JP7132665B1 - Composition for shrinking or disappearing tumors - Google Patents

Abstract

An object of the present invention is to provide a new composition and its use for shrinking or eliminating tumors. A composition comprising a compound represented by the following formula or a salt thereof, or an anti-inflammatory agent as a component is provided. JPEG0007132665000011.jpg58130 {R1 is NH or CH2, R2 is N-R8 or CH-R8, R3 is F, Cl, Br, I, R5 is S or O, R6 is , is a carboxyl group. } [Selection] None

Description

The present disclosure relates to compositions for shrinking or eliminating tumors.

Tumors (or neoplasms) result from the autonomous excessive proliferation of cells against in vivo control. Tumors include malignant and benign tumors. Malignant tumors are generally called cancers (including carcinomas arising from epithelial cells and "cancers").

Treatment methods that have long been used for tumors include surgical excision, administration of anticancer drugs, and radiation therapy. In recent years, new therapeutic methods utilizing the immune system have been developed. As an example, a new method using dendritic cells is attracting attention. Dendritic cells play a role in memorizing antigens presented by cells to be attacked and presenting information about the antigens to other immune cells.

Using this mechanism, techniques have been developed to make dendritic cells memorize antigens specific to tumor cells. For example, tumor cells may be presented to exogenously extracted dendritic cells, or dendritic cells may be administered to a site where a tumor exists.

Also, there have been many reports on the relationship between inflammatory reactions and tumors. It has also been proposed that there is a positive feedback hypothesis that, in general, inflammatory responses further promote tumor growth, and that tumors further provoke inflammatory responses (Non-Patent Document 1). On the other hand, attention to the microenvironment (TME: Tumor Micro Environment) inside the tumor often induces an anti-inflammatory response as well. This is because if the inflammatory response is promoted too much, the tumor cells will be attacked by cytotoxic T cells and put in a disadvantageous situation, so the induction of anti-inflammatory responses by TME also plays an important role in tumor growth. (Non-Patent Document 2). It has been suggested that the seemingly contradictory balance between inflammation and anti-inflammatory is important for tumor growth.

Therefore, research is also being conducted on the possibility of suppressing tumor growth (anti-tumor) by intentionally disrupting the balance between inflammation and anti-inflammatory, but to date, anti-inflammatory agents have been used as anti-tumor treatments. It is not the first-choice drug, and it is usually used only for alleviating cancer-related pain. Furthermore, it has been pointed out that anti-inflammatory agents are incompatible with the above-mentioned immunotherapy, and there is also a report that it is actually the inflammatory response by tumor-specific Th1 cells that suppresses tumors (Non-Patent Document 3). . In addition, in anti-tumor treatment by administration of immune checkpoint inhibitors, which is a type of immunotherapy, the survival rate of patients who had been administered high doses (10 mg or more) of the immunosuppressant prednisone was lower at low doses (10 mg or less). There is also a report that the survival rate is lower than that of the patients who received the drug, suggesting that combined use with immunotherapy is not necessarily highly effective (Non-Patent Document 4).

https://www.nature.com/articles/nature07205 https://www.hindawi.com/journals/jir/2012/948098/ https://www.nature.com/articles/ncomms1239 https://ascopubs.org/doi/pdf/10.1200/JCO.19.00189

There remains a need for new methods to shrink or eliminate tumors. Accordingly, an object of the present disclosure is to provide a new composition and its use for shrinking or eliminating tumors.

As a result of extensive studies by the present inventors, when an anti-inflammatory agent or desired compound was administered to a tumor in a mouse body together with immature dendritic cells, tumor shrinkage was observed.

The invention has been completed based on the above findings, and the present disclosure includes the following inventions in one aspect.

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸ （Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。
｝
（発明８）
発明７の組成物であって、前記化合物が、以下の式で表される、組成物。

｛ただし、
Ｒ¹は、ＮＨであり、
Ｒ²は、Ｎ－Ｒ⁸ （Ｒ⁸：Ｃ１～Ｃ３までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣＨ₃であり、
Ｒ⁵は、Ｏであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂であり、
ｎは、１－１１である。
｝
（発明９）
発明１～８いずれか１つに記載の組成物であって、腫瘍に直接投与されるために使用される、組成物。
（発明１０）
発明１～８いずれか１つに記載の組成物であって、
前記腫瘍が転移性腫瘍の場合、
前記組成物が血管投与用である、組成物。
（発明１１）
発明１～１０いずれか１つに記載の組成物であって、未成熟な樹状細胞の投与の前、同時、又は、後に投与される、組成物。
（発明１２）
腫瘍を縮小又は消失させるための組成物であって、
前記組成物が、未成熟樹状細胞を含み、
抗炎症剤を投与する前、同時、又は、後に投与される、組成物。
（発明１３）
腫瘍を縮小又は消失させるための組成物であって、
前記組成物が、抗炎症剤を含み、
未成熟樹状細胞を投与する前、同時、又は、後に投与される、組成物。
（発明１４）
腫瘍を縮小又は消失させるための組成物であって、
前記組成物が、未成熟樹状細胞を含み、
以下の式で表される化合物又はその塩を投与する前、同時、又は、後に投与される、組成物。

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸ （Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。
｝
（発明１５）
腫瘍を縮小又は消失させるための組成物であって、
前記組成物が、以下の式で表される化合物又はその塩を含み、
未成熟樹状細胞を投与する前、同時、又は、後に投与される、組成物。

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸ （Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。
｝ (Invention 1)
A composition for shrinking or eliminating tumors, comprising:
immature dendritic cells;
A composition comprising an anti-inflammatory agent as an ingredient.
(Invention 2)
The composition of invention 1, wherein said anti-inflammatory agent is a steroidal anti-inflammatory agent.
(Invention 3)
The composition of invention 1, wherein said anti-inflammatory agent is a non-steroidal anti-inflammatory agent.
(Invention 4)
The composition of Invention 3, wherein the non-steroidal anti-inflammatory agent is a COX2 (Cyclooxygenase-2) inhibitor.
(Invention 5)
The composition of invention 1, wherein the anti-inflammatory agent is an antibody that inhibits the IL-6 signaling pathway.
(Invention 6)
The composition of invention 1, wherein the anti-inflammatory agent is an antibody that inhibits the TNFα signaling pathway.
(Invention 7)
A composition for shrinking or eliminating tumors, comprising:
immature dendritic cells;
Including a compound represented by the following formula or a salt thereof as a component,
Composition.

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
}
(Invention 8)
A composition according to invention 7, wherein the compound is represented by the following formula.

{however,
R ¹ is NH;
R ² is NR ⁸ (R ⁸ is an alkyl group of C1 to C3);
R3 is F ^, Cl, Br, or I;
each ^R4 is independently H or _CH3 ;
^R5 is O;
^R6 is a carboxyl group,
^R7 is _NH2 ;
n is 1-11.
}
(Invention 9)
9. A composition according to any one of inventions 1-8, wherein the composition is used for direct administration to a tumor.
(Invention 10)
A composition according to any one of Inventions 1 to 8,
if said tumor is a metastatic tumor,
A composition, wherein the composition is for intravascular administration.
(Invention 11)
11. The composition of any one of inventions 1-10, administered before, concurrently with, or after administration of immature dendritic cells.
(Invention 12)
A composition for shrinking or eliminating tumors, comprising:
the composition comprises immature dendritic cells;
A composition administered prior to, concurrently with, or after administration of an anti-inflammatory agent.
(Invention 13)
A composition for shrinking or eliminating tumors, comprising:
the composition comprises an anti-inflammatory agent;
A composition administered prior to, concurrently with, or after administration of immature dendritic cells.
(Invention 14)
A composition for shrinking or eliminating tumors, comprising:
the composition comprises immature dendritic cells;
A composition administered prior to, concurrently with, or after administration of a compound represented by the following formula or a salt thereof.

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
}
(Invention 15)
A composition for shrinking or eliminating tumors, comprising:
The composition comprises a compound represented by the following formula or a salt thereof,
A composition administered prior to, concurrently with, or after administration of immature dendritic cells.

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
}

In one aspect, the invention relates to a composition comprising a desired compound or anti-inflammatory agent. Tumors can be shrunk by administration of this.

The size (weight) of the tumor in the body of the mouse after a certain period of time has passed since the tumor was implanted. A significant difference occurs between control and anti-inflammatory agent + DC. p in the graph indicates a p-value (probability value; also called risk ratio) (test method: one-way ANOVA with bonferroni post hoc test). The size (weight) of the tumor in the body of the mouse after a certain period of time has passed since the tumor was implanted. A significant difference occurs between control and anti-inflammatory agent + DC. p in the graph indicates a p-value (probability value; also called risk ratio) (test method: one-way ANOVA with bonferroni post hoc test). The size (weight) of the tumor in the body of the mouse after a certain period of time has passed since the tumor was implanted. There is a significant difference between control and AS1842856+DC. p in the graph indicates a p-value (probability value; also called risk ratio) (test method: one-way ANOVA with bonferroni post hoc test). The results of inflammatory reaction by LPS stimulation after differentiation of cell line THP-1 are shown. It also shows that AS1842856 exerts an anti-inflammatory effect on the inflammatory response. Figure 1 shows the effect of anti-inflammatory agents and AS1842856 on lung and liver metastases. The effect of the order of administration of dendritic cells and anti-IL-6 receptor antibody is shown.

Specific embodiments for carrying out the invention will be described below. The following description is intended to facilitate understanding of the invention. it is not intended to limit the scope of the invention.

1. Definitions As used herein, the term "tumor" refers to a mass of tissue in which cells proliferate autonomously and excessively against in vivo control. Tumors include malignant tumors, benign tumors, and the like.

Also, as used herein, the term "neoplasm" is used interchangeably with tumor.

In addition, the term "cancer" used herein means a malignant tumor. "Cancer" also includes tissue masses that develop within the epithelium.

The term “immature dendritic cells” used herein refers to dendritic cells in which the expression of any one or more of the marker genes CD80, CD83, and CD86 cannot be detected. These marker genes are used in the art in verifying dendritic cell (DC) maturity.

Also, as used herein, the term "anti-inflammatory agent" means an agent that exerts an anti-inflammatory effect. For example, what satisfies any of the following conditions can be called an anti-inflammatory agent.

(1) Drugs containing glucocorticoids or derivatives thereof, generally called SAIDs (steroidal anti-inflammatory drugs), which are known to act on glucocorticoid receptors.
(2) Those commonly called NSAIDs (non-steroidal anti-inflammatory drugs), which can suppress inflammatory reactions without mediated by glucocorticoid receptors.
(3) those that specifically bind to inflammatory cytokines or their receptors and suppress inflammatory cytokine-receptor binding and activation of downstream signaling pathways; They are often antibodies, but may also be chimeric proteins that fuse the extracellular domain of a receptor with the Fc domain of an antibody (such as the TNFα inhibitor etanercept).
(4) Substances containing extracellular domains of receptors that bind to inflammatory cytokines.

In another embodiment, the term "anti-inflammatory agent" as used herein includes agents whose efficacy is anti-inflammatory, eg, as described in a pharmaceutical package insert. In yet another embodiment, the term "anti-inflammatory agent" as used herein is classified as "anti-inflammatory agent" in the KEGG DRUG DATABASE (https://www.kegg.jp/brite/jp08330). and drugs classified as “immunological agents/TNF inhibitors” and “immunological agents/interleukin inhibitors.”

2. Compositions In one embodiment, the present disclosure relates to compositions for shrinking or eliminating tumors. "Tumor shrinking" can mean, for example, an event in which comparative administration results in a statistically significant difference in tumor weight. Also, "eliminate a tumor" may mean, for example, a reduction in size to an imperceptible level.

2-1. Anti-Inflammatory Agent In one embodiment, the composition comprises an anti-inflammatory agent as an ingredient. The definition of anti-inflammatory agent is as explained above.

Anti-inflammatory agents are broadly classified into "steroidal anti-inflammatory agents", "non-steroidal anti-inflammatory agents", and antibody drugs that recognize, bind to, and inhibit inflammatory cytokines or their receptors.

Steroidal anti-inflammatory agents include glucocorticoids and their derivatives. For example, steroidal anti-inflammatory agents can include compounds belonging to the group designated DG01955 in the KEGG DRUG DATABASE. Specific examples of steroidal anti-inflammatory agents include, but are not limited to: hydrocortisone, hydrocortisone succinate, prednisolone, methylprednisolone, methylprednisolone succinate, triamcinolone, triamcinolone acetonide, dexamethasone (DEX).

Non-steroidal anti-inflammatory agents include compounds with basic skeletons different from glucocorticoids and their derivatives. For example, non-steroidal anti-inflammatory agents can include compounds belonging to the group designated DG01504 (non-steroidal anti-inflammatory drugs (NSAIDs)) in the KEGG DRUG DATABASE.

Non-steroidal anti-inflammatory agents include COX-2 (Cyclooxygenase-2) selective substances and COX-2 non-selective substances. Examples of COX-2 selective non-steroidal anti-inflammatory agents include the following substances.
Enolic acids (e.g. meloxicam)
Acetates (e.g. etodolac)
- Coxibs (e.g., celecoxib, rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, pormacoxib, tilmacoxib, deracoxib, phyrocoxib, mabacoxib, apricoxib)

Examples of COX-2 non-selective non-steroidal anti-inflammatory agents include the following substances.
- Propionates (e.g. loxonin, zaltoprofen, pranoprofen, oxaprozin, tiaprofenic acid, naproxen, ibuprofen)
Enolic acid-based (e.g., lornoxicam, ampiroxicam, piroxicam)
Phenylacetic acids (e.g. diclofenac sodium, nabumetone)
Indoleacetic acid series (e.g. indomethacin farnesel, sulindac, indomethacin)
- isoxazole acetic acid (e.g. mofezolac)
Anthranilic acids (e.g. mefenamic acid (Pontal))
Salicylates (e.g. aspirin)

Representative examples of agents that specifically bind to inflammatory cytokines or their receptors and suppress inflammatory cytokine-receptor binding and activation of downstream signal transduction pathways include the following.
- Anti-IL-6 antibody (e.g., EBI-031 (Eleven Biotherapeutics), siltuximab, orokizumab, clazakizumab, silkumab, ercilimomab, jerilimuzumab, OPR-003, MEDI-5117, PF-04236921)
- Anti-TNFα antibody (e.g., adalimumab, infliximab, etanercept, certolizumab pegol, golimumab, etc.)
-Anti-IL-6 receptor (IL-6R) antibodies (e.g., tocilizumab, sarilumab, bovalilizumab (ALX-0061), SA-237, etc.)

2-2. In one embodiment by compound , the composition comprises as a component a compound represented by Formula I below, or a salt thereof:

｛ただし、
Ｒ¹は、ＮＨ又はＣＨ₂であり、
Ｒ²は、Ｎ－Ｒ⁸又はＣＨ－Ｒ⁸ （Ｒ⁸：Ｈ、又はＣ１～Ｃ４までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣ１～Ｃ２までのアルキル基であり、
Ｒ⁵は、Ｓ又はＯであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂、又はＣ１～Ｃ２までのアルキル基であり、
ｎは、１－１１である。
｝

{however,
^R1 is NH or _CH2 ;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
^R5 is S or O;
^R6 is a carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
}

The salt form is not particularly limited, and may be sodium salt, potassium salt, or the like.

In a preferred embodiment, the compound may be a compound represented by Formula II below or a salt thereof.

｛ただし、
Ｒ¹は、ＮＨであり、
Ｒ²は、Ｎ－Ｒ⁸ （Ｒ⁸：Ｃ１～Ｃ３までのアルキル基）であり、
Ｒ³は、Ｆ、Ｃｌ、Ｂｒ、又はＩであり、
Ｒ⁴は、それぞれ独立して、Ｈ又はＣＨ₃であり、
Ｒ⁵は、Ｏであり、
Ｒ⁶は、カルボキシル基であり、
Ｒ⁷は、ＮＨ₂であり、
ｎは、１－１１である。
｝

{however,
R ¹ is NH;
R ² is NR ⁸ (R ⁸ is an alkyl group of C1 to C3);
R3 is F ^, Cl, Br, or I;
each ^R4 is independently H or _CH3 ;
^R5 is O;
^R6 is a carboxyl group,
^R7 is _NH2 ;
n is 1-11.
}

In a most preferred embodiment, the compound may be a compound represented by Formula III below (CAS No. 836620-48-5) or a salt thereof.

2-3. Other component compositions may further include ingredients known in the art, in addition to the anti-inflammatory agents and compounds described above. For example, any one or more of preservatives, excipients, antibiotics, pH buffers, osmotic pressure regulators, anticancer agents, etc. may be included. Also, the composition may be in a liquid, solid, or semi-solid (eg, gel) state. Also, in the case of solids, they may be of any shape (eg, granules, tablets, capsules, tablets). In the case of solids, if necessary, a solvent (eg, water) may be added to redissolve.

2-4. Subjects, Methods of Administration, etc. In one embodiment, the composition is administered directly to a tumor. Direct administration, as referred to herein, means administration at or near the site where the tumor is present. Therefore, direct administration is different from administration from a site distant from the tumor through intravenous injection and blood circulation. Moreover, when administering, it can be administered using known means such as a catheter and an injection needle. Examples of dosage forms include subcutaneous injection, intramuscular injection, and the like.

In another embodiment, the composition may be administered by intravascular administration (eg, intravenous administration) and the like. Intravascular administration is preferred when the tumor is metastatic.

The subject of administration of the composition is a tumor tissue, preferably a malignant tumor tissue, more preferably a malignant tumor tissue present in the epithelium, or a malignant tumor tissue that may invade from the epithelium and metastasize. be.

By administering a composition containing the anti-inflammatory agent and/or the compound as an active ingredient, the action of dendritic cells can be enhanced, and tumor shrinkage or disappearance can be promoted. In other words, the anti-inflammatory agent and/or the compound alone do not have a significant effect on tumor shrinkage. Also, dendritic cells alone can have some effect on tumor shrinkage. However, the synergistic effect of the anti-inflammatory agents and/or compounds and dendritic cells can have a significant effect on tumor shrinkage or disappearance.

Although the following description is not intended to limit the scope of the invention, it is believed that the composition prevents inflammation from inhibiting the function of dendritic cells.

For this reason, in a further embodiment the composition may further comprise immature dendritic cells. By including the anti-inflammatory agent and/or compound and immature dendritic cells in the composition, a synergistic effect of both can be obtained.

Of course, when administered into the body, the anti-inflammatory agent and/or compound and immature dendritic cells may be administered separately. Accordingly, in another embodiment, the present disclosure is directed to a composition comprising immature dendritic cells, wherein the composition administers an anti-inflammatory agent and/or the compound It may be administered before, at the same time, or after. Furthermore, in another embodiment, the present disclosure relates to a composition, the composition comprising an anti-inflammatory agent and/or the compound, the composition administering immature dendritic cells It may be administered before, at the same time, or after.

3. Use of the Composition In one embodiment, the present disclosure relates to use of the composition. The compositions may be used alone or combined with other therapeutic modalities and the like.

Examples of other therapeutic methods include irradiation, anticancer drug administration, immunotherapy, and the like. Examples of immunotherapy include antibody therapy and lymphocyte therapy. As a treatment in combination with the above composition, a combination with lymphocyte therapy is preferred. Examples of lymphocyte therapy include cytotoxic T cells (CTL) and γδT cells. Among these, CTLs in particular recognize tumors as attack targets by receiving antigen presentation from dendritic cells, and are expected to have a synergistic effect with the composition of the present disclosure. Concomitant therapeutic methods can be performed at any time and may be performed prior to administration of the composition, may be performed after administration of the composition, or may be performed simultaneously with administration of the composition. You may However, lymphocyte therapy is desirably performed after administration of the present composition, as efficiency is increased by exploiting antigen presentation of dendritic cells.

Preferably, the timing of administering the dendritic cells in vivo is before, at the same time as, or after the administration of the anti-inflammatory agent and/or the compound. More preferably, the timing of in vivo administration of dendritic cells is within 72 hours, preferably within 48 hours, and more preferably within 12 hours from before and after administration of the composition. The reason for this is that the composition in one embodiment of the present disclosure is believed to contribute to enhancement of dendritic cell function. In other words, within a period of time in which the dendritic cells are sufficiently alive and within a period of time in which the anti-inflammatory agent and/or the compound acts effectively after being administered in vivo, the dendritic cells and the It is preferable to administer the anti-inflammatory agent and/or the compound under conditions that allow it to exist in vivo.

4. Manufacture of Compositions In one embodiment, the present disclosure relates to manufacture of compositions for shrinking or eliminating tumors. The composition may be prepared by combining at least one or more of the anti-inflammatory agent, the compound of Formula I (e.g., the compound of Formula II), and immature dendrites described above. Accordingly, in one embodiment, the present disclosure relates to the use of at least one of an anti-inflammatory agent, a compound of Formula I (eg, a compound of Formula II), and immature dendrites as described above. The purpose of such use may be the manufacture of a composition for shrinking or eliminating tumors.

Specific examples of the embodiments described above are described in the following examples, which are not intended to limit the scope of the invention.

1. Overview The effect on tumors can be verified in the following experimental system. First, a specimen such as a mouse is prepared. The specimen is exposed to radiation to temporarily extinguish the immune system. Human-derived CD34+ hematopoietic stem cells are then transplanted into the specimen. Since the immune system has been extinguished once, rejection does not occur. Through the transplantation, a human immune system based on human CD34+ hematopoietic stem cells is constructed in the specimen (hereinafter referred to as a humanized mouse). Then, a human-derived tumor is implanted. This allows us to create a situation in mice that is similar to the situation with tumors and immune systems that occurs in humans. Such mice are commercially available (https://www.criver.com/products-services/research-models-services/animal-models/mice/humanized-mice?region=28).

2. Preparation of various cells (Example 1)
2-1. Preparation of cancer cells Human breast cancer-derived cells MDA-MB-231 (ATCC, HTB-26) were mixed with RPMI-1640 (Gibco, 11875-093) + 10% FBS (Fetal Bovine Serum; SIGMA, F7524) in a T175 flask (CellBIND). Corning, 3292) and allowed to stand in an incubator (37° C., 5% CO ₂ ) for culturing. Cells that reached semiconfluence were detached using an enzyme solution (TrypLE Express), diluted with PBS(−), and subjected to centrifugation (400×g for 5 minutes). The sedimented cells were resuspended in culture medium, seeded again in T175 flasks, and the cells continued to be subcultured to coincide with the timing of the animal experiments.

2-2. Preparation of Dendritic Cells After blood was collected from the humanized mice prepared in 0050, a peripheral blood mononuclear cell fraction was separated using Ficoll-Paque PLUS (17144002, Cytiva). The protocol followed that recommended by Cytiva. RPMI-1640+10% FBS was then used to inoculate T75 flasks (CellBIND; Corning, 3290). After 1 hour, the culture medium was collected and discarded because the cells that did not stick to the flask were lymphocytic cells. The remaining cells sticking to the flask were enriched with human monocytes, and these cells were spiked with RPMI-1640 + 10% FBS + 20 ng/ml IL-4 (AF-200-04; PeproTech) + 50 ng/ml GM-CSF ( AF-300-03; PeproTech) for 1 week. The medium was replaced on the third day on the way. After culturing, the cells were collected and confirmed to be differentiated into dendritic cells using a marker. Dendritic cells obtained by differentiating monocytes in this way are so-called "immature" dendritic cells that have not yet encountered antigens. It has the ability to take up a portion and present information to other immune cells such as cytotoxic T cells through antigen presentation. On the other hand, the collected peripheral blood mononuclear cell fraction also contains mature dendritic cells, but these are mature dendritic cells that have already presented antigens or that have lost their ability to present antigens. However, it was not used because it was not suitable for the purpose of use of the present invention.

3. Changes in tumor size in mice (Example 2)
1×10 ⁶ cells of MDA-MB-231 suspended in 100 μL of PBS(−) were added subcutaneously to the humanized mouse prepared as described above, and 100 μL of Matrigel (354230; Corning) was added. The mixture was mixed to increase viscosity so that it would remain subcutaneously and administered by subcutaneous injection using a 1 mL syringe. After 1 to 2 weeks, after the appearance of subcutaneous tumor formation became visible, an anti-inflammatory agent (DEX or celecoxib) was added at 1 mg (DEX) or 10 mg (celecoxib) per kg body weight of the mouse. ), or 1×10 ⁶ cells of dendritic cells were administered directly to the tumor by subcutaneous injection. In the case of the pattern of administering an anti-inflammatory agent, a pattern of administering dendritic cells two days after administration and a pattern of not administering dendritic cells were further provided. About two weeks later, the abdomen was opened and the tumor tissue was weighed. Five humanized mice were used for each control and each administration group. Similar numbers of individuals were used for each group for the following examples.

The results are shown in FIG. Here, when the anti-inflammatory agent was administered, a slight trend toward tumor tissue shrinkage was observed (approximately 70 mg and approximately 80 mg for DEX and celecoxib, respectively) compared to controls (approximately 90 mg). In addition, when dendritic cells (DC) were administered, a tendency toward tumor tissue shrinkage was observed (approximately 60 mg) compared to the control. Furthermore, there was a statistically significant reduction (approximately 20 mg and approximately 25 mg for DEX and celecoxib, respectively) when anti-inflammatory agents and dendritic cells were administered in combination. From the above results, it has become clear that the combination of an anti-inflammatory agent and dendritic cells can significantly reduce or eliminate tumors.

Here, DEX and celecoxib have completely different structures as compounds. Nevertheless, it has been shown that similar effects can be achieved in terms of tumor shrinkage or elimination. This indicates that synergistic action with dendritic cells can lead to reduction or disappearance of tumors, irrespective of differences in structure, if a common effect of anti-inflammatory effect is exhibited.

4. Effect of antibody drug (Example 3)
The same tests were performed as in the procedures of Examples 1-2 described above. However, the compounds were replaced with anti-IL-6R antibody (tocilizumab; 5 mg/kg) and anti-TNFα antibody (etanercept; 5 mg/kg). The results are shown in FIG. When the anti-IL-6R antibody and the anti-TNFα antibody were administered alone, almost no tumor tissue shrinkage was observed compared to the control (approximately 170 mg). However, when administered in combination with dendritic cells (DC), there was a statistically significant reduction (approximately 40 mg each). From the above results, it was clarified that a combination of an antibody drug with anti-inflammatory effect and dendritic cells can significantly reduce or eliminate tumors.

5. Effects of Compound AS1842856 (Example 4)
The same tests were performed as in the procedures of Examples 1-2 described above. However, the anti-inflammatory agent was replaced with AS1842856 (a compound represented by CAS number 836620-48-5). Since AS1842856 is hardly soluble in aqueous solution, it was dissolved in DMSO (dimethylsulfoxide). Therefore, groups not administered AS1842856 were administered DMSO as a control instead. The results are shown in FIG. A trend toward tumor tissue shrinkage was observed when AS1842856 was administered (approximately 60 mg) compared to the DMSO control (approximately 90 mg). Also, when dendritic cells (DC) were administered, a tendency toward tumor tissue shrinkage was observed compared to the control (approximately 60 mg). Furthermore, when AS1842856 and dendritic cells were administered in combination, there was a statistically significant reduction (approximately 35 mg). From the above results, it was revealed that the combination of AS1842856 and dendritic cells can significantly reduce or eliminate tumors.

6. Anti-inflammatory action by compound AS1842856 (Example 5)
Next, the anti-inflammatory action of compound AS1842856 was verified. Specifically, a cell line THP-1 was prepared, 100 nm of PMA was administered, and the human monocytic cell line THP-1 was differentiated into macrophages. Twenty-four hours after the administration, desired substances were further administered in the following four patterns.
DMSO (as control)
DMSO (as control) + AS1842856 1 μM
LPS (lipopolysaccharide) 1 μg/mL
LPS (lipopolysaccharide) 1 μg/mL + AS1842856 1 μM

After administration, culturing was continued for 24 hours, and then RNA was collected from the cells. More specifically, after culturing, total RNA was extracted from the cells using the ReliaPrep RNA Miniprep system (Promega, Z6012). After extraction, cDNA synthesis (PrimeScript RT Master Mix; Takara, RR036A) was performed using 500 ng of RNA, and quantitative PCR (Thunderbird Sybr qPCR Mix; TOYOBO, QPS-201X5) was performed.

A mixture for cDNA synthesis was prepared according to the following composition.
2 μl of 5x PrimeScript RT Master Mix (1x final concentration)
Total RNA 500ng
Adjusted to 10 μl total RNase free H ₂ O

The above mixture was processed using a Veriti 96 well Thermal Cycler manufactured by Applied Biosystems under the following conditions.
37°C 15 minutes ↓
85°C 5 seconds ↓
4°C ∞

The synthesized cDNA (10 µl) was diluted 10-fold with 90 µl of TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0). The dilutions were subjected to quantitative PCR.

As a specific procedure for quantitative PCR, the dilutions were mixed under the following conditions.
10 μl of 2x THUNDERBIRD Probe qPCR Mix
0.4 μl of 5 mM Forward Primer
0.4 μl of 5 mM Reverse Primer
8.2 μl of H ₂ O
cDNA (10-fold dilution) 1 μl

cDNA was amplified from the above mixture using Bio-Rad's CFX-Connect. The PCR cycle conditions were as follows.
1.95°C 1 minute (initial denaturation)
2.95°C 15 seconds (denaturation)
3.60°C 30 seconds (elongation)
(Repeat steps 2-3 40 times, detect fluorescence signal each time step 3 is completed)
4. Raise the temperature from 65°C to 95°C in increments of 0.5°C, hold the temperature for 5 seconds, and then detect the fluorescence signal.

The PCR product was detected in the above cycle, and the identity of the PCR product was confirmed by melting curve.

GAPDH ( Glyceraldehyde 3- phosphate dehydrogenase) was used as an internal standard ( a housekeeping gene expressing the same expression in all cells and conditions ) .
The primer sequences for detecting each gene were as follows.
GAPDH Forward primer: 5'-agccacatcgctcagacac-3'
GAPDH Reverse primer: 5'-gcctaatacgaccaaatcc-3'
TNFα Forward primer: 5'-cagcctcttctccttcctgat-3'
TNFα Reverse primer: 5' - gccagagggctgattagaga - 3'
IL-1β Forward primer: 5' - tacctgtcctgcgtgttgaa - 3'
IL-1β Reverse primer: 5' - tctttgggtaatttttgggatct - 3'
IL-10 Forward primer: 5'-gatgccttcagcagagtgaa-3'
IL-10 Reverse primer: 5' - gcaacccaggtaacccttaaa - 3'

All primers were purchased from Fasmac Co., Ltd. (reverse phase column purification grade).
The expression level of each gene indicates the standardized value obtained by dividing the expression level of each gene obtained by quantitative PCR by the expression level of GAPDH. It was confirmed by a melting curve that the PCR products amplified using the above primers were all single (that is, multiple types of sequences were not amplified with the same primers).

The results are shown in FIG. Cells differentiated into macrophages exhibit an inflammatory response in the presence of lipopolysaccharide present on the surface of bacteria. Specifically, they express the inflammatory cytokines TNFα and IL-1β. Supporting this, LPS administration (vehicle) increased the expression levels of TNFα and IL-1β compared to the control (vehicle). However, the expression levels of TNFα and IL-1β increased by LPS administration were suppressed by administration of AS1842856.

In contrast to inflammatory cytokines, administration of AS1842856 markedly increased the expression level of IL-10, which is an anti-inflammatory cytokine. Compared with the control (vehicle), the degree of increase was more marked when LPS was administered.

From the above, it was shown that AS1842856 has the effect of shrinking tumors. It was also shown that there is a high possibility that tumor shrinkage is caused by an action similar to that of an anti-inflammatory agent (in other words, an action that suppresses inflammation).

7. Effect on metastatic tumor (Example 6)
The same tests were performed as in the procedures of Examples 1-2 described above. However, an experimental system simulating a systemic metastatic tumor was used by administering MDA-MB-231 cells not subcutaneously but through the tail vein. 1×10 ⁶ cells of MDA-MB-231 were suspended in 200 μL of PBS(−) and administered to the tail vein. One week later, DEX at 1 mg/kg, celecoxib at 10 mg/kg, tocilizumab at 5 mg/kg, or etanercept at 5 mg/kg was administered through the tail vein. Alternatively, AS1842856 was administered intraperitoneally at 100 mg/kg. The route of administration of AS1842856 is intraperitoneal because AS1842856 is dissolved in DMSO, which is a type of organic solvent, and the risk of direct administration into blood vessels is considered. As a control for intraperitoneal administration, the same volume of DMSO was administered. Two hours after the administration of various components, after confirming that there were no symptoms such as immobility or rough breathing, 1×10 ⁶ dendritic cells were administered through the tail vein. Two weeks later, the mice were dissected and the number of tumor-derived nodules formed in the lungs and liver was counted.

The results are shown in FIG. Compared to the number of nodules formed in the lung and liver in control animals administered only MDA-MB-231, the administration of various anti-inflammatory agents or combination administration of AS1842856 and dendritic cells significantly reduced the number of nodules formed. was observed. From these results, anti-inflammatory agents, or combinations of dendritic cells that can induce anti-inflammatory effects, are effective against tumors that metastasize throughout the body, including dissemination, when administered through blood vessels. was found to be possible.

8. Effect of Administration Procedure (Example 7)
The same tests were performed as in the procedures of Examples 1-2 described above. However, 1×10 ⁶ cells of MDA-MB-231 were administered subcutaneously for 1 week. After that, 1×10 ⁶ dendritic cells and an anti-IL-6R antibody (tocilizumab; 5 mg/kg) were administered to the tumor in the following three orders.
(1) Administration of anti-IL-6R antibody ⇒ 1 day lapse ⇒ administration of dendritic cells (2) Simultaneous administration of dendritic cells and anti-IL-6R antibody (3) Administration of dendritic cells ⇒ 1 day lapse ⇒ anti-IL Administer -6R antibody

Results are shown in FIG. Similar to the results of Examples 1 and 2 described above, in Example 7, although there was a slight difference from the control, the anti-IL-6R antibody administered either before, at the same time, or after The combination of dendritic cells was able to significantly shrink tumors. From this, it was shown that the order of administration of the anti-inflammatory agent exhibited similar effects whether before, at the same time, or after the administration of dendritic cells.

Specific embodiments of the invention have been described above. The above embodiments are only specific examples, and the present invention is not limited to the above embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Also, unless otherwise stated, for a particular method, some steps may be interchanged with other steps, and additional steps may be added between two particular steps. The scope of the invention is defined by the claims.

Claims (8)

A composition for shrinking or eliminating tumors, comprising:
immature dendritic cells;
A composition comprising an anti-inflammatory agent as an ingredient,
wherein the anti-inflammatory agent is any one or more of an anti-IL-6 antibody and an anti-IL-6 receptor (IL-6R) antibody ,
In the composition, only one or more of an anti-IL-6 antibody and an anti-IL-6 receptor (IL-6R) antibody are included as anti-inflammatory agents, or
In the composition, the anti-inflammatory agents include any one or more of an anti-IL-6 antibody and an anti-IL-6 receptor (IL-6R) antibody, and one or more selected from the following: is only a combination of
Composition.
・Steroidal anti-inflammatory agents
・Non-steroidal anti-inflammatory agents
- A compound represented by the following formula or a salt thereof

{however,
R1 is NH or ^CH2 _;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
R5 is ^S or O;
R6 is a ^carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
} A composition for shrinking or eliminating tumors, comprising:
immature dendritic cells;
A composition comprising an anti-inflammatory agent as an ingredient,
wherein the anti-inflammatory agent is any one or more of an anti-IL-6 antibody and an anti-IL-6 receptor (IL-6R) antibody,
A composition that does not contain an anti-TNF antibody. 3. The composition of any one of claims 1-2, wherein the anti-inflammatory agent is tocilizumab. A composition according to any one of claims 1-3, used for direct administration to a tumor. A composition according to any one of claims 1 to 3,
if said tumor is a metastatic tumor,
A composition, wherein the composition is for intravascular administration. 6. The composition of any one of claims 1-5, administered prior to, concurrently with, or after administration of immature dendritic cells. A composition for shrinking or eliminating tumors, comprising:
the composition comprises immature dendritic cells;
A composition to be administered prior to, concurrently with, or after administration of an anti-inflammatory agent,
The anti-inflammatory agent is
Only one or more of anti-IL-6 antibody and anti-IL-6 receptor (IL-6R) antibody, or
Any one or more of anti-IL-6 antibodies and anti-IL-6 receptor (IL-6R) antibodies in combination with one or more selected from the following only :
Composition.
・Steroidal anti-inflammatory agents
・Non-steroidal anti-inflammatory agents
- A compound represented by the following formula or a salt thereof

{however,
R1 is NH or ^CH2 _;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
R5 is ^S or O;
R6 is a ^carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
} A composition for shrinking or eliminating tumors, comprising:
the composition comprises an anti-inflammatory agent;
A composition administered before, concurrently with, or after administration of immature dendritic cells,
The anti-inflammatory agent is
Only one or more of anti-IL-6 antibody and anti-IL-6 receptor (IL-6R) antibody, or
Any one or more of anti-IL-6 antibodies and anti-IL-6 receptor (IL-6R) antibodies in combination with one or more selected from the following only :
Composition.
・Steroidal anti-inflammatory agents
・Non-steroidal anti-inflammatory agents
- A compound represented by the following formula or a salt thereof

{however,
R1 is NH or ^CH2 _;
R ² is N—R ⁸ or CH—R ⁸ (R ⁸ : H or a C1-C4 alkyl group);
R3 is F ^, Cl, Br, or I;
each R ⁴ is independently H or a C1-C2 alkyl group;
R5 is ^S or O;
R6 is a ^carboxyl group,
R ⁷ is NH ₂ or a C1-C2 alkyl group;
n is 1-11.
}

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