BR112017017064B1 - PHARMACEUTICAL COMPOSITION AND USE OF THE COMPOUND OF FORMULA II OR ITS PHARMACEUTICALLY ACCEPTABLE SALT - Google Patents

Abstract

PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CANCER, AND USE OF THE COMPOUND OF FORMULA I. The present invention relates to a pharmaceutical composition for preventing or treating cancer and a method of preventing or treating cancer with the use thereof. Cancer can be effectively prevented or treated with the use of the pharmaceutical composition and the method.

Description

FIELD OF THE INVENTION

[001] The present disclosure relates to a pharmaceutical composition for preventing or treating cancer and a treatment method using the same.

BACKGROUND OF THE INVENTION

[002] Cancer immunotherapy refers to a therapy that treats cancer with the use of the immune system in the body. Cancer cells can have cell surface molecules, for example proteins or carbohydrates, which can often be detected by the immune system. Cancer immunotherapy can induce the immune system to attack cancer cells by targeting these antigens. One way to activate anti-tumor immunity is to block immune checkpoint pathways. The immune checkpoint pathway refers to intracellular signaling pathways that maintain self-tolerance and protect tissues from an excessive immune response that causes damage. Some immunological checkpoints are primary immunological evasion mechanisms for tumor cells. Therefore, inhibition or blocking of the immune checkpoint can induce T cell activation and thereby antitumor immunization can be enhanced.

[003] Transforming growth factor (TGF)-β is a cytokine that regulates cell proliferation and differentiation, wound healing, extracellular matrix production, or the like. The TGF-β family belongs to the TGF-β superfamily, and this TGF-β superfamily includes activins, inhibins, bone morphogenetic proteins, and anti-Mullerian hormone. Tumor cells and stromal cells within tumors in the advanced stages of many cancers often overexpress TGF-β. TGF-β would lead to stimulation of angiogenesis and cell motility, suppression of the immune system, and increased interaction of tumor cells with the extracellular matrix. TGF-β receptors are serine/threonine receptors, and they are divided into TGF-β receptor 1, TGF-β receptor 2 and TGF-β receptor 3. Among them, the TGF-β receptor 1 is also called an activin A kinase type II-like receptor (ALK5).

[004] Consequently, for the effective prevention or treatment of cancer, there is a need for a pharmaceutical composition capable of effectively inhibiting a TGF-β signaling pathway, as well as enhancing antitumor immunity.

SHORT DESCRIPTION OF THE INVENTION

[005] A pharmaceutical composition is provided for preventing or treating cancer, the pharmaceutical composition including a compound having a TGF-β signaling pathway inhibiting activity and a T lymphocyte activating factor.

[006] A method of treating or preventing cancer is provided with the use of a compound that has a TGF-β signaling pathway inhibition activity and a T lymphocyte activating factor.

[007] A pharmaceutical composition for preventing or treating cancer, according to an aspect, and a method of preventing or treating cancer using the same can be used to effectively prevent or treat cancer.

BRIEF DESCRIPTION OF THE FIGURES

[008] These and/or other aspects will become more apparent and more readily understood from the description of the modalities, taken together with the attached figures, in which:

[009] Figures 1A to 1C are graphs showing tumor volume (mm3) depending on combinations of TEW-7197, anti-CTLA4 antibody, anti-PD-L1 antibody or anti-PD1 antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0010] One aspect provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition including a compound represented by Formula I, a pharmaceutically acceptable salt, solvate or stereoisomer thereof, or a combination thereof:

[0011] where, in Formula I, Ra can each independently be hydrogen (H), halo, C1-6 alkyl, C1-6 haloalkyl, C3-6 cycloalkyl, OH, -O-C1-6 alkyl, -O-C1-6 haloalkyl, -O-C3-6 cycloalkyl, -NH2, -NH-C1-6 alkyl, -NH-C1-6 haloalkyl, -NH-C3-6 cycloalkyl, -S-C1-6 alkyl , -S-haloC1-6alkyl, -S-C3-6cycloalkyl, -CN or -NO2;

[0012] m can be 0, 1, 2, 3 or 4;

[0013] either of A1 and A2 may be N, and the other is NR1, wherein R1 may be H, OH, C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl;

[0014] X can be a bond, -(CH2)p-, -NR2-, -O- or -S-, where p can be 0 or 1 and R2 can be H or C1-3 alkyl;

[0015] Rb can each independently be H, halo, C1-6 alkyl, C1-6 haloalkyl, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, -(CH2)q-OR3, -( CH2)q-NR3R4, -(CH2)q-SR3, -(CH2)q-NO2, -(CH2)q-CONHOH, -(CH2)q-CN, -(CH2)q-COR3, -(CH2) q-CO2R3, -(CH2)q-CONR3R4, -(CH2)q-tetrazole, -(CH2)q-CH=CH-CN, -(CH2)q-CH=CH-CO2R3, -(CH2)q- CH=CH-CONR3R4, -(CH2)q- CH=CH-tetrazole, -(CH2)q-NHCOR3, -(CH2)q-NHCO2R3, -(CH2)q-CONHSO2R3, -(CH2)q-NHSO2R3, -(CH2)q-C=C-CN, -(CH2)q-C=C-CO2R3, - (CH2)q-C=C-CONR3R4, -(CH2)q-C=C-tetrazole, -(CH2)q-SOR5, -( CH2)q-SO2R5 or -(CH2)r-(OR3)2, wherein R3 and R4 can each independently be H, C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl or, taken together with a nitrogen atom bonded thereto to form a monocyclic ring, for example imidazole, pyrrolidine, piperidine, morpholine, piperazine and homopiperazine, R5 may be C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl, q may be 0 , 1, 2, 3 or 4, r can be 1, 2, 3 or 4; It is

[0016] n cannot be 0, 1, 2, 3, 4 or 5; It is

[0017] a T lymphocyte activating factor.

[0018] The alkyl group may be linear or branched. Examples of the alkyl group may include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and n-hexyl. The alkyl group may be replaced by at least one selected from alkoxy, cycloalkoxy, amino, nitro, carboxy, cyano, halo, hydroxyl, sulfo, mercapto or a combination thereof.

[0019] The cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0020] The halo can be fluorine, chlorine, bromine or iodine.

[0021] The alkenyl group may be linear or branched. The alkenyl group can be vinyl, allyl, isoprenyl, 2-butenyl or 2-hexenyl. The alkenyl group can be replaced by alkoxy, cycloalkoxy, amino, nitro, carboxy, cyano, halo, hydroxyl, sulfo, mercapto or a combination thereof.

[0022] The alkynyl group may be linear or branched. The alkynyl group can be ethynyl, propargyl or 2-butynyl. The alkynyl group can be replaced by alkoxy, cycloalkoxy, amino, nitro, carboxy, cyano, halo, hydroxy, sulfo, mercapto or a combination thereof.

[0023] The compound may be a compound represented by Formula II:

[0024] The compound of Formula II may be N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl) - 1H-imidazol-2-yl)methyl)-2-fluoroaniline.

[0025] The compound of Formula I, for example, the compound of Formula II can selectively inhibit the TGF-β 1 receptor (ALK5) and/or the activin receptor type 1B (ACVR1B or ALK-4).

[0026] The pharmaceutically acceptable salt may be a salt that cannot cause significant irritation in an organism to which a compound is administered and cannot nullify the biological activity and properties of the compound. The salt may be, for example, an inorganic acid salt, an organic acid salt or a metal salt. The inorganic acid salt may be a salt of hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid or disulfuric acid. The organic acid salt can be a salt of formic acid, acetic acid, propionic acid, lactic acid, oxalic acid, tartaric acid, malic acid, maleic acid, citric acid, fumaric acid, besylic acid, cansylic acid, edisilic acid, trichloroacetic acid , trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, ethanesulfonic acid, benzenesulfonic acid, p-tonulenesulfonic acid or aspartic acid. The metal salt can be a calcium salt, a sodium salt, a magnesium salt, a strontium salt or a potassium salt.

[0027] The solvate can be a compound formed by attractive forces between solute and solvent molecules. The solvate can be a hydrate.

[0028] Stereoisomer refers to molecules that have the same molecular formula and connectivity of their atoms, but differ in the spatial arrangement of atoms. The stereoisomer can be a diastereomer or an enantiomer of the compound of Formula I.

[0029] T lymphocyte activating factor refers to a molecule that induces T cell activation. T lymphocyte activating factor can be an immune checkpoint inhibitor, an immune stimulating molecule, a factor inhibitor immunosuppressant or a combination thereof.

[0030] Immune checkpoint refers to a molecule in the immune system that either increases a T cell signal (costimulatory molecules) or decreases a T cell signal. The immune checkpoint molecule can be an immune stimulus molecule or an immune inhibiting molecule. The immune stimulus molecule can be an agonist that induces T cell activation. The immune stimulus molecule can be CD27, CD40, OX40, GITR, CD137, CD28, an inducible T cell costimulator (ICOS) or a combination thereof .

[0031] An inhibition or blockage of the immune checkpoint may refer to the inhibition of immune tolerance or activation of an immune system. Immune checkpoint blockade can include any of the signaling pathways that activate T cells for increased anti-tumor immune response. Immune checkpoint inhibitor can refer to a molecule that inhibits or blocks inhibitory checkpoint molecules from activating T cells. Immune checkpoint inhibitor can be an antagonist release inhibition of T cells.

[0032] The T lymphocyte activating factor can be an agonist that can inhibit signal transduction or reduce the expression of a T cell receptor selected from the group consisting of a programmed cell death protein 1 (PD1), a cytotoxic T lymphocyte-associated antigen 4 (CTLA4), a B and T lymphocyte attenuator (BTLA), a killer cell immunoglobulin-like receptor (KIR), a lymphocyte activation gene 3 (LAG3), a cell membrane protein T cell 3 (TIM3) and an adenosine A2a receptor (A2aR). PD1, CTLA4, BTLA, KIR, LAG3, TIM3 and A2aR are T cell receptors that transmit inhibitory T cell signals. In this sense, T lymphocyte activating factor can inhibit signal transduction or reduce the expression of PD1, CTLA4 , BTLA, KIR, LAG3, TIM3, A2aR or a combination thereof to activate T cells.

[0033] The T lymphocyte activating factor can be an antagonist that can inhibit signal transduction or reduce the expression of a surface protein of an antigen presenting cell (APC) selected from the group consisting of a ligand of programmed death 1 (PD-L1 or PDL1), a programmed death 2 ligand (PD-L2 or PDL2), a differentiation cluster (CD) 80, CD86, B7-H3, B7-H4, a mediator of virus entry herpes virus (HVEM) and galectin 9 (GAL9). PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, GAL9 or a combination thereof is an APC surface protein that can bind to a T cell receptor that transmits signals from inhibition of T cells. In this sense, the T lymphocyte activation factor can inhibit signal transduction or reduce the expression of PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, GAL9 or a combination thereof to activate T cells.

[0034] The T lymphocyte activating factor can be an agonist that activates a T lymphocyte receptor. The T lymphocyte activating factor can be an agonist that can induce signal transduction or increase the expression of a T lymphocyte receptor selected from the group consisting of CD28, ICOS, CD137, OX40 and CD27. CD28, ICOS, CD137, OX40 and CD27 are T cell receptors that transmit T cell activation signals. T cells can be activated through signal transduction activation or increased T cell receptor expression. stimulatory checkpoint molecule can activate signal transduction or increase expression of D28, ICOS, CD137, OX40, CD27 or a combination thereof to activate T cells.

[0035] Immunosuppressive factor refers to a molecule that inhibits or blocks antitumor immune responsiveness. The immunosuppressive factor can be transforming growth factor β (TGFβ), interleukin (IL)-10, prostaglandin E2 (PGE2), CCL2 (also known as macrophage chemoattractant protein 1: MCP-1), Fas receptor (FasR) /ligand (FasL), cytotoxic T lymphocyte antigen-4 (CTLA-4), B7 homologue 1 (B7-H1), programmed death ligand 1 (PD-L1) or a combination thereof. The immunosuppressive factor inhibitor can induce T cell activity. The immunosuppressive factor can be one selected from the group consisting of TGFβ, IL-1, IL-6, IL-10, IL-12 and IL18 or an agonist that regulates the one selected from the group. Regulation can be suppression or activation of functions or a decrease or increase in expression. TGFβ, IL-1, IL-6, IL-10, IL-12, and IL-18 are cytokines, and cytokine inhibition can activate T cells.

[0036] The immunosuppressive factor agonist, antagonist or inhibitor may be a polypeptide, sugar, nucleic acid, a low molecular weight compound or a combination thereof. The polypeptide may be an antibody or an antigen-binding fragment thereof. The term "antibody" may be used interchangeably with the term "immunoglobulin (Ig)". A complete antibody has a structure that has two full-length light chains and two full-length heavy chains, each light chain being linked to the heavy chain via a disulfide bond (SS bond). The antibody can be IgA, IgD, IgE, IgG or IgM. The term "antigen-binding fragment" refers to a fragment of a complete Ig structure, i.e., a polypeptide portion that includes a portion to which an antigen can bind. The antigen-binding fragment can be F(ab') 2 , Fab', Fab, Fv or scFv. The antibody can be a monoclonal antibody or a polyclonal antibody. Examples of the nucleic acid may include microRNA (miRNA) and small interfering RNA (siRNA).

[0037] The T lymphocyte activating factor may be a PD-L1 inhibitor, a PD1 inhibitor, a CTLA4 inhibitor or a combination thereof. Examples of the PD-L1 inhibitor may include BMS-936559 (MDX1105, Bristol Myers Squibb), MEDI4736 (MedImmune, AstraZeneca), MPDL3280A (Roche) and MSB0010718C (Merck). Examples of the PD1 inhibitor may include AMP-224 (Amplimmune, GlaxoSmith Klein), AMP-514 (MEDI0680, Amplimmune, GlaxoSmith Klein), nivolumab (Opdivo, Bristol Myers Squibb), Pembrolizumab (Keytruda, Merck) and Pidilizumab (Cure Tech) . Examples of the CTLA4 inhibitor can include ipilimumab (Yervoy, Bristol Myers Squibb) and tremelimumab (Pfizer).

[0038] The cancer may be a solid cancer or a non-solid cancer. Solid cancer refers to the occurrence of a cancerous tumor in organs, for example, the liver, lung, breast, skin or the like. Non-solid cancer refers to cancer that occurs in the blood and is also referred to as a blood cancer. The cancer may be carcinoma, sarcoma, hematopoietic cell-derived cancer, germ cell tumor, or blastoma. Carcinoma can be a cancer derived from epithelial cells. Sarcoma can be cancer derived from connective tissues (ie, bone, cartilage, adipose tissue, and nerves) that can develop from cells derived from mesenchymal cells outside the marrow. Cancer derived from hematopoietic cells can be derived from hematopoietic cells that tend to mature in lymph nodes and in blood away from the bone marrow. Germ cell tumor may be pluripotent cell derived cancer. The pluripotent cell can often be present in testes or ovaries. The blastoma can be derived from an immature precursor cell or from embryonic tissue. The cancer may be selected from the group consisting of melanoma, cerebrospinal fluid brain tumor, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, cancer liver, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, spermatocytoma, thyroid cancer, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS ), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, sarcoma and skin cancer.

[0039] The term "prevention" means all actions whereby the occurrence of cancer is delayed by the administration of the pharmaceutical composition. The term "treatment" means all actions whereby the symptoms of cancer were directed towards an improvement or were favorably modified by the administration of the pharmaceutical composition.

[0040] The pharmaceutical composition may additionally include a pharmaceutically acceptable carrier. The carrier can mean the inclusion of excipients, diluents or supplements. The carrier may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone , water, physiological saline, a buffer such as PBS, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like. The composition can include a filler, an anticoagulant agent, a lubricant, a humectant, a flavor, an emulsifier, an antiseptic or the like.

[0041] The pharmaceutical composition can be prepared in any formulation based on conventional methods. The composition can be formulated into an oral dosage form (eg powder, tablet, capsule, syrup, pill or granule) or a parenteral dosage form (eg injectable formulation). Furthermore, the composition can be prepared in systemic dosage forms or topical dosage forms.

[0042] The pharmaceutical composition may be a single composition or individual compositions. For example, the pharmaceutical composition of the compound of Formula I, a pharmaceutically acceptable salt, solvate, stereoisomer thereof or a combination thereof can be a composition in an oral dosage form and the T lymphocyte activating factor can be a composition in an abdominal dosage form.

[0043] The pharmaceutical composition may include the compound of Formula I, a pharmaceutically acceptable salt, solvate, stereoisomer thereof or a combination thereof; and T lymphocyte activating factor in an effective amount. The term "effective amount" means an amount sufficient to show a prevention or treatment effect when administered to a subject in need of prevention or treatment. The effective amount may suitably be selected by one of ordinary skill in the art depending on the cell or individual selected. The effective amount can be determined according to factors including the severity of a disease, the patient's age, weight, health conditions, sex, drug sensitivity, time of drug administration, route of administration , discharge rate, treatment period, and the drugs that are mixed or used in combination with the composition, and other factors that are well known in the field of medicine. The effective amount can be in a range of about 0.5 µg to about 2 g, about 1 µg to about 1 g, about 10 µg to about 500 mg, about 100 µg to about 100 mg or about 1 mg to about 50 mg based on the pharmaceutical composition.

[0044] An administration dose of the pharmaceutical composition can be, for example, in a range from about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg or about 0.1 mg/kg to about 1 mg/kg for adults, once a day, a few times a day, one to four times a month, or one to twelve times a year.

[0045] Another aspect provides a method of preventing or treating cancer, the method including administering the compound of Formula I, according to one aspect, a pharmaceutically acceptable salt, solvate or stereoisomer thereof or a combination thereof and a T lymphocyte activating factor to an individual.

[0046] The compound of Formula I, a pharmaceutically acceptable salt, solvate or stereoisomer thereof, the T lymphocyte activating factor, cancer prevention and treatment are the same as defined above.

[0047] The individual can be human, cattle, horses, pigs, dogs, sheep, goats or cats. The individual may be an individual who has cancer or a high possibility of getting cancer.

[0048] The administration of the compound, a salt, solvate, pharmaceutically acceptable stereoisomer thereof or a combination thereof, and the T lymphocyte activating factor can be carried out directly by any suitable method, for example, oral, intravenous administration , intramuscular, transdermal, mucosal, intranasal, intratracheal or subcutaneous. The compound, a pharmaceutically acceptable salt, solvate, stereoisomer thereof or a combination thereof, and the T lymphocyte activating factor can be administered systemically or topically individually or together with another pharmaceutically active compound.

[0049] The compound, a pharmaceutically acceptable salt, solvate, stereoisomer thereof or a combination thereof and the T lymphocyte activating factor may be administered simultaneously, individually or sequentially.

[0050] A preferred administration dose of the compound, a pharmaceutically acceptable salt, solvate, stereoisomer thereof or a combination thereof, and the T-lymphocyte activating factor may differ depending on conditions and a patient's body weight, severity disease, drug formulation, route and period of administration, but it can be properly selected by an individual of ordinary skill in the art. The dose of administration can be, for example, in a range from about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg or about 0.1 mg /kg to about 1 mg/kg for adults, once a day, a few times a day, one to four times a month, or one to twelve times a year.

[0051] Reference will now be made in detail to the embodiments, examples of which are illustrated in the accompanying drawings, in which reference numerals refer to similar elements throughout them. In this sense, the present modalities may have different forms and should not be interpreted as being limited to the descriptions presented in the present document. Accordingly, the embodiments are described below, referring to the figures, merely to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0052] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

[0053] Example 1. Testing of antitumor effects in a mouse model xenotransplanted with melanoma cell

1. Preparation of mouse model and drug

[0054] In order to validate the antitumor effects in vivo, 5-week-old female C57BL/6 SPF mice were obtained from Koatech Co. (South Korea). These mice were kept in a controlled room at a temperature of about 22°C while freely provided with food and water. 1 week later, 5 x 10 4 cells of B16F10 melanoma cell line (ATCC, CRL-6475™) were injected into these 6 week old mice by subcutaneous injection. 8-week-old mice, 2 weeks after injection of melanoma cell line, were used in the experiment.

[0055] As a carrier, 7 ml of 37% (v/v) gastric acid, 2.0 g of NaCl, 3.2 g of pepsin (Sigma-Aldrich) and distilled water were mixed together to prepare 1,000 ml of artificial gastric fluid. As a delivery drug, N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazole -2-yl)methyl)-2-fluoroaniline (hereinafter referred to as "TEW-7197") (Syngene, India) was dissolved in the carrier to prepare a 2.5 mg/ml solution of TEW-7197.

[0056] As another administration drug, anti-PD-L1 monoclonal antibody (prepared by request to ANR therapeutics co., Ltd., host cell: HEK293F), anti-PD1 hamster monoclonal antibody (BioXCell, Cat. No. BE0033 -2), anti-CTLA4 hamster monoclonal antibody (BioXCell, Cat. # BE0131), human immunoglobulin G (IgG) (Thermo Scientific, Cat. # 31154), and monoclonal hamster IgG (BioXCell, Cat. # BE0091) were prepared.

2. Drug administration for mouse model and measurement of weight caused by drug administration

[0057] The mouse models prepared in 1. were administered orally with 2.5 mg/ml of a TEW-7197 solution at a dose of 25 mg/kg weight once a day for 5 consecutive days of a week. Then, during the last 2 days, the TEW-7197 solution was not administered to them. These mice were administered antibody at a dose of about 100 µg per mouse once every 2 days by abdominal injection. As a control group for TEW-7197, a carrier administered group was used. As a control group for the antibody, a group given human IgG and a group given hamster IgG were used.

[0058] The drugs administered to the mouse models are shown in Table 1.

[0059] [Table 1]

[0060] From the day the drugs were administered (start date) to the 11th day, the weights of the mice were measured every 2 days. Changes in mice (%) (mean (%) ± standard deviation) caused by drug administration are shown in Table 2.

[0061] [Table 2]

[0062] T-TEST: ** p < 0.01 (vs. human IgG-administered group)

[0063] As shown in Table 2, the mice had almost no significant change in weight caused by anti-PD-L1 antibody, anti-PD1 antibody, anti-CTLA4 antibody, TEW-7197 and a combination thereof.

3. Test of tumor inhibitory effects of drug in mouse model

[0064] As described in 2., drug administration was performed, and then, 20 minutes after the last drug dose, the mice were sacrificed, the tumors removed from the mice. Tumor volume was measured with calipers and calculated using Equation 1:

[0065] [Equation 1] Tumor volume (mm3) = [length (mm) x width (mm) x height (mm)]/2

[0066] The difference between the initial tumor volume (V0) at the start date and the tumor volume after drug administration (Vt) was calculated to calculate the change in tumor volume (Δt = Vt - V0) . From the calculated tumor volume, the tumor inhibition rate (%) by drug administration was calculated. Their results are shown in Table 3.

[0067] [Table 3]

[0068] t-TEST: * p < 0.05, ** p < 0.01, *** p < 0.001 (vs. vehicle-administered group, human IgG-administered group, or hamster IgG-administered group), t: Δt = Vt - V0, and t: tumor inhibition rate (%) (vs. vehicle-administered group, human IgG-administered group, or hamster IgG-administered group)

[0069] As shown in Table 3, on the 11th day, the tumor inhibition rate of the group administered with anti-PD-L1 antibody and TEW-7197 (group 7) was about 2.5 times greater than that of the group administered with anti-PD-L1 antibody (group 4) and about 1.9 times greater than that of the group administered with TEW-7197 (group 10). On the 11th day, the tumor inhibition rate of the group administered with anti-PD1 antibody and TEW-7197 (group 8) was about 3.5 times greater than that of the group administered with anti-PD1 antibody (group 5) and about 1.3 times greater than that of the TEW-7197-administered group (group 10). On the 11th day, the tumor inhibition rate of the group administered with anti-CTLA4 antibody and TEW-7197 (group 9) was about 3.2 times greater than that of the group administered with anti-CTLA4 antibody (group 6) and about 1.3 times greater than that of the TEW-7197-administered group (group 10).

[0070] Consequently, the combination of anti-PD-L1 antibody, anti-PD1 antibody and anti-CTLA4 antibody as immune checkpoint inhibitors and TEW-7197 as a TGF-β receptor inhibitor was found to have an inhibitory effect of significant tumor compared to each of anti-PD-L1 antibody, anti-PD1 antibody, anti-CTLA4 antibody, and TEW-7197.

4. Test of synergistic combination of drugs in a mouse model

[0071] A test of tumor inhibitory effect by synergistic combination of immune checkpoint inhibitors and TGF-β receptor inhibitor was performed.

[0072] In detail, as described in 1., C57BL/6 mice were prepared and 1 x 10 4 B16F10 melanoma cell line cells (ATCC, CRL-6475™) were injected into each mouse. When the average tumor size of each group reached about 26.0 mm3 drug administration was performed.

[0073] As described in 1., TEW-7197, anti-CTLA4 antibody, anti-PD-L1 antibody, anti-PD1 antibody, human IgG and hamster IgG have been prepared as drugs.

[0074] The prepared mice were administered orally with a solution of TEW-7197 at a dose of 10 mg/weight in kg once a day for 5 consecutive days of one week. So, during the last 2 days, the TEW-7197 solution was not administered. These mice were administered antibody at a dose of about 100 µg per mouse once every 2 days by abdominal injection. In a negative control group, a vehicle was administered for TEW-7197 and human IgG or hamster IgG was administered for the antibody. 11 days after drug administration, tumor volume was measured with calipers and calculated as described in 3.

[0075] Tumor volume, according to combinations of TEW-7197 and anti-CTLA4 antibody, anti-PD-L1 antibody or anti-PD1 antibody, is shown in Figures 1A to 1C.

[0076] As shown in Figure 1A, the tumor volume of the group administered with anti-CTLA4 antibody, the group administered with TEW-7197, and the group administered with a combination of anti-CTLA4 antibody and TEW-7197 was decreased by about 14.5%, about 10.1% and about 27.1 compared to the tumor volume of the negative control group, respectively.

[0077] As shown in Figure 1B, the tumor volume of the group administered with anti-PD-L1 antibody, the group administered with TEW-7197, and the group administered with a combination of anti-PD-L1 antibody and TEW-7197 was decreased by about 15.0%, about 9.2% and about 28.8% compared to the tumor volume of the negative control group, respectively.

[0078] As shown in Figure 1C, the tumor volume of the group administered with anti-PD1 antibody, the group administered with TEW-7197, and the group administered with a combination of anti-PD1 antibody and TEW-7197 was decreased by about 9.6%, about 8.1% and about 23.3% compared to the tumor volume of the negative control group, respectively.

[0079] Consequently, the combination of anti-PD-L1 antibody, anti-PD1 antibody and anti-CTLA4 antibody as immune checkpoint inhibitors and TEW-7197 as a TGF-β receptor inhibitor was found to have an inhibitory effect of synergistic tumor compared to each of anti-PD-L1 antibody, anti-PD1 antibody, anti-CTLA4 antibody and TEW-7197.

[0080] It should be understood that the embodiments described in this document are to be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each modality should typically be considered as available for other similar features or aspects in other modalities.

[0081] Although one or more embodiments have been described with reference to the figures, it will be understood by individuals of ordinary skill in the art that various changes in form and detail can be made to them without departing from the spirit and scope of the inventive concept as defined by the following claims.

Claims (5)

1. PHARMACEUTICAL COMPOSITION, characterized by comprising a compound represented by Formula II, or its pharmaceutically acceptable salt: , and a T lymphocyte activating factor; wherein the T lymphocyte activating factor is an anti-programmed cell death protein 1 (PD1) antibody, an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) antibody, or an anti-programmed death ligand 1 (PD) antibody -L1). 2. COMPOSITION, according to claim 1, characterized by the compound of Formula I being for the prevention or treatment of cancer. 3. COMPOSITION, according to claim 2, characterized in that the cancer is selected from the group consisting of melanoma, cerebrospinal fluid brain tumor, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, cancer of the esophagus, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, spermatocytoma, thyroid cancer, ovarian cancer, cancer cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, and skin cancer. 4. COMPOSITION according to any one of claims 1 to 3, characterized in that it is a single composition or individual compositions. 5. USE OF THE COMPOUND OF FORMULA II OR ITS PHARMACEUTICALLY ACCEPTABLE SALT, as defined in claim 1, and a T lymphocyte activating factor, characterized in that it is to produce a drug to prevent or treat cancer in an individual, in which the factor of activation of T lymphocyte activation is an anti-programmed cell death protein 1 (PD1) antibody, an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) antibody, or an anti-programmed death ligand 1 (PD-L1) antibody.