WO2025033330A1 - Antitumor therapy by combined use of chymase inhibitor and immunotherapy - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition which is for treating a tumor, and which includes a chymase inhibitor as an active ingredient, said pharmaceutical composition being characterized by being used in combination with at least one selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12); a pharmaceutical composition which is for treating a tumor, and which includes, as an active ingredient, at least one selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), said pharmaceutical composition being characterized by being used in combination with a chymase inhibitor; and the like.

Description

Antitumor therapy using chymase inhibitors in combination with immunotherapy

RELATED APPLICATIONS This application claims priority based on Japanese Patent Application No. 2023-127528 (filed August 4, 2023), the contents of which are incorporated herein by reference.
TECHNICAL FIELD The present invention relates to a novel antitumor therapy characterized by combining a chymase inhibitor with immunotherapy using interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, interleukin-12 (IL-12), or the like.

Chymase is a neutral serine protease found mainly in mast cells. Human chymase is known to be involved in hypertension and arteriosclerosis by producing angiotensin II using angiotensin I as a substrate. There have been reports showing that chymase correlates with angiogenesis around tumor tissues in lung cancer, colon cancer, and pancreatic cancer (Non-Patent Documents 1-3), but there have been no reports showing the direct effect of chymase on tumors.

Interleukin-18 (IL-18) is a cytokine discovered by Okamura et al. as an interferon-gamma (IFNγ) production inducer. IL-18 binds to the IL-18 receptor expressed on the cell surface of cells in which IFNγ production is induced in the presence of IL-12, enhancing IFNγ production and enhancing Th1-type immune responses. On the other hand, in the absence of IL-12, IL-18 enhances Th2-type immune responses.

IL-18 is expected to have an antitumor effect, and cancer treatment drugs that combine IL-18 with rituximab or Herceptin (Patent Document 1) and with a molecular target antibody have been reported (Patent Document 2). Recently, it has been reported that a strong antitumor effect was achieved by using CAR-T cells that secrete IL-18 to secrete IL-18 in a tumor-specific manner (Non-Patent Document 4).

IL-18 is produced as an IL-18 precursor containing a propeptide, which is cleaved by caspases and released outside the cell as active IL-18. A method for producing IL-18 has been reported in which the IL-18 precursor is cleaved by chymase instead of caspase (Patent Document 3). This report does not show the effect of chymase inhibitors on active (mature) IL-18, but suggests that chymase inhibitors can inhibit IL-18 activity by inhibiting the conversion of the IL-18 precursor to the active form.

PD-1 and PD-L1 are representative immune checkpoint molecules, and antibodies targeting these molecules have been shown to have significant anti-cancer effects in a variety of cancer types, and are currently being developed as medicines. However, the remission rate for solid cancers with anti-PD-1/PD-L1 antibody therapy is low, and the response rate cannot be said to be sufficient.

WO2006/116423 WO2016/021720 JP2007-151414

Ibaraki et al., The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I non-small cell lung cancer. Eur J Cardiothorac Surg. 2005;28(4):617-621. Kondo et al., Expression of chymase-positive cells in gastric cancer and its correlation with the angiogenesis. J Surg Oncol. 2006;93(1):36-43. Laface et al., Chymase-positive mast cells correlate with tumor angiogenesis: first report in pancreatic cancer patients. Eur Rev Med Pharmacol Sci. 2021;25(22):6862-6873. Svoboda et al., Interleukin-18 Secreting Autologous Anti-CD19 CAR T-Cells (huCART19-IL18) in Patients with Non- Hodgkin Lymphomas Relapsed or Refractory to Prior CAR T-Cell Therapy. Blood (2022) 140 (Supplement 1): 4612-4614

The objective of the present invention is to provide a novel method for treating tumors.

The inventors have found that the antitumor effect of immunotherapy using IL-18, anti-PD-1/PD-L1 antibody (anti-PD-1 antibody or anti-PD-L1 antibody), or the like can be enhanced by combining the immunotherapy with a chymase inhibitor that does not have an antitumor effect by itself. The present application is based on the above findings, and provides the following [1] to [8] as a first aspect.
[1] A pharmaceutical composition for treating a tumor, comprising a chymase inhibitor as an active ingredient, in combination with one or more selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12).
[2] A pharmaceutical composition for treating a tumor, comprising a combination of one or more selected from interleukin-18 (IL-18), PD-1/PD-L1 antibody, and interleukin-12 (IL-12) and a chymase inhibitor.
[3] A pharmaceutical composition for treating a tumor, comprising as an active ingredient any one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), wherein the pharmaceutical composition is used in combination with a chymase inhibitor.
[4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound The pharmaceutical composition according to any one of [1] to [3], which is selected from the group consisting of 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445.
[5] The pharmaceutical composition according to any one of [1] to [4], wherein one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12 are of human origin.
[6] The pharmaceutical composition according to any one of [1] to [5], wherein one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12 and a chymase inhibitor are administered simultaneously or sequentially.
[7] A pharmaceutical composition for enhancing the effect of any one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and IL-12 in tumor treatment, comprising a chymase inhibitor as an active ingredient.
[8] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-am ino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide) The pharmaceutical composition according to [7], which is any one selected from SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445.

As a second aspect, the present application provides the following [1] to [6].
[1] A method for treating a tumor, comprising administering to a subject a combination of a chymase inhibitor and one or more selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12).
[2] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-am ino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide) The method according to [1], wherein the compound is any one selected from SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445.
[3] The method according to [1], wherein one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12 are administered simultaneously with a chymase inhibitor.
[4] The method according to [1], wherein one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12 and a chymase inhibitor are administered sequentially.
[5] A method for enhancing the anti-tumor effect of any one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), comprising administering a chymase inhibitor to a subject administered or who has been administered IL-18, an anti-PD-1/PD-L1 antibody, and IL-12.
[6] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, (2-[5-amino-2-(4-fluoroph enyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), ASB17061, ONO-8020435, and ONO-2220445.

As a third aspect, the present application provides the following [1] to [7].
[1] An agent for use in treating a tumor, comprising a chymase inhibitor, wherein the agent is used in combination with any one or more selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12).
[2] A drug for use in treating a tumor, comprising a combination of one or more selected from interleukin-18 (IL-18), PD-1/PD-L1 antibody, and interleukin-12 (IL-12) and a chymase inhibitor.
[3] A drug for use in treating a tumor, comprising any one or more selected from interleukin-18 (IL-18), an anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), characterized in that the drug is used in combination with a chymase inhibitor.
[4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445.
[5] The composition for use according to any one of [1] to [4], wherein IL-18, the anti-PD-1/PD-L1 antibody, and IL-12 are of human origin.
[6] The drug for use according to any one of [1] to [5], wherein one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12 and a chymase inhibitor are administered simultaneously or sequentially.
[7] The agent for use according to any of [1] to [6], wherein the chymase inhibitor enhances the antitumor effect of any one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12.

As a fourth aspect, the present application provides the following [1] to [7].
[1] Use of one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), together with a chymase inhibitor, in the manufacture of a medicine for treating a tumor.
[2] Use of a chymase inhibitor in the manufacture of a medicament for treating a tumor, characterized in that the medicament is used in combination with one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12).
[3] Use of one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12) in the manufacture of a medicament for treating a tumor, characterized in that the medicament is used in combination with a chymase inhibitor.
[4] Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic acid, N-[1-Benzyl-2,3-dioxo-6-(2-pyridyloxy)hexyl]-2-[2-phenyl-5-(formylamino)-6-oxo-1,6-dihydropyrimidine-1-yl]acetamide, and (2-[5-amino-2-(4-fluorop henyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), SUN-C8257, SUN13834, BCEAB, Compound The use according to any one of [1] to [3], wherein the compound is selected from the group consisting of 17, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Y-40079, ASB17061, ONO-8020435, and ONO-2220445.
[5] The use according to any one of [1] to [4], wherein IL-18, the anti-PD-1/PD-L1 antibody, and IL-12 are of human origin.
[6] The use according to any one of [1] to [5], wherein one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12 and a chymase inhibitor are administered simultaneously or sequentially.
[7] The use according to any one of [1] to [6], wherein the chymase inhibitor enhances the antitumor effect of any one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12.

The present invention provides novel clinical applications of chymase inhibitors and immunotherapeutic agents such as IL-18, anti-PD-1/PD-L1 antibodies, and L-12.

FIG. 1 shows survival curves for the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18/chymase inhibitor combination group 100 days after tumor (CT26) cell transplantation. FIG. 2 shows photographs of the tumor transplant site of a mouse that survived 100 days after tumor (CT26) cell transplantation (top: IL-18, bottom: combined use of IL-18 and chymase inhibitor). FIG. 3 shows survival curves for the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18/chymase inhibitor combination group, 110 days after tumor (CT26) cell transplantation. FIG. 4 shows the percentage (A), absolute number (B), cell size (C), and size of intracellular granules (D) of mast cells in ascites after tumor (CT26) cell transplantation. FIG. 5 shows the number of CD4 ⁺ T cells (A), the number of CD8 ⁺ T cells (B), and the ratio of CD4 ⁺ T cells/CD8 ⁺ T cells (C) after tumor (CT26) cell transplantation. FIG. 6 shows the percentage of NK cells in ascites after tumor (CT26) cell transplantation (A), the absolute number of NK cells (B), and the percentage of DX5 ⁺ B220 ⁺ NK cells (C). FIG. 7 shows the change in the numbers of immune cells (mast cells, CD8 ⁺ T cells, NK cells, and DX5 ⁺ B220 ⁺ NK cells) in the ascites after tumor (CT26) cell transplantation. Figure 8 shows the results of comparing T cells in ascites in the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18/chymase inhibitor combination group. The ratio of CD4 ⁺ T cells/CD8 ⁺ T cells (A), CD4 ⁺ T cell count (B), CD8 ⁺ T cell count (C), and CD44high CD8 ⁺ T cell count (D). Figure 9 shows the results of comparing the number of NK cells in ascites in the placebo group, IL-18 administration group, chymase inhibitor administration group, and IL-18/chymase inhibitor combined administration group. DX5 ⁺ NK cell count (A), DX5 ⁺ B220 ⁺ NK cell count (B) Figure 10 shows survival curves for the placebo group, the anti-PD-1 antibody administration group, the chymase inhibitor administration group, and the anti-PD-1 antibody/chymase inhibitor combination group 34 days after tumor (CT26) cell transplantation.

1. Chymase Inhibitor In this specification, a chymase inhibitor means a drug capable of inhibiting the activity of chymase. Chymase is a neutral serine protease, and human chymase is known to be involved in hypertension and arteriosclerosis by producing angiotensin II from angiotensin I. The chymase inhibitor of the present invention is preferably a human chymase inhibitor.

As the chymase inhibitor, a known chymase inhibitor can be used. Examples of known chymase inhibitors include compounds that have already been reported for clinical application or validation in preclinical models, such as SUN-C8257, SUN13834, BCEAB, Compound 17, NK3201, TEI-E548, RO5066852, JNJ-10311795 (RWJ-355871), Suc-Val-Pro-Phe ^P (OPh) ₂ , Y-40613, Y-40079, TY-51469, Chymostatin, Fulacimstat (BAY1142524), ASB17061, ONO-8020435, and ONO-2220445 (Heuston and Hyland, Br J Pharmacol. 2012 Oct; 167(4): 732-740.doi: 10.1111/j.1476-5381.2012.02055.x etc.)

Or, WO2016/132483, JP 2016-150915, JP 2016-535095, JP 2012-516335, JP 2012-502034, JP 2012-121898, JP 2010-536768, WO2010/147133, JP 2010-515699, JP 2010-505900, WO Chymase inhibitors disclosed in WO 2005/018672, WO03/018061, JP 2004-507539, WO01/062294, WO01/062293, WO01/062292, WO00/005204, WO99/032459, JP 11-001479, and JP 10-087493 can also be used.

2. Interleukin-18 (IL-18)
Interleukin-18 (IL-18) is a cytokine that was discovered as an IFN-γ production inducer. IL-18, together with IL-12, acts on CD4 ⁺ CD8 ⁺ T cells and NK cells to induce IFN-γ production.

IL-18 is produced as an inactive precursor (pro-IL-18) containing the propeptide, and is released as active IL-18 following cleavage of the propeptide by caspase 1, caspase 4, etc. In the present invention, the source of IL-18 is not particularly limited, but it is preferable that the IL-18 is derived from the same species as the subject to be administered, or a closely related species. Therefore, when administered to humans, mammalian IL-18, particularly human IL-18, is preferred.

Human IL-18 precursor consists of 193 amino acids (24 KDa), and mature (active) human IL-18 without the propeptide consists of 157 amino acids (18 KDa). The amino acid sequence of human IL-18 is known, and an example of the amino acid sequence of human IL-18 is the amino acid sequence shown in SEQ ID NO: 1. Human IL-18 having an amino acid sequence that has 80%, 85%, 90%, 95%, 96%, 98%, or 99% or more sequence identity with this amino acid sequence can also be used as the IL-18 of the present invention as long as it has the activity of IL-18. IL-18 activity means the physiological activity of IL-18, such as IFNγ production-inducing activity.

An example of human IL-18 is the following protein (1) or (2):
(1) A protein comprising the amino acid sequence shown in SEQ ID NO:1, or
(2) A protein having 80% or more, preferably 90% or more, and more preferably 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO:1 and having IL-18 activity (such as IFNγ production-inducing activity).

IL-18 exerts its physiological activity by binding to a receptor on the cell membrane, but since there is an excess of IL-18-specific IL-18-binding proteins in the body, much of the secreted IL-18 loses its activity upon binding to the IL-18-binding proteins. Recently, it has been reported that the antitumor effect of IL-18 is enhanced by using modified IL-18 with increased stability and by secreting IL-18 from Car-T cells. Such modified IL-18 and new delivery systems are also within the scope of the present invention, so long as they do not contradict the objectives of the present invention.

3. Anti-PD-1/PD-L1 antibody (anti-PD-1 antibody, anti-PD-L1 antibody)
In the present invention, the term "anti-PD-1/PD-L1 antibody" refers to an antibody that specifically inhibits PD-1 or PD-L1, an anti-PD-1 antibody or an anti-PD-L1 antibody.

The "anti-PD-1 antibody" used in the present invention is not particularly limited as long as it can bind to PD-1 and inhibit its function as an immune checkpoint. Anti-PD-1 antibodies include those that have already been approved and sold as medicines and those that are under development, and these can be used preferably. Examples of such "anti-PD-1 antibodies" include nivolumab (Opdivo (GSK/Ono Pharmaceutical)), pembrolizumab (Merck), which is a humanized IgG4 antibody, and dipirizumab, but are not limited to these.

The "anti-PD-L1 antibody" used in the present invention is not particularly limited as long as it can bind to PD-L1 and inhibit its function as an immune checkpoint. PD-L1 antibodies include those that have already been approved (sold) as medicines and those that are under development, and these can be used preferably. Examples of such "anti-PD-L1 antibodies" include, but are not limited to, atezolizumab (Roche/Chugai), durvalumab (AstraZeneca), avelumab (Merck), and MED10680/AMP-514.

The antibody may be produced according to a conventional method. In this case, the antibody is preferably a chimeric antibody, a humanized antibody, or a fully human antibody in order to reduce heterologous antigenicity to humans. The antibody may be an antibody fragment so long as it has a function as an immune checkpoint inhibitor. Examples of the antibody fragment include F(Ab') ₂ , Fab', Fab, Fv, and scFv.

4. Interleukin-12 (IL-12)
Interleukin-12 (IL-12) is a cytokine produced mainly by macrophages, polymorphonuclear leukocytes, dendritic cells, etc., and is known to induce the production of IFN-γ in NK cells and T cells, and to enhance the activity of NK cells and cytotoxic T cells.

It has been reported that IL-12 preferentially activates Th1 cells and activates cellular immunity by suppressing Th2 cells, and is therefore expected to be used in the treatment of Th2-driven immune disorders and in the treatment of cancer and infectious diseases by activating cellular immune function. As mentioned above, IL-12, along with IL-18, acts on CD4 ⁺ CD8 ⁺ T cells and NK cells to induce the production of IFN-γ. Therefore, IL-12 and IL-18 may be used in combination in the treatment of cancer and infectious diseases.

5. Pharmaceutical compositions (medicines, drugs)
The pharmaceutical composition of the present invention is characterized by effectively treating tumors by combining one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12 with a chymase inhibitor that does not exhibit anti-tumor activity alone.

In a first embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating a tumor, comprising a chymase inhibitor as an active ingredient, and characterized in that it is used in combination with one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12.

In a second embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating a tumor, comprising a combination of a chymase inhibitor and one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12. In this case, the chymase inhibitor and one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12 may be formulated together, or may be formulated separately as a kit formulation.

In a third embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating tumors, comprising one or more active ingredients selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12, and is characterized in that it is used in combination with a chymase inhibitor.

In a fourth embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for enhancing the action of one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12 in tumor treatment, comprising a chymase inhibitor as an active ingredient.

The pharmaceutical composition of the present invention may contain pharmacologically acceptable carriers and additives. Examples of such carriers and additives include excipients, binders, lubricants, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, isotonicity agents, stabilizers, soothing agents, preservatives, antioxidants, flavoring agents, colorants, buffers, and flow promoters, but are not limited to these, and other commonly used carriers and additives can be used as appropriate.

Examples of excipients include organic excipients such as sugars such as lactose, glucose, and D-mannitol, starches, and celluloses such as crystalline cellulose, and inorganic excipients such as calcium carbonate and kaolin.

Binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, etc.

Examples of the lubricant include stearic acid, fatty acid salts such as stearates, talc, silicates, and the like.
Examples of the solvent include purified water, physiological saline, and phosphate buffer.

Disintegrants include low-substituted hydroxypropyl cellulose, chemically modified cellulose, and starches.

Solubilizing agents include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

Suspending agents or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc.

Isotonicity agents include sodium chloride, potassium chloride, sugars, glycerin, urea, etc.

Stabilizers include polyethylene glycol, sodium dextran sulfate, and other amino acids.
Examples of the soothing agents include glucose, calcium gluconate, and procaine hydrochloride.

Preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

Antioxidants include the water-soluble antioxidants ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite, the fat-soluble antioxidants ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol, and the metal chelating agents citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

Flavors include sweeteners and fragrances that are commonly used in the pharmaceutical field, and colorants include colorants that are commonly used in the pharmaceutical field.

The administration route of the pharmaceutical composition of the present invention is not particularly limited, and may be oral or parenteral. Specific examples of parenteral administration include injection, nasal administration, pulmonary administration, and transdermal administration. Examples of injection include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, intramedullary injection, intrathecal injection, and intradermal injection. The administration method can be appropriately selected depending on the age and symptoms of the patient.

The pharmaceutical compositions of the present invention are administered to a subject in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount of a therapeutic agent that is useful for alleviating a selected condition. The therapeutically effective amount is determined as appropriate depending on the intended use, route of administration, age and symptoms of the patient, etc.

Tumors to which the pharmaceutical composition of the present invention can be applied include, for example, leukemias such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, malignant lymphomas such as Hodgkin's lymphoma and non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome, head and neck cancer, tongue cancer, pharyngeal cancer, oral cancer, esophageal cancer, esophageal adenocarcinoma, gastric cancer, colorectal cancer, colon cancer, rectal cancer, liver cancer, gallbladder/bile duct cancer, biliary tract cancer, and pancreatic cancer. , thyroid cancer, lung cancer such as non-small cell lung cancer and small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, renal cancer, adenocarcinoma, urothelial carcinoma, prostate cancer, testicular tumor, bone and soft tissue sarcoma, uveal malignant melanoma, malignant melanoma, skin cancer such as Merkel cell carcinoma, malignant tumors such as neuroblastoma, glioblastoma, brain tumor, and pleural mesothelioma, as well as benign tumors such as angiomyolipoma, but are not limited to these. Preferably, the tumor is a malignant tumor such as colorectal cancer.

The pharmaceutical composition of the present invention may be used in combination with other drugs or treatment methods as long as the purpose of the present invention is not impaired.

6. Treatment Method The present application also provides a method for treating a tumor, comprising administering to a subject a chymase inhibitor in combination with one or more selected from IL-18, an anti-PD-1/PD-L1 antibody, and IL-12.

The present application also provides a method for enhancing the antitumor effect of any one or more selected from IL-18, anti-PD-1/PD-L1 antibodies, and IL-12, comprising administering a chymase inhibitor to a subject to which any one or more selected from IL-18, anti-PD-1/PD-L1 antibodies, and IL-12 are administered or which has been administered to a subject.

In the above method, the chymase inhibitor and one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and IL-12 may be administered simultaneously or sequentially. There are no particular limitations on the administration route for each, and the method described in the previous section can be applied.

The present invention will be specifically explained below using examples, but the present invention is not limited to these examples.

Example 1: Antitumor effect of combined use of peptidic chymase inhibitor and IL-18 Method: Colon cancer cells (CT26: 5 x ¹⁰⁴ cells) were administered intraperitoneally to 7-week-old male BALB-c mice (purchased from Japan SLC), and administration was started on the third day after administration. The placebo group (n=8) received 0.17% dimethyl sulfoxide (drug solvent: placebo) intraperitoneally once a day. The IL-18 administration group (n=8) received 2 μg/body of IL-18 (mouse IL-18: obtained from GSK) intraperitoneally once every two days. The chymase inhibitor administration group (n=8) received 3 mg/kg of Suc-Val-Pro-Phe ^p (OPh) ₂ (peptidic chymase inhibitor: synthesized at Peptide Institute) intraperitoneally once a day. The IL-18/chymase inhibitor combination group (n=8) received 2 μg/mouse IL-18 once every two days and 3 mg/kg chymase inhibitor once a day intraperitoneally. Medication was terminated 30 days after cell transplantation, and analysis was performed 31 and 100 days after cell transplantation.

Observation: Mice were observed twice a day, morning and evening, for survival.
Analysis method: Significance tests for survival curves of each group were performed using the Kaplan-Meier method, with a P value of 5% or less considered significant.

Results: At 31 days after cell transplantation, there was no significant difference in survival time between the placebo group and the IL-18 group (P = 0.5024) or the chymase inhibitor group (P = 0.2068). On the other hand, there was a significant difference between the placebo group and the IL-18/chymase inhibitor combination group (P = 0.0125), indicating that the IL-18/chymase inhibitor combination group had a significantly longer survival time than the placebo group (Figure 1). At 100 days after cell transplantation, there were no surviving mice in the chymase inhibitor group, and one mouse in the IL-18 group was surviving, but there was no significant difference in survival time between the placebo group and the IL-18 group (P = 0.2916) or the chymase inhibitor group (P = 0.2507) (Figure 1). On the other hand, a significant difference was observed between the survival curves of the placebo group and the IL-18/chymase inhibitor combination group (P = 0.0126, Figure 1). Furthermore, the tumors in the surviving mice were macroscopically clear (Figure 2).

In this experiment, administration of a chymase inhibitor alone did not result in an extension of survival time after cancer transplantation, but administration of IL-18 in combination with a chymase inhibitor extended survival time compared to administration of IL-18 alone.

Example 2: Antitumor effect of a combination of a commercially available chymase inhibitor and IL-18 Method: Colon cancer cells (CT26: 5 x ¹⁰⁴ cells) were administered intraperitoneally to 7-week-old male BALB-c mice (purchased from Japan SLC), and administration was started on the third day after administration. The placebo group (n=10) received 0.17% dimethyl sulfoxide (drug solvent: placebo) intraperitoneally once a day. The IL-18 administration group (n=10) received 2μg/body of IL-18 (mouse IL-18 as above) intraperitoneally once every two days. The chymase inhibitor administration group (n=10) received 5mg/kg (100μg/body) of Fulacimstat (chymase inhibitor: MedChemExpress) intraperitoneally once a day. The IL-18 + chymase inhibitor combination group (n=10) received 2 μg/body IL-18 once every two days and 5 mg/kg chymase inhibitor once a day intraperitoneally. Medication was discontinued 30 days after cell transplantation, and analysis was continued up to 110 days after.

After the administration, the mice were checked daily for mortality.
Analysis method: Significance tests for survival curves of each group were performed using the Kaplan-Meier method, with a P value of 5% or less considered significant.

Results: There was no difference in survival rate between the placebo group and the chymase inhibitor group, and all mice died before 40 days in both groups. On the other hand, the IL-18 group and the IL-18/chymase inhibitor combination group showed a clear improvement in survival rate compared to the placebo group, and at 110 days after cell transplantation, the IL-18/chymase inhibitor combination group showed a higher survival rate than the IL-18 group (Figure 3).

　Even when different chymase inhibitors were used, the overall trend was similar to that of peptidic chymase inhibitors. In other words, administration of a chymase inhibitor alone did not result in an extension of survival time after cancer transplantation, but the combination of IL-18 and a chymase inhibitor extended survival time compared to IL-18 alone.

Example 3: Changes in intraperitoneal lymphocyte cells due to tumor antigen stimulation Method: Colon cancer cells (CT26: 5 x ¹⁰⁴ cells) were administered intraperitoneally to 7-week-old male BALB-c mice (purchased from Japan SLC), and ascites was collected 3, 5, 7, 11, 14, 17, and 21 days after cell transplantation (n=4 each time).

To collect ascites, mice were anesthetized with Isoflurane, and chilled PBS was injected intraperitoneally and collected (total 8 ml). The collected ascites was centrifuged and washed twice, and the number of cells was counted using a trypan blue exclusion assay test. The collected cells were applied to a chamber and subjected to HE and TB staining. Cell subsets were also analyzed by flow cytometry.

Results: Mast cells showed a tendency to increase from 7 days after cell transplantation, but granular mast cells decreased (Fig. 4). Mast cells also tended to gather around the tumor tissue (data not shown). Tumor cell transplantation increased CD4 ⁺ T cells and CD8 ⁺ T cells, while the ratio of CD4 ⁺ T cells/CD8 ⁺ T cells decreased (Fig. 5). This is presumably due to an increase in CD8 ⁺ killer cells. NK cells, especially activated killer NK cells (DX5 ⁺ B220 ⁺ NK cells), also increased after tumor cell transplantation (Fig. 6).

Analysis of immune cell subsets in the peritoneal cavity showed that NK cells increased early after transplantation up to day 10, CD8 ⁺ T cells increased from days 10 to 21, and mast cells gradually increased after day 7 (Figure 7).

Example 4: Changes in intraperitoneal antitumor lymphocytes by combined use of a peptide chymase inhibitor and IL-18 Method: Colon cancer cells (CT26: 5 x ¹⁰⁴ cells) were administered intraperitoneally to 7-week-old male BALB-c mice (purchased from Japan SLC), and administration was started on the third day after administration. The control group received 0.17% dimethyl sulfoxide (drug solvent: placebo) intraperitoneally once a day. The IL-18 alone administration group received 2μg/body of IL-18 (see above mouse IL-18) intraperitoneally once every two days. The chymase inhibitor alone administration group received 3mg/kg (100μg/body) of Suc-Val-Pro-Phe ^p (OPh) ₂ intraperitoneally once a day. The IL-18/chymase inhibitor combination group received intraperitoneal administration of 2 μg/body of IL-18 once every two days and 3 mg/kg of chymase inhibitor once a day. Medication was terminated 28 days after cell transplantation.

Following Example 3, ascites was collected from the mice 21 to 28 days after cell transplantation, and the cell subsets in the ascites were analyzed.

Results: In both the IL-18 group and the IL-18/chymase inhibitor combination group, the ratio of CD4 ⁺ T cells/CD8 ⁺ T cells in peritoneal exudate cells (PEC) was significantly lower than that in the placebo group, and the number of CD8 ⁺ T cells, especially CD44highCD8 ⁺ T cells, increased (Fig. 8). In the IL-18 group and the IL-18/chymase inhibitor combination group, the number of NK cells, especially B220 ⁺ killing NK cells, was significantly higher than that in the placebo group (Fig. 9).

The composition of immune cell subsets in the ascites also suggested that the combined use of IL-18 and chymase enhanced the antitumor effect compared to IL-18 alone.

Example 5: Antitumor effect of combination of chymase inhibitor and anti-PD-1 antibody Method: Colon cancer cells (CT26: 5 x ¹⁰⁴ cells) were administered intraperitoneally to 7-week-old male BALB-c mice (purchased from Japan SLC), and administration was started on the third day after administration. The placebo group (n=10) received 0.17% dimethyl sulfoxide (drug solvent: placebo) intraperitoneally once a day. The anti-PD-1 antibody administration group (n=10) received 200μg/body of anti-PD-1 antibody (manufactured by BioXCell) intraperitoneally three times a week (total 12 times). The chymase inhibitor administration group (n=10) received 100μg/body of Fulacimstat (chymase inhibitor: MedChemExpress) intraperitoneally once a day (total 30 times). The anti-PD-1 antibody/chymase inhibitor combination group (n=10) received intraperitoneal administration of 200μg/body of anti-PD-1 antibody three times a week (total 12 times) and 100μg/body of chymase inhibitor once a day (total 30 times). Medication was completed 30 days after cell transplantation, and analysis was continued up to 34 days after that.

Observation: Mice were observed twice a day, morning and evening, for survival.
Analysis method: Significance tests for survival curves of each group were performed using the Kaplan-Meier method, with a P value of 5% or less considered significant.

Results: 34 days after cancer cell transplantation, the group receiving the combination of anti-PD-1 antibody and chymase inhibitor showed a higher survival rate (50%) than not only the placebo group (20%) and the group receiving chymase inhibitor alone (20%), but also the group receiving anti-PD-1 antibody alone (10%), demonstrating the combined effect of adding a chymase inhibitor to an anti-PD-1 antibody (Figure 10).

In this experiment, administration of a chymase inhibitor alone did not result in an extension of survival time after cancer transplantation, but the combination of an anti-PD-1 antibody and a chymase inhibitor extended survival time compared to anti-PD-1 antibody alone.

The present invention is useful in treating tumors.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (6)

A pharmaceutical composition for treating a tumor, comprising a chymase inhibitor as an active ingredient, the pharmaceutical composition being used in combination with one or more of interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12). A pharmaceutical composition for treating a tumor comprising a combination of one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12) with a chymase inhibitor. A pharmaceutical composition for treating tumors, comprising as an active ingredient one or more selected from interleukin-18 (IL-18), anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12), which is used in combination with a chymase inhibitor. Chymase inhibitors include Suc-Val-Pro-PheP(OPh) ₂ , Chymostatin, Fulacimstat, 2-[4-[[(5-Fluoro-3-methylbenzo[b]thien-2-yl)sulfonyl]amino]-3-(methylsulfonyl)phenyl]-4-thiazolecarboxylic The pharmaceutical composition according to any one of claims 1 to 3, wherein the compound is any one selected from the group consisting of (2-[5-amino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide), (2-[5-amino-2-(4-fluorophenyl)-1,6-dihydro-6-oxo-1-pyrimidinyl]-N-[1-[(5-methoxycarbonyl-2-benzoxazolyl)carbonyl]-2-phenylethyl]acetamide). The pharmaceutical composition according to any one of claims 1 to 3, wherein IL-18, anti-PD-1/PD-L1 antibody, and IL-12 are derived from humans. The pharmaceutical composition according to any one of claims 1 to 3, wherein the chymase inhibitor and any one or more selected from IL-18, anti-PD-1/PD-L1 antibody, and interleukin-12 (IL-12) are administered simultaneously or sequentially.

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