the tumor cytotoxic activity was higher in ^{CD8 +} T cells supplemented with BiTE and the test drug than in ^{CD8 + T cells supplemented with BiTE alone (p = 0.05).} .. This suggests that tetracycline compounds have the effect of enhancing the tumor cytotoxic activity of ^{CD8 + T cells.}

IV. Example 3: Verification of increase in the abundance ratio of granzyme B-expressing CD8 ^{+ T cells by tetracycline compounds I.} 2. Using demethylchlortetracycline (DMC: final concentration 2.5 μM) as a test drug according to the method described in the above section, the abundance ratio ^{of granzyme B-expressing CD8 + T cells in each PBMC collected from 4 healthy subjects.} The effects of tetracycline compounds were verified. ^{As shown in FIG. 4, the abundance ratio of granzyme B-expressing CD8 +} T cells was higher in PBMC supplemented with both BiTE and the test drug than in PBMC supplemented with BiTE alone (p = 0.01). ). This suggests that tetracycline compounds ^{increase granzyme B-expressing CD8 +} T cells.

V. Example 4: Verification of enhancement of ^{CD8 + T cell proliferation by tetracycline compounds I.} 3. 3. Tetracycline system for the proliferative capacity ^{of CD8 +} T cells in each PBMC collected from 5 healthy subjects using demethylchlortetracycline (DMC: final concentration 2.5 μM) as a test drug according to the method described in The effect of the compound was verified. ^{As shown in FIG. 5, the proliferative capacity of CD8 +} T cells was higher in PBMC to which BiTE and the test drug were added in combination than in PBMC to which BiTE was added alone (p = 0.007). This suggests that tetracycline compounds enhance the proliferative capacity of ^{CD8 + T cells, which exhibit tumor cytotoxic activity.}

VI. Example 5: Verification of the effect of tetracycline compounds on enhancing the induction of CMV-specific cytotoxic T cells. 4. The effect of tetracycline compounds on inducing CMV-specific cytotoxic T cells by stimulating PBMC with CMV antigen was examined according to the method described in. Demethylchlortetracycline (DMC: final concentration 2.5 μM) was used as the test drug. PMBC was collected from one healthy person. CMV-specific cytotoxic T cells were identified by FACS analysis. As shown in FIG. 6A, the induction of CMV-specific cytotoxic T cells (fractionated into compartment P7 in FIG. 6A) is still 7 days after stimulation of CMV-specific cytotoxic T cells with an inducing antigen. Although not observed, induction of CMV-specific cytotoxic T cells was observed on day 14. Comparing the group without DMC (DMC-) and the group with DMC (DMC +), DMC + was significantly more CMV-specific cytotoxic T cells (CMV Tetramer + CD8 ⁺ T cells) than DMC-. ) Increased (Fig. 6B; p <0.05). This suggests that tetracycline compounds enhance the proliferative capacity of antigen-specific T cells. It was also shown that it can induce the proliferation of T cells that recognize viral antigens as well as tumor antigens.

VII. Example 6: Verification of the effect of the tetracycline compound on enhancing the tumor cytotoxic activity of T cells in lung cancer tissue. 5. T cells in the tumor tissue of a lung cancer patient were collected according to the method described in the above, and the tumor cell-damaging activity was measured using the cells to verify the effect of the tetracycline compound. In addition, the above I. 5. T cells in the tumor tissue of a lung cancer patient were collected according to the method described in the above I.I. 6. ^{The abundance ratio of IFNγ-producing CD8 +} T cells was measured according to the method described in the above, and the effect of the tetracycline compound was verified.

Demethylchlortetracycline (DMC) was used as a test drug, and the cytotoxic activity measurement of n was 5 (independent patients), and ^{the abundance ratio measurement of IFNγ-producing CD8 +} T cells was 3 (independent patients).

In the BiTE and test drug combination group, the antitumor activity of T cells was enhanced as compared with the case where BiTE was added alone (Fig. 7A).

In the BiTE and test drug combination group, ^{the abundance ratio of IFNγ-producing CD8 +} T cells was higher than when BiTE was added alone (Fig. 7B, C). This suggests that tetracycline compounds ^{increase IFNγ-producing CD8 +} T cells.

VIII. Example 7: Effect of tetracycline compound in vivo 7. The inhibitory effect of tetracycline compounds on tumor growth in vivo was verified according to the method described in. Compared with the Vehicle group, the test drug-administered group tended to suppress the increase in tumor diameter (Fig. 8B).

From the above results, it was shown that tetracycline compounds have the effect of activating the antitumor effect of T cells.

IX. Example 8: Verification of the combined effect of the tetracycline compound and the anti-PD-L1 antibody in vivo It was verified that the tetracycline compound can enhance the antitumor effect of the immune checkpoint inhibitor in vivo.

1. 1. Method CT26WT cells were thawed 6 days before inoculation and cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. Two days prior to administration to the tumor, CT26WT cells were trypsinized and passaged.
The hair on the right dorsal side of Balb / c 6-week-old mice was trimmed and ^{then inoculated into the skin at the trimmed site of 3 × 10 5} cells / 50 μL / animal (Day 0). On Day 6, the body weight and tumor diameter of the mice were measured, and 9 mice were divided into 5 groups so that they were uniform. BioXCell's InVivoMAb anti-mouse PD-L1 was used as the anti-PD-L1 antibody.

The breakdown of each group is as follows.
Group to administer control IgG and water (control IgG + H2O group)
Group to administer anti-PD-L1 antibody and water (aPD-L1 + H2O group)
Group receiving anti-PD-L1 antibody and DMC (300 mg / kg) (aPD-L1 + DMC (300 mg / kg) group) Group receiving anti-PD-L1 antibody and DMC (100 mg / kg) (aPD-L1 + DMC) (100 mg / kg) group) Anti-PD-L1 antibody and DMC (30 mg / kg) administration group (aPD-L1 + DMC (30 mg / kg) group)

Introduction DMC administration was started on Day 7. After dissolving DMC in physiological saline, 200 μl / animal was orally administered using an oral sonde. In the DMC non-administered group, 200 μL / animal of distilled water was orally administered. DMC was administered once daily from Day 7 to Day 12.

Next, on Day 10, anti-PD-L1 antibody or control IgG was intraperitoneally administered at 200 μg / 200 μL / animal.
Tumor diameter was measured on Day 10 and Day 13. The tumor volume was ^{calculated by the formula (minor axis 2} x major axis) / 2.

2. Results Fig. 9 shows the measurement results. In the control IgG + H2O group, the tumor volume increased from about ^{40 mm 3} ^{at the start of DMC administration to about 160 mm 3.} On the other hand, the tumor volume of the aPD-L1 + H2O group was only about ^{90 mm 3.} Furthermore, in the group to which DMC was pre-administered, the increase in tumor volume was suppressed more than in the aPD-L1 + H2O group. In particular, in the aPD-L1 + DMC (30 mg / kg) group, the effect of suppressing the increase in tumor volume was observed.
From this, it was shown that the administration of tetracycline compounds enhances the antitumor effect of immune checkpoint inhibitors.

X. Example 9: Verification that the enhancing effect of the anti-PD-L1 antibody on the antitumor effect of the ^{tetracycline-based compound in vivo depends on CD8 +} T cells. The immune checkpoint inhibitor exhibited by the tetracycline-based compound in vivo. To verify whether the antitumor effect of CD8 ⁺ T cells depends on the antitumor effect of CD8 ⁺ T cells, tetracycline compounds are used as immune checkpoint inhibitors in the presence and absence of CD8 + T cells. The effect of T cell on antitumor effect was investigated.

1. 1. Method CT26WT cells were cultured in the same manner as in Example 8 and inoculated subcutaneously into Balb / c 6-week-old mice (Day 0). On Day 6, the body weight and tumor diameter of the mice were measured, and 9 mice were divided into 6 groups so that they would be uniform.
Group to administer control IgG and water (IgG + H2O group)
Group to administer anti-PD-L1 antibody and water (aPD-L1 + H2O group)
Group to administer anti-PD-L1 antibody, water and anti-CD8 antibody (aPD-L1 + H2O + CD 8 depletion group)
Group to administer anti-PD-L1 antibody, DMC (30 mg / kg) and anti-CD8 antibody (aPD-L1 + DMC + CD8 depletion group)
Group to administer control IgG and DMC (30 mg / kg) (IgG + DMC group)
Group to administer anti-PD-L1 antibody and DMC (30 mg / kg) (aPD-L1 + DMC group)

Prior to administration of DMC or water and anti-PD-L1 antibody, 400 μg / 200 μL / animal of anti-CD8 antibody (InVivoMAb anti-mouse CD8a, BioXCell) was intraperitoneally administered to the CD8 depletion group on Day6. Control IgG 400 μg / 200 μL / animal was administered to the other groups.

Next, DMC administration was started on Day 7. After dissolving DMC in physiological saline, 200 μL / animal was orally administered using an oral sonde. In the DMC non-administered group, 200 μL / animal of distilled water was orally administered. DMC was administered once daily from Day 7 to Day 12.

Next, on Day 10, anti-PD-L1 antibody or control IgG was intraperitoneally administered at 200 μg / 200 μL / animal.
Tumor diameter was measured on Day 10 and Day 13. The tumor volume was calculated in the same manner as in Example 8.

2. Results Figure 10 shows the results. In the IgG + H2O group, the tumor volume on Day 13 was about ^{150 mm 3.} In contrast, the tumor volume of the aPD-L1 + H2O group on Day 13 was about ^{120 mm 3.} In addition, the increase in tumor volume was suppressed in the IgG + DMC group compared to the aPD-L1 + H2O group, and the tumor volume on Day 13 was about ^{100 mm 3.} Furthermore, in the aPD-L1 + DMC group, the tumor volume on Day 13 was about ^{80 mm 3.} On the other hand, when anti-CD8 antibody was administered, the tumor volume increased faster than in the IgG + H2O group, and in the aPD-L1 + H2O + CD 8 depletion group and aPD-L1 + DMC + CD8 depletion group, the tumor volume increased on Day 10 compared to the IgG + H2O group. Was there. ^{Furthermore, on Day 13, the tumor volume was around 300 mm 3 in} both the aPD-L1 + H2O + CD 8 depletion group and the aPD-L1 + DMC + CD8 depletion group, even if anti-PD-L1 antibody or anti-PD-L1 antibody and demethylchlortetracycline were present. , The increase in tumor volume was accelerating. On Day 13, the aPD-L1 + DMC group showed a significant decrease in tumor volume compared to the aPD-L1 + H2O group, whereas the aPD-L1 + DMC + CD 8 depletion group and the aPD-L1 + H2O + CD8 depletion group showed a significant difference in tumor volume. There wasn't.

From this, it was shown that the enhancing effect of the immunocheckpoint inhibitor of the tetracycline compound on the antitumor effect ^{depends on CD8 + T cells.}

XI. Example 10: Verification of effect of tetracycline-based compound on cancer antigen-specific CD8 ⁺ T cells in vivo Due to the antitumor effect-enhancing effect of tetracycline-based compound on immune checkpoint inhibitors, cancer antigen-specific CD8 in vivo ^It was verified whether or not + T cells increased.

1. 1. Method Blood was collected from the fundus of the mice of the IgG + H2O group, aPD-L1 + H2O group, and aPD-L1 + DMC group of Example 9 on Day 13. 1 ml of Pharmalyse was added to the tube containing the collected blood to hemolyze the erythrocytes, and the supernatant was removed after centrifugation. HBSS medium containing 2% FBS and 10 mM HEPES (hereinafter referred to as “HBSS + 2% FBS + 10 mM HEPES”) was added to a tube containing the sediment to suspend the sediment, and then the mixture was centrifuged again to remove the supernatant. Gp70 Tetramer [T-Select H-2Ld MuLV gp70 Tetramer-SPSYVYHQF-PE, MBL] was added to the tube containing the sediment and reacted at 4 ° C. for 30 minutes. As a control, β-galactosidase Tetramer [T-Select H-2Ld β-galactosidase Tetramer-TPHPARIGL-PE, MBL] was added to a tube containing sediment and reacted at 4 ° C. for 30 minutes. HBSS + 2% FBS + 10 mM HEPES was added to the tube after completion of the reaction and mixed. The tube was centrifuged again and the supernatant was removed. Anti-CD8 antibody was added into the tube, and after staining, the cells were washed and subjected to FACS measurement, and cells positive for gp70 Tetramer and CD8 were counted.

2. Results Counting results are shown in FIG. Compared with the IgG + H2O group, the aPD-L1 + DMC group had an increased abundance ratio of ^{cancer antigen-specific CD8 + cells.} From this, it was clarified that ^{tetracycline compounds increase cancer antigen-specific CD8 +} T cells when enhancing the antitumor effect of immune checkpoint inhibitors.

XII. Example 11: Evaluation of Tumor Cell Injury Activity of Tetracycline Compound In order to verify that the tetracycline compound does not have the activity of directly injuring cells, how much the tumor cell was injured by the tetracycline compound (tumor). The direct tumor cytotoxic activity of each test drug was evaluated by measuring the cytotoxic activity). DMC was used as a tetracycline compound. In addition, the antitumor agents entinostat and olaparib were used as positive controls for compounds having tumor cytotoxic activity.

1. 1. Tumor cytotoxic activity when each test drug is administered alone 1-1. Method (1) After culturing the U251 cell line in RPMI1640 medium supplemented with 10% FBS (RPMI1640 medium supplemented with 10% FBS), peel off with trypsin, and RPMI1640 added with 10% FBS so as to be ^{1 × 10 5 cells / mL.} A cell suspension was prepared by suspending in a medium. The cell suspension was seeded on a 96-well plate to 100 μL / well and incubated at 37 ° C. for 18 hours in a wet incubator in the presence of 5% carbon dioxide.

(2) To the 96-well plate after the incubation in (1), 100 μL of the medium containing each test drug was added per well. Each test drug was prepared to have a final concentration of 5 μM, 2.5 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM. As a control, a well to which only the medium was added was prepared in a 96-well plate after incubation in (1). The amount of RPMI medium with 10% FBS per well was finally adjusted to a total volume of 200 μL. The 96-well plate after adding the test drug was incubated at 37 ° C. for 48 hours.

(3) After completion of the incubation, the medium of each well was collected. 200 μL of RPMI medium containing 10% FBS was added to each well, and the inside of the well was washed, and the medium used for washing was collected. This operation was repeated 3 times and the inside of the well was washed 3 times. To each well from which the medium was removed after washing, 100 μL of RPMI medium containing 10% FBS and 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Reagent (MTS reagent: Promega) were added. Then, the 96-well microplate was incubated at 37 ° C. for about 30 minutes to 1 hour in a wet incubator in which 5% carbon dioxide gas was present.

(4) For the 96-well microplates that had been incubated in (3), the absorbance of each well at 492 nm was measured using a microplate reader. Subsequently, the tumor cytotoxic activity was calculated according to the following formula.
Absorbance of wells containing only U251 cell line = A
Absorbance of well containing U251 cell line and test drug = B
Tumor cytotoxic activity (%) = [(AB) / A] x 100

1-2. Results The results are shown in FIGS. 12A to 12C. Regarding DMC, the tumor cytotoxic activity was slightly increased by the addition of 1 μM and 0.1 μM, but it was about several percent (Fig. 12A). The positive subject, entinostat (Fig. 12B), showed tumor cytotoxic activity with the addition of 5 μM and 2.5 μM. Olaparib (Fig. 12C) had a slight increase in tumor cytotoxic activity with the addition of 5 μM.

2. Evaluation of each test drug in tumor cell injury activity and IFNγ production using peripheral blood mononuclear cells In vitro, peripheral blood mononuclear cells (PBMC) and tumor cells were referred to as BiTE®. The antitumor activity of T cells in PBMC was evaluated by measuring how much the tumor cells were damaged by PBMC (tumor cell injury activity) after contacting and culturing through an engager such as).

2-1. Method

(2) Peripheral blood (heparin blood sampling) was collected from healthy subjects, and PBMC was recovered using Lymphoprep TM (Alere Technologies AS) according to the attached protocol. Specifically, the peripheral blood collected in Lymphoprep TM placed in a tube was slowly layered, centrifuged at 400 g for 50 minutes, and then the lymphocyte layer was collected. RPMI1640 medium containing 10% FBS was added to the recovered solution and centrifuged, and then the supernatant was removed. Cell pellets were suspended in RPMI 1640 medium supplemented with 10% FBS to adjust to ^{1 × 10 6 / mL.} The recovery of PBMC was performed immediately before the step (3) described later.

(3) EphA2-CD3 BiTE®, which has an antigen-binding site for EphA2 and an antigen-binding site for CD3 in one molecule, was prepared at 400 ng / mL in RPMI1640 medium supplemented with 10% FBS (“BiTE solution”). "). In addition, the stock solution of each test drug (DMC, entinostat, olaparib) was prepared with the BiTE solution so as to be 20 μM, 10 μM, 4 μM, 0.4 μM, 0.04 μM, 0.004 μM. To the 96-well plate after incubation in (1), 50 μL of PBMC cell suspension and 50 μL of BiTE solution containing each test drug were added per well. BiTE final concentrations were 100 ng / mL, and the final concentrations of each test drug were added to the wells at 5 μM, 2.5 μM, 1 μM, 0.1 μM, 0.01 μM, and 0.001 μM.
As controls, 50 μL of PBMC cell suspension and 50 μL of BiTE solution containing no test drug are added into a 96-well plate that has been incubated in (1), and a 96-well plate that has been incubated in (1). A well was prepared to which 50 μL of PBMC cell suspension and 50 μL of RPMI1640 medium supplemented with 10% FBS were added. The amount of RPMI medium with 10% FBS per well was finally adjusted to a total volume of 200 μL. A 96-well plate after the addition of PBMC and EphA2-CD3 BiTE® or after the addition of PBMC and EphA2-CD3 BiTE® and the study drug was incubated at 37 ° C. for 48 hours.

(4) After completion of the incubation, 80 μM of the medium in each well was collected and stored at -20 ° C. Further, the remaining medium was collected together with PBMC. Further, 200 μL of RPMI medium containing 10% FBS was added to each well to wash the inside of the well, and the medium used for washing was collected. This operation was repeated 3 times and the inside of the well was washed 3 times. At this time, the medium collected first and the medium collected at the time of washing were put together in one tube for each well. After washing, 100 μL of 10% FBS-added RPMI medium and 20 μL of CellTiter 96 (registered trademark) AQueous One Solution Reagent (MTS reagent) were added to each well from which the medium and PBMC had been removed. Then, the 96-well microplate was incubated at 37 ° C. for about 30 minutes to 1 hour in a wet incubator in which 5% carbon dioxide gas was present.

(5) For the 96-well microplates that had been incubated in (4), the absorbance of each well at 492 nm was measured using a microplate reader. Subsequently, the tumor cytotoxic activity was calculated according to the following formula.
Absorbance of wells containing only U251 cell line and PBMC = A
Absorbance of wells containing U251 cell line, PBMC and EphA2-CD3 BiTE®, or well containing U251 cell line, PBMC, EphA2-CD3 BiTE® and test drug = B tumor cytotoxic activity ( %) = [(AB) / A] x 100

(6) The IFNγ concentration of the medium stored at -20 ° C in (4) was measured using Human IFN-gamma Quantikine ELISA Kit (R & D systems).

2-2. Results Tumor cytotoxic activity is shown in FIGS. 12 D to F. Although DMC did not show cytotoxic activity by itself, tumor cytotoxic activity was observed at the addition of 0.1 μM or more by adding PBMC and BiTE at the time of contact with the U251 cell line. Concentration-dependent increases in tumor cytotoxic activity were observed with the addition of 0.1 μM to 0.001 μM DMC, and these increases in tumor cytotoxic activity were simply due to contact between PBMC and BiTE. It is considered that the phenomenon occurred depending on the action of DMC rather than the reaction. In addition, entinostat (FIG. 12E) showed a concentration-dependent increase in tumor cytotoxic activity with an addition of 1 μM or more as compared with the case of single administration shown in FIG. 12B. Olaparib (Fig. 12F) had increased tumor cytotoxic activity with the addition of 2.5 μM or more as compared with the case of single administration shown in FIG. 12C.

The IFNγ concentration in the culture supernatant is shown in G to I in FIG. For DMC, the production of IFNγ concentration was observed when the addition was 1 μM or more, but the production of IFNγ concentration was not observed when the addition was 0.1 μM or less.

Combined with the results of XI.1-2. Above, it was considered that the tetracycline compound induces the activation of T cell-mediated immunity.

It was also shown that tetracycline compounds can induce activation of T-cell-mediated immunity even at a low concentration of 0.1 μM.

XIII. Example 12: Effect of long-term administration of tetracycline compound In order to verify the effect of long-term administration of the tetracycline compound, the tetracycline compound was administered for a longer period than in Example 8 and the effect of the tetracycline compound was observed.
1. 1. Method Six days before inoculation, EMT6 cells, which are breast cancer culture cell lines, were thawed and cultured in DMEM + 10% FBS + 1% + penicillin / streptomycin medium. EMT6 cells were trypsinized and passaged 2 days prior to tumor cell inoculation.

After trimming the hair on the right dorsal side of BALB / c mice, 3 × 10 ⁵ cells / 50 ul / animal of EMT6 cells were intradermally inoculated (Day 0). On the 6th day (Day 6) after inoculation of tumor cells, the body weight and tumor diameter of the mice were measured, and the mice were divided into the following four groups so as to be uniform (n = 9 each).
Group to administer control IgG and water (IgG + H2O group)
Group to administer anti-PD-L1 antibody and water (aPD-L1 + H2O group)
Group to administer control IgG and DMC (30 mg / kg) (IgG + DMC group)
Group to administer anti-PD-L1 antibody and DMC (30 mg / kg) (aPD-L1 + DMC group)

In the DMC-administered group, DMC was dissolved in physiological saline to adjust to 3.0 mg / mL, and 200 μL / animal was orally administered using an oral sonde so as to be 30 mg / kg / day. In the water-administered group, only water was orally administered at 200 μL / animal using an oral sonde. Administration of DMC or saline was performed daily from 7 days to 28 days after tumor inoculation. The DMC-administered group and the physiological saline-administered group were further divided into two groups, and PD-L1 administration or IgG administration was performed. On the 10th day (Day 6) and the 13th day (Day 13) after the administration of the tumor cells, anti-PD-L1 antibody or control IgG was intraperitoneally administered so as to be 200 μg / 200 μL / animal. Tumor diameter was measured every 3 days from Day 10 (Day10, Day13, Day16, Day19, Day22, Day25, and Day28). The tumor volume was calculated in the same manner as in Example 8.

2. Results Figure 13 shows the results. In the IgG + H2O group, the tumor volume on Day 28 was about ^{1300 mm 3.} In contrast, the tumor volume of the aPD-L1 + H2O group on Day 13 was about ^{350 mm 3.} The increase in tumor volume in the IgG + DMC group was similar to that in the aPD-L1 + H2O group. Furthermore, in the aPD-L1 + DMC group, the tumor volume on Day 28 was about ^{100 mm 3.} In addition, no side effects were observed due to long-term administration of DMC.

From this, it was shown that the tumor growth inhibitory effect of the tetracycline compound and the antitumor effect enhancing effect of the tetracycline compound on the immune checkpoint inhibitor are effective for a long period of time. In addition, since the tetracycline compound alone had an inhibitory effect on tumor growth, it was shown that it is highly likely that the effect will be exhibited even in the case of viral infection.

Claims

A therapeutic or prophylactic composition comprising at least one selected from the group consisting of tetracyclines represented by the following general formula (I) and pharmaceutically acceptable salts thereof, the tetracyclines and their pharmaceuticals. A therapeutic or prophylactic composition for administering an acceptable salt at a dose of 1/10 to 1/2 of the prescribed dose used for the treatment of bacterial infections for 8 days or longer:

[During the ceremony,
R 1 is a group represented by the following general formula (II) (R 8 is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms), or a lower alkyl group having 1 to 3 carbon atoms:

..
R 2 is a group represented by the following general formula (III) (R 9 is a lower alkyl group having 1 to 3 carbon atoms or a hydrogen atom):

..
R 3 is a hydrogen atom, a hydroxyl group, or a lower alkyl group having 1 to 3 carbon atoms.
R 4 and R 5 are hydrogen atoms, hydroxyl groups or lower alkyl groups having 1 to 3 carbon atoms together or independently, or R 4 and R 5 are methylene groups in one.
R 6 is a hydrogen atom, a halogen or a group represented by the following general formula (IV) (R 10 is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms):

..
R 7 is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, -NH-CO-CH 2- NH-C (CH 3 ) 3 or -CH 2- NH-CH 2- C (CH 3 ) 3 . be. ].
The therapeutic or preventive composition according to claim 1, wherein the lower alkyl group having 1 to 3 carbon atoms is a methyl group.
The compound represented by the general formula (I) is at least one selected from the group consisting of demethylchlortetracycline, meclocycline, tetracycline, chlortetracycline, doxycycline, minocycline, and oxytetracycline, according to claim 1. Composition for treatment or prevention of.
The therapeutic or prophylactic composition according to any one of claims 1 to 3, which is used for treating or preventing a viral infection or tumor.
An immunostimulatory agent comprising at least one selected from the group consisting of a tetracycline compound represented by the following general formula (I) and a pharmaceutically acceptable salt thereof, which is a tetracycline compound and its pharmaceutically acceptable salt. An immunostimulatory agent for administering the salt to be administered at a dose of 1/10 to 1/2 of the prescribed dose used for the treatment of bacterial infections for 8 days or more:

[During the ceremony,
R 1 is a group represented by the following general formula (II) (R 8 is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms), or a lower alkyl group having 1 to 3 carbon atoms:

..
R 2 is a group represented by the following general formula (III) (R 9 is a lower alkyl group having 1 to 3 carbon atoms or a hydrogen atom):

..
R 3 is a hydrogen atom, a hydroxyl group, or a lower alkyl group having 1 to 3 carbon atoms.
R 4 and R 5 are hydrogen atoms, hydroxyl groups or lower alkyl groups having 1 to 3 carbon atoms together or independently, or R 4 and R 5 are methylene groups in one.
R 6 is a hydrogen atom, a halogen or a group represented by the following general formula (IV) (R 10 is a hydrogen atom or a lower alkyl group having 1 to 3 carbon atoms):

..
R 7 is a hydrogen atom, a lower alkyl group having 1 to 3 carbon atoms, -NH-CO-CH 2- NH-C (CH 3 ) 3 or -CH 2- NH-CH 2- C (CH 3 ) 3 . be. ].
A composition for treating a viral infection, which comprises the immunostimulatory agent according to claim 5 and an antiviral agent.
A composition for treating a tumor, which comprises the immunostimulatory agent according to claim 5 and a tumor immunological agent.
The composition for treating a tumor according to claim 7, wherein the tumor immunological agent is at least one selected from an immune checkpoint inhibitor, a CAR-T cell drug, a bispecific molecular agent, and a cancer vaccine.
A composition for treating an acid-fast bacillus infection, which comprises the immunostimulatory agent according to any one of claims 1 to 3 and an acid-fast bacillus agent.
A composition for treating a fungal infection, which comprises the immunostimulatory agent according to any one of claims 1 to 3 and an antifungal agent.