Anticancer combination of tidamide and celecoxib

Abstract

The present invention provides a combination of the acid salt form of the histone deacetylase (HDAC) inhibitor chidamide with the base salt form of the nonsteroidal anti-inflammatory drug (NSAID) celecoxib. The present invention also provides a method for significantly modulating the tumor microenvironment, thus dramatically improving anti-cancer activity.

Description

[Technical Field]

[0001] This disclosure relates to the field of cancer treatment. In particular, this disclosure provides combinations comprising tidamide and celecoxib in salt form, and their applications in the modulation of the tumor microenvironment and cancer immunotherapy. [Background technology]

[0002] Cancer immunotherapy is a rapidly growing field that has seen impressive and promising breakthroughs. The discovery of tumor-associated antigens has increased the potential to intervene in tumor growth using the host's immune system. Various mechanisms utilizing both the humoral and cellular arms of the immune system are currently being explored for cancer immunotherapy.

[0003] Several strategies have been proposed to disrupt immune tolerance, including adoptive transfer of immune effectors, immunomodulatory therapy, and vaccination. However, these strategies still fail to halt immune evasion. Major evasion pathways arise within cancer cells, including anti-apoptotic signaling, mitogen-activated protein kinase (MAPK), and cyclic adenosine monophosphate (cAMP)-related mechanisms. The tumor microenvironment is a crucial area of ​​study because it is dynamic and complex in the process of tumor progression. Tumors employ mechanisms to evade immune regulation through a process called immunoediting, which provides selective pressures in the tumor microenvironment that can lead to malignant progression. During the tumor-promoting phase, known as "immune evasion," the immune system can accelerate tumor progression by selecting cancer cells with a higher ability to overcome the host's immune competence, or by modifying the tumor microenvironment in a way that promotes tumor growth. The distinct characteristics of the tumor microenvironment involve various factors, such as hypoxia, acidic pH, vascular architecture, metabolic state, immunosuppressive function of many immune cells, and the involvement of cytokines or chemokines. These factors control immune evasion and reduce the immune response. Therefore, controlling the tumor microenvironment is one of the important strategies for anti-cancer treatment, especially for immunotherapy.

[0004] Immune system homeostasis involves the presence of both stimulative and inhibitory mechanisms to control the balance in immune system responses. Inhibitory mechanisms include cytotoxic T lymphocyte-associated antigen-4 (CTLA-4, CD28 homolog), programmed cell death protein-1 (PD-1) or its ligand (PD-L1), TIM-3 (T cell immunoglobulin-3), BTLA (B and T lymphocyte attenuation factor), VISTA (V-domain Ig inhibitor of T cell activation), and LAG-3 (lymphocyte-activating gene 3). The stimulatory mechanisms include differentiation antigen group 28 (CD28), tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as CD134 or OX40, glucocorticoid-induced TNFR family-related genes (GITR), members of the tumor necrosis factor (TNF) receptor family (CD137;4-1BB), members of the tumor necrosis factor receptor superfamily (CD27), and herpesvirus entry signaling molecules (HVEM). Currently, many immune checkpoint inhibitor monoclonal antibodies, including anti-CTLA-4, anti-PD-1, and anti-PD-L1 antibodies, are approved by the US FDA, EMA, PMDA, and NMPA for therapeutic use in several oncological indications. However, only about 20%–30% of cancer patients show a tumor response to monotherapy with these immune checkpoint inhibitors. Efficacy remains insufficient. The strategy of novel drug combinations with immune checkpoint inhibitors is a recent approach to enhance the response rate of these immune checkpoint inhibitors. This provides an opportunity to evaluate the benefits of immunotherapy in patients with various advanced cancers. On the other hand, drug resistance to immune checkpoint inhibitors can reduce the benefits of treatment compared to expectations. Many promising combination approaches are underway in preclinical and clinical trials. Efforts with these promising combination regimens offer hope for resolving the problem of drug resistance by improving immune response rates and efficacy.

[0005] US20180355042 and US20190211103 provide combinations of HDACi and PD-1 inhibitors useful for treating cancer, including reducing and / or inhibiting cancer metastasis. However, there remains a need to develop therapeutic solutions to control the tumor microenvironment and improve the anti-cancer efficacy of immunotherapy. [Overview of the project]

[0006] This disclosure provides combinations including salts of thidamide and salts of celecoxib, as well as methods for modulating the tumor microenvironment and dramatically improving immune response and anticancer activity by administering thidamide salts in combination with celecoxib salts.

[0007] In one embodiment, the present disclosure provides a combination comprising an acidic salt of thidamide and a basic salt of celecoxib.

[0008] In one embodiment, the amounts of the acidic salt of thidamide and the basic salt of celecoxib are in the range of about 5% (w / w) to about 80% (w / w) and about 95% (w / w) to about 20% (w / w), respectively. In one embodiment, the weight ratios of the acidic salt of thidamide and the basic salt of celecoxib are about 8:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:8.

[0009] In one embodiment, the acidic salt of thidamide and the basic salt of celecoxib are contained in the same dosage form or independently in separate dosage forms. In further embodiments, the dosage form is a tablet or a capsule.

[0010] In one embodiment, the acidic salt of thidamide is a hydrochloride or sulfate. In another embodiment, the acidic salt of thidamide is in crystalline or amorphous form.

[0011] In one embodiment, the hydrochloride salt of tidamide is a crystalline form (Form A) having a powder X-ray diffraction (XRPD) pattern with peaks at 2-theta values of about 16.12 degrees, about 19.02 degrees, about 21.62 degrees, about 23.38 degrees, and about 30.16 degrees. In another embodiment, the XRPD pattern of Form A further has peaks at 2-theta values of about 21.08 degrees, about 23.76 degrees, about 25.58 degrees, about 27.82 degrees, and about 28.18 degrees.

[0012] In yet another embodiment, the hydrochloride salt of tidamide has a Fourier transform infrared spectroscopy (FTIR) pattern with peaks at about 3162 cm -1 , about 3059 cm -1 , about 3036 cm -1 , about 2751 cm -1 , about 2588 cm -1 , about 2359 cm -1 , about 2341 cm -1 , about 1667 cm -1 , about 1658 cm -1 , about 1639 cm -1 , about 1620 cm -1 , about 1610 cm -1 , about 1562 cm -1 , about 1517 cm -1 , about 1508 cm -1 , about 1485 cm -1 , about 1468 cm -1 , about 1444 cm -1 , about 1431 cm -1 , about 1307 cm -1 , about 1282 cm -1 , about 1265 cm -1 , about 1243 cm -1 , about 1220 cm -1 , about 1182 cm -1 , about 1145 cm -1 , about 1074 cm -1 , about 1046 cm -1 and is a crystalline form (Form A).

[0013] In a further embodiment, form A is further characterized by exhibiting substantially the same XRPD pattern as shown in Figure 3(B) or substantially the same FTIR pattern as shown in Figure 4(B).

[0014] In one embodiment, the sulfate of thidamide is a crystalline form (form B) having a powder X-ray diffraction (XRPD) pattern with peaks including 2-theta values ​​of about 21.15°, about 24.65°, about 17.00°, about 18.49° and about 26.69°. In another embodiment, the XRPD pattern of form B further has peaks including 2-theta values ​​of about 14.74°, about 19.45°, about 22.00°, about 23.55° and about 27.94°.

[0015] In one embodiment, the sulfate of thidamide is approximately 3249 cm³. -1 , approx. 3067cm -1 , approx. 2578cm -1 , approx. 2360cm -1 , approx. 1689cm -1 , approx. 1664cm -1 , approx. 1647cm -1 , approx. 1614cm -1 , approx. 1568cm -1 , approx. 1521cm -1 , about 1510cm -1 , approx. 1486cm -1 , approx. 1467cm -1 , approx. 1434cm -1 , approx. 1412cm -1 , approx. 1388cm -1 , approx. 1354cm -1 , approx. 1328cm -1 , approx. 1283cm -1 , approx. 1266cm -1 , approx. 1252cm -1 , approx. 1226cm -1 , approx. 1184cm -1 Approximately 1099cm -1 , approx. 1059cm -1 , approx. 1034cm -1 and approximately 1022cm -1 This is a crystal morphology (morphology B) with an FTIR pattern having a peak.

[0016] In a further embodiment, form B is further characterized by exhibiting substantially the same XRPD pattern as shown in Figure 3(C) or substantially the same FTIR pattern as shown in Figure 4(C).

[0017] In one embodiment, the basic salt of celecoxib is the sodium salt of celecoxib. In another embodiment, the sodium salt of celecoxib is in amorphous or crystalline form. In yet another embodiment, the amorphous form of the sodium salt of celecoxib has substantially the same XRPD pattern as shown in Figure 7(B).

[0018] In one embodiment, the sodium salt of celecoxib is a crystalline form (Form I) having a powder X-ray diffraction (XRPD) pattern with peaks including two theta values ​​of approximately 19.85°, 20.51°, 21.51°, 22.55°, and 18.25°. In another embodiment, the XRPD pattern of Form I further has peaks including two theta values ​​of approximately 10.95°, 14.05°, 14.60°, 17.2°, 25.80°, and 27.30°. In a further embodiment, Form I is further characterized by exhibiting substantially the same XRPD pattern as that shown in Figure 7(C).

[0019] In one embodiment, the combination further includes an immune checkpoint inhibitor and / or a chemotherapeutic agent. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. Certain embodiments of immune checkpoint inhibitors include pembrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, atezolizumab, toripalimab, sintilimab, camrelizumab, and MIHI.

[0020] In one embodiment, the present disclosure provides a method for treating cancer by modulating the microenvironment and improving the immune response, comprising administering an effective amount of tidamide in combination with an effective amount of celecoxib. In further embodiments, tidamide and celecoxib are administered simultaneously, separately, or sequentially.

[0021] In one embodiment, the present disclosure provides a method for modulating the tumor microenvironment in cancer immunotherapy, comprising administering an effective amount of the combination described herein to the target. In one embodiment, the acidic salt of tidamide and the basic salt of celecoxib are administered simultaneously, separately, or sequentially.

[0022] In another embodiment, the Disclosure provides a method for treating cancer, comprising administering an effective dose of the combination described herein to a target. In one embodiment, cancer is treated by modulating the microenvironment and improving the immune response. In one embodiment, the method further comprises administering an immune checkpoint inhibitor. In another embodiment, the combination and immune checkpoint inhibitor of the Disclosure are administered simultaneously, separately, or sequentially. Examples of immune checkpoint inhibitors are described herein.

[0023] In one embodiment, administration of the acidic salt of thidamide and the basic salt of celecoxib results in free thidamide and free celecoxib. base It improves the pharmacokinetic profile compared to the previous version.

[0024] Certain embodiments of cancer include glioblastoma, liver cancer, colorectal carcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, benign prostate hyperplasia, prostate cancer, ovarian cancer, melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), gallbladder cancer, bile duct cancer, bladder cancer, and uterine cancer. [Brief explanation of the drawing]

[0025] [Figure 1-1] This shows the 1H-NMR and 13C-NMR spectra for thidamide-API, thidamide-HCl salt, and thidamide-H2SO4 salt. The 1H-NMR spectrum of thidamide-API (active pharmaceutical ingredient) (A), thidamide-HCl salt (B), and thidamide-H2SO4 salt (C) are shown. The 13C-NMR spectra of thidamide-API (D), thidamide-HCl salt (E), and thidamide-H2SO4 salt (F) are also shown. The 13C-NMR spectral data for different forms of thidamide are compared (G). [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 1-4] This is a continuation of Figure 1-3. [Figure 1-5] This is a continuation of Figure 1-4. [Figure 1-6] This is a continuation of Figure 1-5. [Figure 1-7] This is a continuation of Figure 1-6. [Figure 2-1] This shows the ESI-MS spectra for both positive and negative ions of thidamide-HCl salt and thidamide-H2SO4 salt. The ESI-MS spectra of thidamide-HCl salt in positive ion mode (A) and negative ion mode (B) are shown. The ESI-MS spectra of thidamide-H2SO4 salt in positive ion mode (C) and negative ion mode (D) are shown. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3-1] The powder X-ray diffraction (XRD) spectra for thidamide-API, thidamide-HCl salt, and thidamide-H2SO4 salt are shown. The XRD spectra of thidamide-API (A), thidamide-HCl salt (B), and thidamide-H2SO4 salt (C) are compared, and it is shown that the 2-theta values ​​of thidamide-API, thidamide-HCl salt, and thidamide-H2SO4 salt are different (D). [Figure 3-2] This is a continuation of Figure 3-1. [Figure 3-3] This is a continuation of Figure 3-2. [Figure 4-1] Fourier transform infrared (FTIR) spectra for thidamide-API, thidamide-HCl salt, and thidamide-H2SO4 salt are shown. The FTIR spectra of thidamide-API (A), thidamide-HCl salt (B), and thidamide-H2SO4 salt (C) were analyzed for characterization of thidamide-API, thidamide-HCl salt, and thidamide-H2SO4 salt (D). [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5-1] The ¹H-NMR and ¹³C-NMR spectra for celecoxib-API and celecoxib-Na salt are shown. The ¹H-NMR spectra (400 MHz, CDCl3) of celecoxib-API (active pharmaceutical ingredient) and celecoxib-Na salt are shown in Figures 5A and 5B, respectively. The ¹³C-NMR spectra of celecoxib-API and celecoxib-Na salt are shown in Figures 5C and 5D, respectively. The ¹³C-NMR spectra (100 MHz, DMSO-d6) of celecoxib-API and celecoxib-Na salt are compared in Figure 5E. Celecoxib-Na salt can be prepared in amorphous or crystalline form by different processes. The ¹H-NMR and ¹³C-NMR spectra of amorphous celecoxib-Na salt have the same patterns as those of the crystalline salt form. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 5-4] This is a continuation of Figure 5-3. [Figure 5-5] This is a continuation of Figure 5-4. [Figure 6] The Fast Atom Bombardment Mass Spectrometry (FAB-MS) spectrum of celecoxib-Na salt is shown. The FAB-MS spectrum of amorphous celecoxib-Na salt has the same pattern as that of the crystalline salt form. [Figure 7-1]Powder X-ray diffraction (XRD) spectra of celecoxib-API and celecoxib-Na salt in amorphous and crystalline forms are shown. The XRD spectra of celecoxib-API, celecoxib-Na salt in amorphous and crystalline forms are shown in Figures 7A, 7B, and 7C, respectively. The diffraction peaks differed significantly between the amorphous and crystalline forms. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Figure 8-1] Fourier transform infrared (FTIR) spectra of celecoxib-API and celecoxib-Na salt in amorphous and crystalline forms are shown. The FTIR spectra of celecoxib-API and celecoxib-Na salt in crystalline and amorphous forms are shown in Figures 8A, 8B, and 8C, respectively. The FTIR patterns were characterized between celecoxib-API and celecoxib-Na salt (Figure 8D). [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9-1]Therapeutic responses to tidamide-HCl plus celecoxib-cap in combination with anti-PD-1 antibody were observed in CT26 tumor-carrying mice. BALB / c mice carrying CT26 colon tumors were treated with various therapeutic methods as shown: IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); CD-HCl, tidamide-HCl 12.5, 25, 50 mg / kg; CD-K30, tidamide-K30 (tidamide coated with polyvinylpyrrolidone K30, 50 mg / kg); C-cap50, celecoxib product from capsules (50 mg / kg, Celebrex®). The fold change in total tumor volume and tumor size (A), individual tumor volume (B), body weight of CT26 tumor-carrying mice (C), and animal survival rate (D) were recorded. CT26 tumor-carrying mice were treated as shown, and euthanized after tumor implantation when the tumor volume reached 3000 mm3. Mean and standard deviation are shown. The number of animals used in each experimental arm and the p-value are also shown. *P<0.05 (vs. IgG); #P<0.05 (vs. PD-1). P-values ​​were calculated using Student's t-test comparing tumor size in the indicator group with that of the IgG group. Differences in survival rates between different treatment groups were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 10-1]In mice carrying CT26 tumors, a therapeutic response was observed with tidamide-HCl salt plus celecoxib-Na salt in combination with an anti-PD-1 antibody. BALB / c mice carrying CT26 colon tumors were treated with various therapeutic methods as shown: IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); CD-HCl, tidamide-HCl salt (50 mg / kg); C-Na, amorphous celecoxib-Na salt (12.5, 25, and 50 mg / kg); CD-K30, tidamide-K30 (tidamide coated with polyvinylpyrrolidone K30, 50 mg / kg); C-Capsule 50, celecoxib product from capsules (50 mg / kg, Celebrex®). The multiplier changes in total tumor volume and tumor size (A), individual tumor volumes (B), the percentage of tumor-free mice (C), body weight of CT26 tumor-carrying mice (D), and animal survival rate (E) were recorded. CT26 tumor-carrying mice were treated as shown and euthanized after tumor implantation when the tumor volume reached 3000 mm3. Mean and standard deviation are shown. The number of animals used in each experimental arm and the p-value are also shown. *P<0.05 (vs. IgG); #P<0.05 (vs. PD-1). The p-value was calculated using Student's t-test comparing tumor size in the indicator group with that of the IgG group. Differences in survival rates between different treatment groups were analyzed using one-way ANOVA followed by Tukey's multiple comparison test. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-2. [Figure 11-1]To determine the optimal therapeutic response dose of tidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 antibody, and to evaluate the therapeutic response of tidamide-H2SO4 plus celecoxib-Na in combination with an anti-PD-1 antibody in CT26 tumor-carrying mice, BALB / c mice with CT26 colon tumors were treated with various therapeutic methods, as indicated, with tumor size approximately 300 mm3. IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); CD-HCl, thidamide-HCl salt (12.5, 25, and 50 mg / kg); C-Na, amorphous celecoxib-Na salt (12.5, 25, and 50 mg / kg); C-Na cry, crystalline celecoxib-Na salt (50 mg / kg); CD-H2SO4, thidamide-H2SO4 salt (50 mg / kg); CD-K30, thidamide-K30 (polyvinylpyrrolidone K30 coated thidamide, 50 mg / kg); C-cap, celecoxib product from capsules (50 mg / kg, Celebrex®). The magnification changes in total tumor volume and tumor size (A), individual tumor volumes (B), body weight of CT26 tumor-carrying mice (C), and animal survival rate (D) were recorded. CT26 tumor-carrying mice were treated as shown, and euthanized after tumor implantation when the tumor volume reached 3000 mm3. Mean and standard deviation are shown. The number of animals used in each experimental arm and the p-value are also shown. *P<0.05 (vs. IgG); #P<0.05 (vs. PD-1). P-values ​​were calculated using Student's t-test comparing tumor size in the indicator group with that of the IgG group. Differences in survival rates between different treatment groups were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 11-3] This is a continuation of Figure 11-2. [Figure 12-1]This study demonstrates that resistance to PD-1 checkpoint blockade therapy can be overcome in CT26 tumor-carrying mice by using anti-PD-1 or anti-CTLA-4 Ab in combination with tidamide-HCl plus celecoxib-Na. CT-26-carrying mice (average tumor size approximately 120 mm³) were treated with a first-line anti-PD-1 antibody (2.5 mg / kg) administered twice (twice weekly). Mice were re-enrolled in a second-line treatment trial if the tumor met the first-line treatment failure criteria, defined as a threefold increase in tumor size to an average of approximately 360 mm³, and the tumor volume was <600 mm³. These anti-PD-1 resistant mice were treated with seven different regimens as shown (n=9-11 mice / group): IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); CTLA-4, anti-CTLA-4 monoclonal antibody (2.5 mg / kg); CD-HCl, thidamide-HCl salt (50 mg / kg); C-Na, amorphous celecoxib-Na salt (50 mg / kg); MS275, entinostat (20 mg / kg). The fold change in total tumor volume and tumor size (A), individual tumor volume (B), body weight of CT26 tumor-carrying mice (C), and animal survival rate (D) were recorded. CT26 tumor-carrying mice were treated as shown and euthanized after tumor implantation when the tumor volume reached 3000 mm3. Mean and SD values ​​are shown. The number of animals used in each experimental arm and the P-value are also shown. *P<0.05 (vs. IgG); #P<0.05 (vs. PD-1). P-values ​​were calculated using Student's t-test comparing tumor size in the index group with that of the IgG group. Differences in survival rates between different treatment groups were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 12-3] This is a continuation of Figure 12-2. [Figure 12-4] This is a continuation of Figure 12-3. [Figure 13-1]This study demonstrates that resistance to PD-L1 checkpoint blockade therapy can be overcome in CT26 tumor-carrying mice by using anti-PD-1 or anti-CTLA-4 Ab in combination with tidamide-HCl plus celecoxib-Na. CT-26-carrying mice (average tumor size approximately 160 mm3) were treated with a first-line anti-PD-L1 antibody (2.5 mg / kg) administered twice (twice weekly). Mice were re-enrolled in a second-line treatment trial if the tumor met the first-line treatment failure criteria, defined as a threefold increase in tumor size to an average of approximately 320 mm3, and the tumor volume was <600 mm3. These anti-PD-L1 resistant mice were treated with seven different regimens as shown (n=9-11 mice / group). IgG, anti-IgG control (vehicle, 2.5 mg / kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg / kg); CTLA-4, anti-CTLA-4 monoclonal antibody (2.5 mg / kg); CD-HCl, thidamide-HCl salt (50 mg / kg); C-Na, amorphous celecoxib-Na salt (50 mg / kg); MS275, entinostat (20 mg / kg). The fold change in total tumor volume and tumor size (A), individual tumor volume (B), body weight of CT26 tumor-carrying mice (C), and animal survival rate (D) were recorded. CT26 tumor-carrying mice were treated as shown and euthanized after tumor implantation when the tumor volume reached 3000 mm3. Mean and SD values ​​are shown. The number of animals used in each experimental arm and the P-value are also shown. *P<0.05 (vs. IgG); #P<0.05 (vs. PD-1). P-values ​​were calculated using Student's t-test, comparing tumor size in the indicator group with that of the IgG group. Differences in survival rates between different treatment groups were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 13-3] This is a continuation of Figure 13-2. [Figure 13-4] This is a continuation of Figure 13-3. [Figure 14-1]This shows the PK profiles of thidamide-HCl and celecoxib-Na salt alone or in combination in male Wistar rats. Rats were orally administered thidamide-K30, thidamide-HCl, celecoxib capsules (Celebrex®, celecoxib / cap), or amorphous celecoxib-Na salt at a dose of 50 mg / kg. A comparison of the PK profiles between thidamide-K30 and thidamide-HCl salt was analyzed (A). A comparison of the PK profiles between celecoxib / cap and amorphous celecoxib-Na salt was analyzed (B). A comparison of the thidamide PK profiles of thidamide-K30 plus celecoxib / cap versus thidamide-HCl salt plus amorphous celecoxib-Na salt is shown (C). A comparison of the celecoxib PK profiles of thidamide-K30 plus celecoxib / cap versus thidamide-HCl salt plus celecoxib-Na salt is shown in (D). A comparison of the thidamide PK profiles of thidamide-K30 versus thidamide-HCl salt versus thidamide-K30 plus celecoxib / cap versus thidamide-HCl salt plus celecoxib-Na salt is shown in (E). A comparison of the celecoxib PK profiles of celecoxib / cap versus celecoxib-Na salt versus thidamide-K30 plus celecoxib / cap versus thidamide-HCl salt plus celecoxib-Na salt is shown in (F). [Figure 14-2] This is a continuation of Figure 14-1. [Modes for carrying out the invention]

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the invention, but preferred methods and materials are described herein. All publications described herein are incorporated herein by reference.

[0027] References to “approximately” values ​​or parameters in this specification include (and describe) embodiments that apply to the value or parameter itself. For example, a description referring to “approximately X” includes a description of “X.” For example, the term “approximately X°” in the XRPD pattern refers to a 2-theta value of ±0.2 degrees.

[0028] The terms "a" and "an" refer to one or more (i.e., at least one) grammatical objects of articles. For example, "an element" means one or more elements. The use of "or" means "and / or" unless otherwise specifically stated.

[0029] The term "polymorph" refers to a crystalline form of a compound (e.g., compound 1) in a particular crystal packing arrangement, or its hydrate or solvate. All polymorphs of a particular compound have the same elemental composition. The term "crystal," as used herein, refers to a solid form consisting of a regular arrangement of structural units. Different crystalline forms of the same compound, or its hydrate or solvate, arise from different molecular packing in the solid, which results in different crystalline symmetries and / or unit cell parameters. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability, and / or solubility.

[0030] The term "substantially shown" means, for example, when referring to an XRPD pattern, a graph that is not necessarily identical to one shown herein but falls within the range of experimental error or deviation as considered by those skilled in the art.

[0031] As used herein, “subject,” “individual,” and “patient” are to be used interchangeably to refer to vertebrates, preferably mammals, more preferably humans. Examples of mammals, but not limited to, include mice, monkeys, humans, domestic animals, sport animals, and pets. Tissues, cells, and their offspring of biological entities obtained or cultured in vitro are also included.

[0032] As used herein, “therapeutic dose” means an amount sufficient to treat a subject suffering from a disease (e.g., a neurodegenerative disease) or to alleviate symptoms or complications associated with that disease.

[0033] As used herein, terms such as “to treat,” “treating,” and “treatment” refer to reducing or restoring a disorder and / or its associated symptoms. It will be recognized, though not excluded, that treating a disorder or condition does not require the complete elimination of the disorder, condition, or its associated symptoms.

[0034] As used herein, the term “immunotherapy” means treatment of a subject who is suffering from a disease, is at risk of developing a disease, or is suffering from a relapse of a disease, by means of a method including inducing, enhancing, suppressing, or otherwise modifying an immune response.

[0035] As used herein, the term “programmed cell death protein 1 (PD-1)” refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed in vivo on previously activated T cells and binds to two ligands, PD-L1 and PD-L2. As used herein, the term “PD-1” includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found in GenBank accession number U64863.

[0036] As used herein, the term “programmed death-ligand 1 (PD-L1)” refers to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2), which, upon binding to PD-1, downregulate T cell activation and cytokine secretion. The term “PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank accession number Q9NZQ7.

[0037] As used herein, “antibody” and “antigen-binding fragment” include spontaneously occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA, IgE, etc.) and non-spontaneous immunoglobulins, such as single-chain antibodies, chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), Fab', F(ab').sub.2, Fab, Fv, and rIgG, etc. As used herein, “antigen-binding fragment” refers to a portion of a full-length antibody that retains the ability to specifically recognize an antigen, as well as various combinations of such portions.

[0038] As used herein, the term “cancer” refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells within the body. Uncontrolled cell division and growth result in the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body via the lymphatic system or bloodstream. As used herein, “cancer” refers to primary, metastatic, and recurrent cancers.

[0039] The tumor microenvironment is a crucial aspect of cancer biology that contributes to tumor initiation, tumor progression, and response to treatment. It consists of a heterogeneous population of cells, including malignant cells and cells that support tumor growth, invasion, and metastasis despite significant crosstalk. Tumor cells often induce an immunosuppressive microenvironment that favors the development of immunosuppressive populations of immune cells, such as myeloid suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs). Therefore, targets within the tumor microenvironment have been identified that can help direct and enhance the effects of various cancer therapies, particularly immunotherapies that function by enhancing the host's anti-tumor immune response.

[0040] Surprisingly, this invention has found that a combination of a histone deacetylase (HDAC) inhibitor (e.g., thidamide or its acidic salt) and a nonsteroidal anti-inflammatory drug (NSAID) (e.g., celecoxib or its basic salt) significantly improves the immune response, modulates the tumor microenvironment, and therefore dramatically improves anticancer activity. The two active pharmaceutical components are preferably in salt, crystalline, or amorphous form.

[0041] Thidamide (Epidaza®) is a well-known histone deacetylase (HDAC) inhibitor that inhibits class I HDAC1, HDAC2, HDAC3, and class IIb HDAC10. The chemical name of thidamide is 4-(((E)-3-(pyridine-3-yl)acrylamide)methyl)-N-(2-amino-4-fluorophenyl)benzamide, and it has the following structure.

[0042] [ka]

[0043] In particular, celecoxib, sold under the brand name Celebrex (registered trademark), is a COX-2 selective nonsteroidal anti-inflammatory drug (NSAID). The chemical name of celecoxib is 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazole-1-yl]benzenesulfonamide, and it has the following structure.

[0044] [ka]

[0045] In this disclosure, acidic salts of thidamide (e.g., thidamide-HCl or thidamide-H2SO4 salt) and basic forms of celecoxib (e.g., celecoxib-Na salt) are used. Preferably, the salt form of thidamide is crystalline, and the salt form of celecoxib is amorphous.

[0046] In particular, the crystalline form of thidamide-HCl salt (crystalline form A) and the crystalline form of thidamide-H2SO4 salt (crystalline form B) are described herein.

[0047] XRPD and FTIR patterns are shown and described herein for morphology A and morphology B. As used herein, “largest peak” refers to the peak in the diffraction pattern having the highest intensity. As used herein, the term “major intensity peak” includes any peak having intensity in the top 20% of the peaks in a particular powder X-ray diffraction pattern.

[0048] Crystal morphology A has an XRPD pattern with peaks containing 2-theta values ​​as described herein. Alternatively, thidamide hydrochloride is a crystal morphology (morphology A) having a Fourier transform infrared spectroscopy (FTIR) pattern with peaks as described herein. Furthermore, morphology A is further characterized by exhibiting substantially the same XRPD pattern as shown in Figure 3(B) or substantially the same FTIR pattern as shown in Figure 4(B).

[0049] Crystal morphology B has an XRPD pattern with peaks including a 2-theta value as described herein. Alternatively, the sulfate of thidamide is a crystal morphology (morphology B) having an FTIR pattern with peaks as described herein. Furthermore, morphology B is further characterized by exhibiting substantially the same XRPD pattern as shown in Figure 3(C) or substantially the same FTIR pattern as shown in Figure 4(C).

[0050] The basic salt of celecoxib is the sodium salt of celecoxib, which can be in amorphous or crystalline form. In one embodiment, the amorphous form of the sodium salt of celecoxib has substantially the same XRPD pattern as shown in Figure 7(B).

[0051] The sodium salt of celecoxib in crystalline form (form I) has a powder X-ray diffraction (XRPD) pattern having peaks as described herein. In further embodiments, form I is further characterized by exhibiting substantially the same XRPD pattern as that shown in Figure 7(C).

[0052] Thidamide acid salts are prepared under strongly acidic conditions (Arrhenius acid with pKa < 3) during the manufacturing process and through specific processes to produce novel crystalline forms of thidamide-HCl and thidamide-H2SO4 salts. When combined with celecoxib-Na salt and immune checkpoint inhibitors, these salts significantly improve water solubility and pharmacokinetic profiles, greatly enhancing efficacy in immunotherapy. The production processes for the crystalline forms of thidamide-HCl and thidamide-H2SO4 salts are illustrated in the examples herein.

[0053] Celecoxib basic salts are prepared with metal hydrides, such as NaH, during the manufacturing process and through specific processes to produce "anhydrous" amorphous and crystalline forms of celecoxib-Na salt. Amorphous celecoxib-Na salts, when combined with thidamide acid salts and immune checkpoint inhibitors, possess considerable water solubility and a novel pharmacokinetic profile, influencing the enhancement of efficacy in immunotherapy. Similar results have been observed with the crystalline form of celecoxib-Na salt. The production processes for the amorphous and crystalline forms of celecoxib-Na salt are illustrated in the examples herein.

[0054] In some embodiments, the amount of thidamide-HCl or thidamide-H2SO4 salt in the combination is in the range of approximately 5% (w / w) to approximately 80% (w / w), approximately 30% to approximately 80% (w / w), approximately 40% to approximately 80% (w / w), approximately 20% to approximately 60% (w / w), approximately 30% to approximately 60% (w / w), approximately 40% to approximately 60% (w / w), or approximately 35% to approximately 60% (w / w).

[0055] In some embodiments, the amount of celecoxib-Na salt in the combination is in the range of approximately 5% to approximately 80% (w / w), approximately 30% to approximately 80% (w / w), approximately 40% to approximately 80% (w / w), approximately 20% to approximately 60% (w / w), approximately 30% to approximately 60% (w / w), approximately 40% to approximately 60% (w / w), or approximately 35% to approximately 60% (w / w).

[0056] In one embodiment, the combinations of the present disclosure are produced using different ratios of thidamide-HCl salt or thidamide-H2SO4 salt (which may also be referred to as thidamide salt) and celecoxib-Na salt (which may also be referred to as celecoxib salt). The pharmacokinetic properties of thidamide salt and celecoxib salt are improved compared to thidamide-K30 (the original formulation of the thidamide product, Epidaza®) and celecoxib / capsules (the original formulation of the celecoxib product, Celebrex®).

[0057] Furthermore, when combined with an immune checkpoint inhibitor, the combination (thidamide salt plus celecoxib salt) dramatically improved anticancer activity compared to thidamide-K30 plus celecoxib / capsules. Treatment with the combination of the present disclosure combined with an immune checkpoint inhibitor significantly increased efficacy in inhibiting tumor growth compared to the immune checkpoint inhibitor alone, thidamide-K30 plus celecoxib / capsules, and when both are further combined. Moreover, the combination of the combo and the immune checkpoint inhibitor significantly eradicated tumors and increased survival rates to approximately 80-100%.

[0058] Immune checkpoint inhibitors can be used in combination with the combinations described herein to stimulate the immune system against cancer cells and treat cancer. Suitable immune checkpoint inhibitors for use in this disclosure include antagonists of inhibitory receptors that inhibit PD-1, PD-L1, CTLA-4, T cell immunoglobulin-3 (TIM3), B and T lymphocyte attenuation factor (BTLA), V-domain Ig inhibitor of T cell activation (VISTA), or lymphocyte activation gene 3 (LAG3) pathway, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, anti-BTLA antibodies, anti-VISTA antibodies, and anti-LAG-3 antibodies. Examples of PD-1 or PD-L1 inhibitors, but not limited to these, include humanized antibodies that block human PD-1, such as pembrolizumab (anti-PD-1 Ab, trademark Keytruda®), nivolumab (anti-PD-1 Ab, trademark Opdivo®), or pidilizumab (anti-PD-1 Ab, CT-011), tripalimab (anti-PD-1 Ab, trademark Tuo Yi®), cintilimab (anti-PD-1 Ab, trademark Tyvyt®), camrelizumab (anti-PD-1 Ab), Bavencio® (anti-PD-L1 Ab, avelumab), and Imfinzi® (anti-PD-L1 Examples include PD-L1 Ab (durvalumab) and Tecentriq (registered trademark) (anti-PD-L1 Ab, atezolizumab), as well as fully human antibodies, such as nivolumab (anti-PD-1 Ab, trademark Opdivo®) and semiprimab-rwlc (anti-PD-1 Ab, trademark Libtayo®). Other PD-1 inhibitors may include presentation of soluble PD-1 ligands, which include, without limitation, PD-L2 Fc fusion proteins also known as B7-DC-Ig or AMP-244, and other PD-1 inhibitors currently under investigation and / or development for therapeutic use. In addition, immune checkpoint inhibitors may include, without limitation, humanized or fully human antibodies that block PD-L1, such as durvalumab and MIH1, and other PD-L1 inhibitors currently under investigation.In some embodiments, the amount of immune checkpoint inhibitor is in the range of approximately 0.5% (w / w) to approximately 15% (w / w), 0.5% (w / w) to approximately 10% (w / w), 0.5% (w / w) to approximately 5% (w / w), 1.0% (w / w) to approximately 20% (w / w), 1.0% (w / w) to approximately 15% (w / w), 1.0% (w / w) to approximately 10% (w / w), or 1.0% (w / w) to approximately 5% (w / w).

[0059] In some embodiments of this disclosure, thidamide-HCl or thidamide-H2SO4 salt, celecoxib-Na salt, and an immune checkpoint inhibitor are administered simultaneously. In some embodiments, thidamide-HCl or thidamide-H2SO4 salt, celecoxib-Na salt, and an immune checkpoint inhibitor are administered sequentially, either in one order or alternately.

[0060] The pharmaceutically active substances of the present invention can be formulated together with a “carrier.” As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antimicrobial and / or antifungal agent, isotonic agent, absorption retarder, buffer, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutically active substances is well known in the art. For example, pharmaceutical combinations can be specially formulated for administration in solid or liquid form, including those adapted to: (1) oral administration, e.g., oral tablets (aqueous or non-aqueous solutions or suspensions), lozenges, sugar-coated tablets, capsules, pills, tablets (e.g., oral buccal, sublingual, and those targeting internal absorption), boluses, powders, granules, and pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or sustained-release formulations; (3) topical application, e.g., as creams, lotions, gels, ointments, or controlled-release patches or sprays applied to the skin; (4) vaginal or rectal administration, e.g., as pessaries, creams, suppositories, or foaming agents; (5) sublingual; (6) ocular; (7) transdermal; (8) transmucosal; or (9) transnasal.

[0061] The combinations of the present disclosure can be used to modulate the tumor microenvironment and in cancer immunotherapy. Examples of cancers, but not limited to, include glioblastoma, liver cancer (e.g., hepatocellular carcinoma), colorectal cancer, glioblastoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, renal cell carcinoma, benign prostatic hyperplasia, prostate cancer, ovarian cancer, melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), gallbladder cancer, cholangiocarcinoma, bladder cancer, and uterine cancer.

[0062] The pharmaceutically acceptable combinations of this disclosure may be provided as a single formulation. In other embodiments, the pharmaceutically acceptable combinations of this disclosure may be provided as separate formulations. The pharmaceutically acceptable combinations may be formulated into various and / or more forms adapted to one or more preferred routes of administration. Thus, the pharmaceutically acceptable combinations may be administered via one or more known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutically acceptable combination or a part thereof may be administered by means of a mucosal surface, for example, by administration to the nasal or respiratory mucosa (e.g., by a spray or aerosol). The pharmaceutically acceptable combination or a part thereof may also be administered via sustained release or delayed release.

[0063] The pharmaceutically acceptable combinations of the present disclosure may be conveniently presented in unit dosage forms or may be prepared by methods well known in the pharmaceutical art. Methods for preparing combinations with pharmaceutically acceptable carriers include the step of associating the pharmaceutically acceptable combinations of the present disclosure with carriers comprising one or more minor components. Generally, the pharmaceutically acceptable combinations of the present disclosure can be prepared uniformly and / or densely by associating the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, forming the product into a desired formulation.

[0064] In some embodiments, the method may include administering an amount of the pharmaceutically acceptable combination of the present disclosure sufficient to provide doses ranging from, for example, about 10 mg / kg to about 1,000 mg / kg.

[0065] The present invention is illustrated by the following examples. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein. [Examples]

[0066] Materials and methods Materials and equipment. Thidamide-API, thidamide-K30, thidamide-HCl salt, thidamide-H2SO4 salt, and celecoxib-Na salt were supplied by GNT Biotech & Medicals Co. Ltd (Taiwan). Celecoxib-API was purchased from Aarti Drugs Ltd (India). Celecoxib capsule product (Celebrex®, 200 mg) was purchased from Pfizer (Taiwan). The following antibodies and reagents were used in animal experiments: mouse anti-PD-L1 (B7-H1) monoclonal antibody (10F.9G2; Bio X Cell), mouse anti-PD-1 (CD279) monoclonal antibody (RMP1-14; Bio X Cell), mouse anti-CTLA4 (CD152) monoclonal antibody (BE0164; Bio X Cell), and rat anti-IgG2a isotype control monoclonal antibody (2A3; Bio X Cell). LC / MS-grade methanol, HPLC-grade acetonitrile, sodium 1-heptanesulfonate, talc, and ethylenediaminetetraacetic acid were all purchased from JT Baker® (USA). Formic acid, sodium chloride, lactose, magnesium stearate, polyvinylpyrrolidone, and sodium tribasic dodecahydrate were purchased from Sigma-Aldrich (USA). Sodium lauryl sulfate was purchased from Showa Chemical Co., Ltd. (Japan). Distilled water was purified using a Milli-Q distillation system (Merck Millipore®, France). Hydrochloric acid SG (HCl) was purchased from Fisher Chemical, USA. Sodium hydride (NaH) and THF 99.5% molecular sieves were purchased from Acros, Belgium. Anhydrous ethyl ether was purchased from ECHO chemical co., LTD, Taiwan. Filter paper was purchased from Toyo Roshi Kaisha, LTD, Japan.1 1H NMR and 13 ¹³C NMR was recorded using a Bruker AVANCE 400MHz PLUS instrument. FTIR spectra were recorded using a Perkin Elmer Spotlight 200i Sp2 (Perkin Elmer IR spectrophotometer) with an AutoATR system. Powder X-ray diffraction measurements were performed using a PANalytical EMPYREAN X-ray diffractometer. Electrospray ionization masses were recorded using a Bruker microTOF. Fast atomic impact masses were recorded using a JEOL JMS-700. Gibco RPMI 1640 and DMEM, along with L-glutamine, were purchased from Invitrogen Life Technologies. HyClone FBS was purchased from Thermo Scientific.

[0067] Preparation of thidamide-HCl salt. 1 gram of thidamide-API (active pharmaceutical ingredient) was placed in a flask, and 3-5 ml of 6-8 N HCl (aqueous solution) was added. The mixture was stirred until it was completely dissolved by visual inspection. A solid precipitate was then formed without stirring. The solid precipitate was separated by suction filtration, and the slurry was formed four times to further purify it by removing impurities containing diethyl ether. The pure solid was condensed and concentrated to dryness. The solid product was then dried in an oven at 50-60°C for 16 hours, ground into a powder, and passed through a 100-mesh sieve. The thidamide-HCl salt was prepared and subjected to HPLC. 1 H-NMR, 13 Further characterization was achieved through analyses such as 1C-NMR, XRD, saturation solubility, MS, and FTIR. The thidamide-HCl salt was also prepared by the following process.

[0068] 65 mg of thidamide-API was suspended in 50–150 ml of EtOH, MeOH, DCM, THF, or H2O, and then 2–6 drops of 37% HCl were added while stirring until completely dissolved. The mixture was concentrated, and the solvent was removed until 1 ml of liquid remained, which was then added dropwise to 50 ml of ether, at which point a solid salt precipitated.

[0069] 500 mg of thidamide-API was added to 4-10 ml of 4-8 N HCl (aqueous solution) and stirred until completely dissolved. Then 10-20 ml of ethoh was added, followed by 10-20 ml of ether until a cloudy appearance was formed. The crystallization process was continued at 4°C for 12 hours. The salt was collected by filtration, washed with ether, and then dried in an oven at 60°C for 5 hours.

[0070] Preparation of thidamide-H2SO4 salt. 1 gram of thidamide-API was placed in a flask, 3-5 ml of 3-5 M H2SO4 (aqueous solution) was added, and the mixture was stirred until completely dissolved by visual inspection. The solution was slowly added dropwise to 150-200 ml of ethanol, and a solid precipitated. The solid was separated by suction filtration and rinsed three times with ethanol. The solid was purified through three slurry processes using ethanol, and excess water was further removed with diethyl ether. The pure solid was condensed and concentrated to dryness. The solid product was then dried in an oven at 50-60°C for 16 hours, ground into a powder, and passed through a 100-mesh sieve. The thidamide-H2SO4 salt was prepared and subjected to HPLC. 1 H-NMR, 13 Further characterization was achieved through analyses such as C-NMR, XRD, saturation solubility, MS, and FTIR.

[0071] Preparation of celecoxib-Na salt. 5 grams of celecoxib-API were placed in a round-bottom flask, and 150-200 ml of THF was added in the presence of nitrogen gas under airless conditions. The compound was completely dissolved by visual inspection. 450-500 mg of NaH (sodium hydride) was added to the solution and vigorously stirred. A solid precipitate formed in approximately 70-90 minutes. The THF was removed by suction filtration, and the solid was rinsed three times with 20 ml of THF. The solid was then dissolved in 300 ml of methane dichloride (DCM), and the solution was filtered by suction to remove any undissolved material. The filtrate was collected, then condensed, and concentrated to dryness using a rotary evaporator at a pressure of 30-50 mbar and a spin speed of 140 rpm to produce a solid. The pure solid was dried at 60°C for 16 hours, ground into a powder, and passed through a 100-mesh sieve. An anhydrous amorphous celecoxib-Na salt was prepared, 1 H-NMR, 13 Further analysis was performed using spectra such as C-NMR, XRD, MS, and FTIR.

[0072] Another process for producing anhydrous amorphous celecoxib-Na salt is described below. 1 gram of celecoxib-API was placed in a round-bottom flask, and 6 ml of THF was added in the presence of nitrogen gas under airless conditions. The compound was completely dissolved by visual inspection. 75–100 mg of NaH (sodium hydride) was added to the solution and vigorously stirred. A solid precipitate formed in approximately 40–80 minutes. The THF was removed by suction filtration, and the solid was rinsed three times with diethyl ether. The solid was purified through three slurry processes using diethyl ether. The solid was then dissolved in 150–200 ml of methane dichloride (DCM), and the solution was filtered by suction to remove any undissolved material. The filtrate was collected, then condensed and concentrated to dryness. During the condensation process, the initial pressure was set to 400–430 mbar until no distillate remained. The pressure was then set to 10–30 mbar until the solid salt precipitated. The pure solid was dried at 60°C for 16 hours, ground into a powder, and passed through a 100-mesh sieve. Amorphous celecoxib-Na salt was prepared and subjected to HPLC. 1 H-NMR, 13Further characterization was achieved through analyses such as C-NMR, XRD, saturation solubility, MS, and FTIR.

[0073] Anhydrous crystalline celecoxib-Na salts were also prepared using the process described above, except that the pressure during the condensation process was set to 10-30 mbar until the solid salt precipitated.

[0074] Determination of saturation solubility of thidamide-HCl, thidamide-H2SO4, and celecoxib-Na salt. A 5 mg sample of thidamide-HCl, thidamide-H2SO4, or celecoxib-Na salt was added to a 5 ml volumetric flask containing ddH2O and incubated at 25°C for 90 minutes at 100 rpm. The resulting suspension was filtered through a 0.22 μm filter. The concentrations of thidamide-HCl, thidamide-H2SO4, and celecoxib-Na salt were determined by spectrophotometric analysis at 256 nm, 256 nm, and 253 nm, respectively. The saturation solubility of each sample was determined by triple-repeated tests, and the mean and standard deviation were reported. Calibration curve preparation is described below. Stocks of thidamide and celecoxib were prepared in 99.99% MeOH. λ max These were found to be 256 nm and 253 nm, respectively. The standard curve shows a correlation equal to 0.9998 (R) over the concentration range of Beer from 0 to 20 μg / ml. 2 It exhibited good linearity characterized by the coefficient of ).

[0075] Cell line. CT26 (CRL-2638; mouse colorectal adenocarcinoma) was purchased from ATCC. The CT26 tumor cell line was grown in McCoy's 5A infusion supplemented with 10% (vol / vol) FBS at 37°C and 5% CO2.

[0076] Anticancer activity in animal models. Animal studies were approved and monitored by the Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, NO:LAC-2018-0340). Male BALB / C mice (BioLASCO Taiwan) aged 6-8 weeks were used in all animal experiments. CT26 (5×10) 6 Cancer cells were inoculated into the right flank of each mouse using sc. The tumors were left to grow for 10-11 days before randomization and treatment (tumor size approximately 200-300 mm). 3 ) were grown. CT26-retaining mice were conjugated by intravenous administration of 2.5 mg / kg of anti-IgG (lot #65481701), anti-PD-1 (lot #640517M1 and lot #717918D1), anti-PD-L1 (lot #720619F1), or anti-CTLA-4 (lot #702418A2B) antibodies on days 11, 14, 17, 20, 23, and 26 after tumor implantation. All antibodies were diluted to appropriate concentrations in 100 μL of sterile PBS (pH 7.4) (In Vitrogen Life Technology). Thidamide-K30, thidamide-HCl salt, thidamide-H2SO4 salt 、 Celecoxib (capsules / Celebrex®, 200 mg) and celecoxib-Na salt (amorphous or crystalline form) were orally administered on day 11 after tumor implantation. Tumor-carrying mice were treated daily from day 11 to day 26 with orally administered thidamide-K30, thidamide-HCl salt, and thidamide-H2SO4 salt at various doses of 12.5, 25, and 50 mg / kg. Daily treatment with celecoxib (capsules / Celebrex®, 200 mg) or celecoxib-Na salt at various doses of 12.5, 25.0, and 50 mg / kg was performed from day 11 to day 26. From the start of treatment, the tumor volume reached 3,000 mm³. 3 Anticancer activity was measured until the tumor volume reached a certain level (length × width). 2 The calculation was performed using a multiplier of 0.5.

[0077] Survival rates in animal models. Antibody or drug administration was performed from day 11 to day 25 or day 26. Tumors continued to grow in tumor-carrying mice. Tumor volume in mice was measured once every 3 or 4 days (twice a week). Tumor volume of 3,000 mm 3 When the tumor-carrying mouse reached a certain point, it was considered dead. All treatment groups were recorded and analyzed.

[0078] To overcome resistance to first-line PD-1 checkpoint blocker therapy. Animal studies were approved and monitored by the Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, NO:LAC-2018-0340). Male BALB / C mice (Biolasco, Taiwan) aged 6-8 weeks were used for all animal experiments. CT26 (5×10) 6 Cancer cells were inoculated into the right flank of each mouse using sc. The tumors were allowed to grow for 8 days before the first-line treatment (2.5 mg / kg) of anti-PD-1 antibody, administered in two doses (with a 3-day interval between doses) (average tumor size approximately 120 mm). 3 During first-line treatment, after the second dose of anti-PD-1 antibody, the tumor tripled in size over three consecutive days (average tumor size 360 ​​mm). 3 ) The failure criteria are met and the tumor volume is <600 mm 3If this was the case, the mice were re-registered. These mice with resistance to anti-PD-1 Ab were further randomized. Mice resistant to anti-PD-1 Ab were treated with seven different regimens, including: anti-IgG (2.5 mg / kg; lot #65481701), anti-PD-1 Ab (2.5 mg / kg; lot #640517M1), anti-PD-1 Ab (2.5 mg / kg) in combination with entinostat (20 mg / kg), anti-PD-1 Ab (2.5 mg / kg) in combination with thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), and anti-CTLA-4 Ab (2.5 mg / kg; lot #702418A2B) alone or in combination with thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg). Antibodies were administered intraperitoneally (ip) on days 14, 17, 20, 23, 26, and 29 (six treatments with a 3-day interval between treatments). All antibodies were diluted to an appropriate concentration in 100 μL of sterile PBS (pH 7.4) (In Vitrogen Life Technologies). Celecoxib-Na salt, thidamide-HCl salt, and entinostat were administered orally from day 14 to day 29. Celecoxib-Na salt (50 mg / kg) and thidamide-HCl salt (50 mg / kg) were administered daily, while entinostat (20 mg / kg) was administered every two days. Anticancer activity was observed from the start of treatment until the tumor volume reached 3,000 mm³. 3 The measurement was taken until it reached [a certain point]. Tumor volume was measured as length × width 2 The calculation was performed using a multiplier of 0.5. Animal studies designed and demonstrated potential treatment options for failure of first-line anti-PD-1 antibody therapy in human cancer patients who developed primary / secondary resistance to anti-PD-1 antibody treatment.

[0079] To overcome resistance to first-line PD-L1 checkpoint blockade therapy. In vivo animal studies were approved and monitored by the Taipei Medical University Institutional Animal Care and Use Committee (TMU IACUC, NO:LAC-2018-0340). Male BALB / C mice (Biolasco, Taiwan) aged 6-8 weeks were used for all animal experiments. CT26 (5×10) 6 Cancer cells were inoculated into the right flank of each mouse using sc. The tumors were allowed to grow for 8 days before receiving the first-line treatment (2.5 mg / kg) of anti-PD-L1 antibody, administered twice (with a 3-day interval between doses) (average tumor size approximately 160 mm). 3 ). Following the last administration of anti-PD-L1 (lot #720619F1) antibody, the tumor doubled in size over three consecutive days (average tumor size 320 mm). 3 ) The failure criteria are met and the tumor volume is <600 mm 3If this was the case, the mice were re-registered. These mice with resistance to anti-PD-L1 Ab were further randomized. Mice resistant to anti-PD-L1 Ab were treated with seven different regimens, including: anti-IgG (2.5 mg / kg; lot #65481701), anti-PD-1 Ab (2.5 mg / kg; lot #717918D1), anti-PD-1 Ab (2.5 mg / kg) in combination with entinostat (20 mg / kg), anti-PD-1 Ab (2.5 mg / kg) in combination with thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), and anti-CTLA-4 Ab (2.5 mg / kg; lot #702418A2B) alone or in combination with thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg). Antibodies were administered intraperitoneally (ip) on days 14, 17, 20, 23, 26, and 29 (six treatments with a 3-day interval between treatments). All antibodies were diluted to an appropriate concentration in 100 μL of sterile PBS (pH 7.4) (In Vitrogen Life Technologies). Celecoxib-Na salt, thidamide-HCl salt, and entinostat were administered orally from day 14 to day 29. Celecoxib-Na salt (50 mg / kg) and thidamide-HCl salt (50 mg / kg) were administered daily, while entinostat (20 mg / kg) was administered every two days. Anticancer activity was observed from the start of treatment until the tumor volume reached 3,000 mm³. 3 The measurement was taken until it reached [a certain point]. Tumor volume was measured as length × width 2 The calculation was performed using a multiplier of 0.5. The animal study designed and demonstrated a potential treatment option for failure of first-line anti-PD-L1 antibody therapy in human cancer patients who developed primary / secondary resistance to anti-PD-L1 antibody therapy.

[0080] Analysis of the PK profiles (pharmacokinetics) of thidamide-HCl and celecoxib-Na in Wistar rats. Pharmacokinetic studies of thidamide, celecoxib, and their salt forms (thidamide-HCl and celecoxib-Na) were conducted in 7-week-old male Wistar rats by oral administration of the compounds at a dose of 50 mg / kg in water. Wistar male rats were purchased from Biolasco (Taiwan). Prior to the pharmacokinetic studies, the animals were allowed free access to water and fasted for 12 hours. Blood samples were collected at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours after administration (n>5 / time point). At each time point, approximately 250 μL of blood was collected from the jugular vein into an EDTA-labeled Microtainer® tube. Blood samples were processed to obtain plasma samples within a scheduled 30-minute sampling time. All plasma samples were stored below -80°C until analysis. Plasma samples were analyzed by liquid chromatography-mass spectroscopy (LC-MS / MS, 6470 Agilent Tech., USA) for treatment with thidamide-k30, thidamide-HCl salt, celecoxib (capsule / Celebrex®, 200 mg), and amorphous celecoxib-Na salt, with quantification limits of 14.2 ng / mL (thidamide) and 45.5 ng / mL (celecoxib). The PK parameters of thidamide-k30, thidamide-HCl, celecoxib / Celebrex®, and celecoxib-Na were calculated using the trapezoidal rule and the validated non-compartmental analysis tool of Phoenix WinNonlin software (version 6.3). Pharmacokinetic studies were conducted at Taipei Medical University and approved by the Institutional Animal Care and Use Committee (IACUC approval number: LAC-2017-0331). Samples were prepared and analyzed as described below.To 50 μL of calibration standard solution or plasma sample, 150 μL of acetonitrile (containing 10% methanol) was added, and the sample was vortexed for 1 minute to precipitate proteins. After centrifugation at 4°C and 21,130 × g for 15 minutes, 5 μL of supernatant was directly injected into the LC-MS / MS for analysis. Analysis was performed using a 6470 series liquid chromatograph (Agilent Technologies, USA) equipped with a quaternary pump (1260 Infinity II Quaternary Pump LC system), degassing unit, autosampler, thermostatic column compartment, and LC-MS / MS-6470 mass spectrometer (Agilent Technologies, USA). Chromatographic separation was achieved using a LiChrospher® 60RP-Select B column (5 μm, 125 × 4.6 mm, Merck, Germany) at 40°C and with the mobile phase gradients listed in the table below. The flow rate was 0.5 mL / min. The total operating time was 10 minutes. The drying gas flow and spray gas flow were set to 6 and 1.5 L / min, respectively. The system's drying gas temperature and capillary voltage were adjusted to 250°C and 3000 V, respectively. LC-MS / MS was performed in multiple reaction monitoring mode using target ions: m / z 391.1 and 265.1 for thidamide with a positive ion electrospray ionization interface, and m / z 380 and m / z 316 for celecoxib with a negative ion electrospray ionization interface.

[0081] [Table 1]

[0082] Statistics. Mean values and standard errors were calculated for all data points from at least four independent experiments. Pairwise comparisons of tumor sizes between each of the experimental conditions and the IgG control group were performed using a Student's two-sample t-test (Systat Software, SanJose, CA, USA). Student's test or ANOVA was performed for the analysis of animal efficacy data. Kaplan-Meier curves and log-rank tests were generated using SigmaStat 3.5 software. All P values < 0.05 were considered statistically significant.

[0083] [Example 1] Characterization of a Novel Crystal Form of Tidamide-HCl Salt In 2014, tidamide was approved by China CFDA (NMPA) for relapsed or refractory peripheral T-cell lymphoma (PTCL). Tidamide (trade name, Epidaza®) is available for oral use as tablets containing 5 mg of tidamide, and the recommended dose is 30 mg twice weekly at intervals longer than 3 days. The tablets contain tidamide-API coated with polyvinylpyrrolidone k30 (PVP-K30) to improve its water solubility and oral bioavailability. In the present invention, the inventors developed a formulation for tidamide API to produce novel crystal forms of tidamide-HCl and tidamide-H2SO4 salts. The properties of tidamide-HCl and tidamide-H2SO4 salts were able to significantly improve water solubility and oral bioavailability. As shown in Figure 1, the structure of the tidamide salts was 1 identified by 1H-NMR and 13 13C-NMR. 1 1H NMR spectra were recorded using a Bruker Avance 400 MHz plus instrument with the solvent dimethyl sulfoxide (DMSO-d6). 13 13C NMR spectra were recorded at 100 MHz. As shown in Figures 1A and B, 1 the 1H-NMR data showed that compared to the tidamide-API, the chemical shift signal δ of the NH2 group of aniline HWe demonstrated that 5.20 disappeared in the thidamide-HCl salt. This result demonstrates that the salt form was formed at the C21-NH2 position. + Cl - Alternatively, it may be described as thidamide-HCl salt. 13 ¹³C-NMR data are shown in Figures 1D and 1E. Detailed chemical shift data for thidamide-API and thidamide-HCl salts are described in Table 1 and Figure 1G. Furthermore, molecular weight was determined using ESI-MS. The mass spectrum of thidamide-HCl salt was recorded using a Bruker microTOF with an ESI source and ionic polarity: positive / negative mode. The positive ion mode ESI-MS spectrum of thidamide-HCl salt was determined and is shown in Figure 2A. The most abundant peak is m / z 391.158 [M+H]. + It has [this characteristic]. However, the negative ion mode ESI-MS spectrum for thidamide-HCl salt was determined and is shown in Figure 2B. The most abundant peak is m / z 425.118 [M+Cl]. -It has. Next, the crystal form of tidamide-HCl salt was characterized by XRD. The comparison of the XRD profiles between tidamide-API and tidamide-HCl salt was analyzed. The XRD measurement was performed using a PANalytical Empyrean X-ray diffractometer. For the X-ray irradiation source, a Cu (λ = 45 kV, 40 mA) anode was used in the range of 2θ between 3° and 40° at a scan speed of 1 / min. The XRD data demonstrated that tidamide-API and tidamide-HCl salt had different XRD profiles as shown in Figure 3A (tidamide-API) and 3B (tidamide-HCl salt). As shown in Figure 3D, the 2-theta values were different between tidamide-API and tidamide-HCl salt. This data indicated that tidamide-HCl salt had a novel crystal form different from that of tidamide-API. Two different crystal forms of tidamide-API and tidamide-HCl salt were analyzed by a saturation solubility test. As shown in Table 2, tidamide-HCl salt was much more water-soluble than tidamide-API and tidamide-K30. Tidamide-API was water-insoluble, and tidamide-K30, that is, the formulation of tidamide tablets (Epiza (registered trademark)), showed low water solubility (about 26.03 μg / mL). Three independent batches of tidamide-HCl salt were tested and showed saturation solubilities of about 554.83, 566.90, and 536.06 μg / mL, respectively. As shown in Table 2, these results demonstrated that tidamide-HCl salt significantly improved water solubility by more than 20 times compared with tidamide-K30. The improvement in the water solubility of tidamide-HCl salt can increase oral bioavailability, which in turn will improve the PK profile and anti-cancer efficacy. The structure of tidamide-HCl salt was further confirmed by FTIR analysis as shown in Figure 4. The FTIR spectrum was recorded on a PerkinElmer Spotlight 200i Sp2 (PerkinElmer IR spectrophotometer) equipped with an auto-ATR system. The FTIR spectrum was scanned over the range of 4000 to 700 cm -1 The range was scanned. As shown in Figure 4B, in the profile of tidamide-HCl salt, the wavenumbers 3275 and 3309 (unit: cm -1In this case, the signal for aniline NH elongation disappeared. A comparison of FTIR data for thidamide-API (Figure 4A) and thidamide-HCl salt is shown in Figure 4D.

[0084] [ka]

[0085] [Table 2]

[0086] [Table 3]

[0087] [Example 2] Characterization of novel crystalline forms of thidamide-H2SO4 salts A second salt form of thidamide was prepared using H2SO4. As shown in Figures 1C and 1F, the structure of the thidamide-H2SO4 salt was 1 H-NMR and 13 Identified by 13C-NMR. Using a Bruker Avance 400 MHz Plus instrument with dimethyl sulfoxide (DMSO-d6) as the solvent, 1 1H NMR was recorded. 13 The 13C NMR spectrum was recorded at 100 MHz. As shown in Figures 1C and 1A, 1 H-NMR data shows the chemical shift signal of the NH2 group in aniline δ H We demonstrated that 5.20 disappeared in the thidamide-H2SO4 salt compared to thidamide-API. This result demonstrates that the salt form was formed at the C21-NH2 position. + HSO4 -Alternatively, it can be described as thidamide-H2SO4 salt. Detailed chemical shift data for thidamide-API and thidamide-H2SO4 salt are shown in Table 1 and Figure 1G. Furthermore, the molecular weight was determined using ESI-MS. The mass spectrum of thidamide-H2SO4 salt was recorded using a Bruker microTOF with an ESI source and ionic polarity: positive / negative mode. The positive ion mode ESI-MS spectrum of thidamide-H2SO4 salt was determined and is shown in Figure 2C. The most abundant peak is m / z 391.16 [M+H]. + It has [this characteristic]. However, the negative ion mode ESI-MS spectrum for thidamide-H2SO4 salt was determined and is shown in Figure 2D. The most abundant peak is m / z 487.12 [M+HSO4]. -Next, the crystalline form of thidamide-H2SO4 salt was characterized by XRD. A comparison of the XRD profiles between thidamide-API and thidamide-H2SO4 salt was analyzed. XRD measurements were performed using a panarithmic empyrian X-ray diffractometer. A Cu (λ=45kV, 40mA) anode was used relative to the X-ray source, with a 2θ range of 3-40° and a scan rate of 1 / min. As shown in Figures 3A (thidamide-API) and 3C (thidamide-H2SO4 salt), the XRD data demonstrated that thidamide-API and thidamide-H2SO4 salt have different XRD profiles. As shown in Figure 3D, the 2-theta values ​​differed between thidamide-API and thidamide-H2SO4 salt. This data indicates that thidamide-H2SO4 salt has a novel crystalline form different from that of thidamide-API. Two different crystalline forms of thidamide-API and thidamide-H2SO4 salt were analyzed by saturation solubility testing. As shown in Table 2, thidamide-H2SO4 salt was far more water-soluble than thidamide-API and thidamide-K30. Thidamide-API was water-insoluble, and thidamide-K30, i.e., the thidamide tablet formulation (Epidaza®), showed low water solubility (approximately 26.03 μg / mL). Three independent batches of thidamide-H2SO4 salt were tested and showed saturation solubility of approximately 597.39, 652.90, and 561.5 μg / mL, respectively. As shown in Table 2, these results demonstrate that thidamide-H2SO4 salt significantly improved water solubility by more than 20 times compared to thidamide-K30. Improving the water solubility of thidamide-H2SO4 salt can increase its oral bioavailability, which in turn improves its PK profile and anticancer efficacy. The structure of thidamide-H2SO4 salt was further confirmed by FTIR analysis, as shown in Figure 4C. FTIR spectra were recorded using a PerkinElmer Spotlight 200i Sp2 (PerkinElmer IR spectrophotometer) with an auto-ATR system. The FTIR spectra were recorded at 4000–700 cm⁻¹. -1 The scan covered the range of 3412 and 3309 wavenumbers (unit: cm). As shown in Figure 4C, the profile of thidamide-H2SO4 salt was 3412 and 3309 wavenumbers (unit: cm). -1The signal for aniline NH elongation was lost in this case. A comparison of FTIR data for thidamide-API (Figure 4A) and thidamide-H2SO4 salt is shown in Figure 4D.

[0088] [Example 3] Characterization of a novel amorphous form of celecoxib-sodium salt The amorphous form is characterized by having a short-range molecular order, which differs from the crystalline form which has a long-range order of molecular packing. Celecoxib is classified as Class II in the BCS (Biologics Classification System). Celecoxib has low solubility and high osmotic properties. Most commercially available drugs have adequate osmotics, and solubility is the rate-limiting step for the absorption of these drugs. On the other hand, solubility has been another important problem in drug discovery. The preparation of the amorphous form provides an efficient solution to the low solubility problem. The inventors designed and tested a unique method for producing the amorphous form of celecoxib-Na salt. A novel amorphous celecoxib-Na salt was produced through several steps, including purification and condensation, by replacing hydrogen from the sulfonamide group of celecoxib-API with Na under strong NaH base conditions. First, celecoxib-API and celecoxib-Na salt 1 The 1H-NMR spectra were compared and are shown in Figures 5A and 5B. As shown in Figure 5B, the disappearance of the two hydrogen signals in the sulfonamide was clearly demonstrated. This suggests that the two Na atoms replaced the two hydrogen atoms from the sulfonamide group to generate a novel celecoxib-Na salt. 1 The 1H NMR data (400 MHz, CDCl3) was recorded as follows: δ 2.36 (3H, s), 4.86 (2H, s), 6.72 (1H, s), 7.09 (2H, dd), 7.16 (2H, d), 7.46 (2H, m), 7.89 (2H, m). Celecoxib-Na salt 1 The 1H NMR data (400 MHz, CDCl3) was recorded as follows: δ 2.09 (3H, s), 6.59 (1H, s), 6.87 (4H, s), 6.94 (2H, d), 7.61 (2H, d). As shown in Figures 5B and 5A, 1H-NMR data shows the chemical shift signal of the NH2 group in sulfonamides. H We demonstrated that 4.86 disappeared in celecoxib-Na salt compared to celecoxib-API. Furthermore, 13 ¹³C-NMR data were demonstrated in Figures 5C, 5D, and 5E. Next, the inventors confirmed the molecular weight of the celecoxib-Na salt as shown in Figure 6 using FAB-MS. The mass spectrum of the celecoxib-Na salt was recorded by using a JEOL JMS-700 equipped with a FAB source and ionic polarity: positive mode. The data showed an measured m / z value of 426.1[M+H + It was demonstrated that celecoxib-Na salt is C 17 H 12 It was suggested that the molecule is F3N3Na2O2S, with a molecular weight of 425.04. 17 H 12 The calculated m / z value for F3N3Na2O2S is 425.04, while the FAB-MS value is 426.1(M+H). +It was found that the data reaffirmed that celecoxib-Na salt contains two sodium atoms replacing two hydrogen atoms. Next, the water solubility of amorphous celecoxib-Na salt was evaluated. As shown in Table 3, celecoxib-API was water-insoluble, while celecoxib-Capsules (Celebrex®) was slightly water-insoluble (approximately 1.19 μg / mL). When three independent batches of the amorphous form of celecoxib-Na salt were tested, they had saturated solubility of approximately 54.72, 54.45, and 56.72 μg / mL, respectively. The water solubility of celecoxib-Na salt was significantly improved compared to celecoxib-API and celecoxib-Capsules. This result suggests that the improved water solubility properties of the amorphous salt form of celecoxib-Na may increase oral bioavailability and therefore therapeutic efficacy. Furthermore, as shown in Figure 7, XRD data showed that while celecoxib-API has a specific crystalline pattern (Figure 7A), amorphous celecoxib-Na salt has an amorphous diffraction pattern as shown in Figure 7B. This result indicates that amorphous celecoxib-Na salt possesses a specific form with a significant improvement in saturated water solubility. Many studies have focused on creating amorphous forms of celecoxib by using different polymers as supports. As shown in Figure 8, the structure of amorphous celecoxib-Na salt was reconfirmed by FTIR analysis. As shown in Figures 8A and 8B, amorphous celecoxib-Na salt exhibits diffraction patterns at wavenumbers 3234 and 3341 (unit: cm). -1 The NH elongation of the sulfonamide in ) was eliminated. A comparison of FTIR data between celecoxib-API and amorphous celecoxib-Na salt is shown in Figure 8D.

[0089] [Table 4]

[0090] [Example 4] Characterization of the crystalline morphology of celecoxib-Na salt Prepare crystalline celecoxib-Na salt, 1 H-NMR,13 Analysis was performed by 13C-NMR, XRD, MS, and FTIR. As shown in Table 3, the water solubility of the crystalline forms of celecoxib-Na salt from three different batches was approximately 111.5, 133.63, and 95.34 μg / mL. As shown in Figures 7C and 7D, the crystal diffraction pattern of crystalline celecoxib-Na salt differed from that of celecoxib-API. This result indicated that crystalline celecoxib-Na salt possesses a specific crystalline form that resulted in a significant improvement in water solubility. The structure of crystalline celecoxib-Na salt was reconfirmed by FTIR analysis as shown in Figure 8C. As shown in Figures 8C and 8D, celecoxib-Na salt showed improved water solubility compared to celecoxib-API at wavenumbers 3234 and 3341 (unit: cm). -1 The NH elongation of the sulfonamide in ) was eliminated.

[0091] [Example 5] Comparison of anticancer activity between thidamide-K30 and thidamide-HCl salt when celecoxib capsules are combined with anti-PD-1 Ab in CT26-retaining mice. To investigate whether the thidamide salt form increases the potential for tumor inhibition, we evaluated the therapeutic effects of thidamide-K30 plus celecoxib capsules versus thidamide-HCl salt plus celecoxib capsules in combination with anti-PD-1 antibody (2.5 mg / kg; lot #640517M1) in CT26-carrying mice. As shown in Figure 9, the tumor size in CT26 tumor-carrying mice was approximately 200-250 mm at day 11. 3The levels increased. Mice were then treated with six different regimens, as shown. As shown in Figure 9A, thidamide-K30 50 mg / kg combined with anti-PD-1 Ab plus celecoxib capsules 50 mg / kg significantly inhibited tumor growth in CT26-carrying mice compared to the anti-PD-1 Ab group. The results for thidamide-HCl salt plus celecoxib capsules 50 mg / kg at doses of 12.5, 25, or 50 mg / kg combined with anti-PD-1 Ab also showed considerable inhibition of tumor growth in CT26 tumor-carrying mice compared to the anti-PD-1 Ab group. To compare the anticancer activity between thidamide salt forms and thidamide-K30, efficacy was evaluated by the following assessment. In this study, the inventors defined complete response (CR, ≤0.5 times tumor growth in tumor-carrying mice at the end of treatment); partial response (PR, >0.5 times tumor size and ≤2 times tumor growth in tumor-carrying mice at the end of treatment); stable disease (SD, 2 to 5 times tumor growth in tumor-carrying mice at the end of treatment); and progressive disease (PD, equal to or greater than 5 times tumor growth in tumor-carrying mice at the end of treatment).

[0092] As shown in Figure 9B, thidamide-HCl 50 mg / kg plus celecoxib capsules 50 mg / kg combined with anti-PD-1 Ab 2.5 mg / kg was even more effective in inhibiting tumor growth in CT26 tumor-carrying mice compared to thidamide-K30 50 mg / kg plus celecoxib capsules 50 mg / kg combined with anti-PD-1 Ab. Treatment with thidamide-K30 50 mg / kg plus celecoxib capsules 50 mg / kg combined with anti-PD-1 Ab achieved CR in 6 mice (60%) and PD in 4 mice with moderate tumor growth. Treatment with thidamide-HCl 50 mg / kg plus celecoxib-HCl 50 mg / kg combined with anti-PD-1 Ab achieved a response rate of 89%, with 5 mice achieving PR and 3 mice achieving CR, and no mice developing PD. These results suggested that the thidamide-HCl salt form was more efficient than thidamide-K30 due to its higher water solubility and oral bioavailability, thus improving therapeutic efficacy. Figures 9A and 9B also showed that thidamide-HCl salt 12.5 mg / kg plus celecoxib-capsules 50 mg / kg combined with anti-PD-1 Ab was sufficient to affect the tumor microenvironment, reactivate cytotoxic T lymphocytes, and kill tumors. As shown in Figure 9C, none of the treated mice lost any body weight. After discontinuation of treatment on day 26, tumors in CT26 tumor-carrying mice grew faster in the IgG control group. However, the thidamide-HCl salt plus celecoxib-Na salt regimen combined with an immune checkpoint inhibitor was very potent in inhibiting tumor growth and thus significantly increased survival (Figure 9D). As shown in Figure 9D, thidamide-HCl 50 mg / kg combined with anti-PD-1 Ab plus celecoxib capsules 50 mg / kg significantly increased survival to approximately 77.7%, while thidamide-K30 50 mg / kg combined with anti-PD-1 Ab plus celecoxib capsules 50 mg / kg achieved only a 60% survival rate in the CT26-retaining tumor mouse model.It is noteworthy that tidamide-HCl 25 mg / kg plus celecoxib capsules 50 mg / kg, combined with anti-PD-1 Ab, significantly increased survival to approximately 66.6%. This result suggests that tidamide-HCl plus celecoxib capsules is more potent than tidamide-K30 plus celecoxib capsules in controlling and regulating the tumor microenvironment and enhancing immunotherapy to some extent.

[0093] This study also demonstrated that thidamide-HCl plus celecoxib capsules, when combined with an immune checkpoint inhibitor, were more potent than thidamide-K30 plus celecoxib capsules in enhancing the anti-cancer immune response. On the other hand, a one-to-one comparison between thidamide-HCl plus celecoxib capsules and thidamide-K30 plus celecoxib capsules when combined with anti-PD-1 Ab demonstrated that the anti-cancer activity of the combination regimen with thidamide-HCl plus celecoxib-Na was better than that of the combination regimen with thidamide-K30 plus celecoxib capsules.

[0094] [Example 6] Comparison of anticancer effects between thidamide-K30 plus celecoxib capsules combined with anti-PD-1 Ab and thidamide-HCl salt plus celecoxib-Na salt in CT26-retaining mice. To demonstrate improved tumor inhibitory activity, the inventors evaluated the therapeutic effect of tidamide-K30 plus celecoxib capsules versus tidamide-HCl salt plus celecoxib-Na salt in CT26-retaining mice when used in combination with an anti-PD-1 antibody (2.5 mg / kg; lot #640517M1). As shown in Figure 10, the tumor size of CT26-retaining mice was approximately 200-250 mm on day 10. 3Each test group was treated when the tumor size increased to a certain level. First, in CT26-retaining mice, thidamide-K30 50 mg / kg combined with anti-PD-1 Ab plus celecoxib capsules 50 mg / kg significantly inhibited tumor growth compared to the anti-PD-1 Ab group (Figure 10A). The results for thidamide-HCl salt 50 mg / kg combined with anti-PD-1 Ab plus amorphous celecoxib-Na salt at various doses of 12.5, 25, and 50 mg / kg showed significant inhibition of tumor growth in CT26-retaining mice compared to the anti-PD-1 Ab group (Figure 10A). Figure 10B demonstrates that 50 mg / kg of thidamide-HCl salt combined with anti-PD-1 Ab 2.5 mg / kg plus different doses of celecoxib-Na salt was even more effective in inhibiting tumor growth in CT26-retaining mice compared to 50 mg / kg of thidamide-K30 combined with anti-PD-1 Ab 2.5 mg / kg plus 50 mg / kg of celecoxib-Capsules. These results suggest that these salt forms are more efficient than thidamide-K30 and celecoxib-Capsules because thidamide-HCl salt and celecoxib-Na salt possess higher water solubility and oral bioavailability, thus improving therapeutic efficacy.

[0095] Figures 10A and 10B show that 50 mg / kg of tidamide-HCl salt combined with 12.5 mg / kg of celecoxib-Na salt was sufficient to affect the tumor microenvironment, reactivate cytotoxic T lymphocytes, and kill tumors. A one-to-one comparison of the anticancer effects of the same dose (50 mg / kg) of tidamide-K30 plus celecoxib capsules (4 mice achieved CR, 50%) combined with anti-PD-1 Ab 2.5 mg / kg, as shown in Figure 10B, showed that the combination regimen with the latter salt form had better tumor growth inhibitory efficacy in CT26-retaining mice, with 7 mice achieving CR (100%). Furthermore, as shown in Figure 10C, the percentage of tumor-free animals (CR) was evaluated in different treatment groups. All salt forms of the regimen combined with anti-PD-1 Ab were more potent in inhibiting tumor growth (higher percentage of tumor-free mice) compared to tidamide-K30 plus celecoxib capsules. These results suggest that, in a CT26-retaining mouse model, celecoxib-Na salt is more potent than celecoxib capsules in inhibiting tumor growth when combined with an immune checkpoint inhibitor. Similar results were demonstrated with tidamide-HCl compared to tidamide-K30. This finding also demonstrated that the dose of tidamide-HCl salt plus celecoxib-Na salt could be reduced when combined with an immune checkpoint inhibitor for potent reactivation of cytotoxic T-lymphocytes in the tumor microenvironment to inhibit tumor growth, as shown in Figures 10A and 10B. None of the treated mice lost any body weight, as shown in Figure 10D.

[0096] After discontinuing treatment on day 25, tumors in CT26-retaining tumor mice grew faster in the IgG (2.5 mg / kg; lot #65481701) control group. As shown in Figure 10E, in the CT26-retaining tumor mouse model, the group receiving 50 mg / kg of thidamide-HCl salt plus 50 mg / kg of celecoxib-Na salt combined with anti-PD-1 Ab significantly increased survival to approximately 100% compared to thidamide-K30 plus celecoxib-capsules (approximately 75%). Survival in the anti-PD-1 group was only 37.5%. These results suggest that thidamide-HCl salt plus celecoxib-Na salt is more potent than thidamide-K30 plus celecoxib-capsules in controlling and regulating the tumor microenvironment and enhancing the immune response to some extent. In conclusion, the thidamide-HCl plus celecoxib-Na regimen combined with an immune checkpoint inhibitor was highly potent in inhibiting tumor growth and thus significantly increased survival (Figure 10E). This study demonstrated that thidamide-HCl plus celecoxib-Na combined with an immune checkpoint inhibitor is more potent in enhancing the anti-cancer immune response than thidamide-K30 plus celecoxib capsules. On the other hand, a one-to-one comparison between thidamide-HCl plus celecoxib-Na and thidamide-K30 plus celecoxib capsules when combined with anti-PD-1 Ab demonstrated that the anti-cancer activity of the thidamide-HCl plus celecoxib-Na combination regimen was better than that of the thidamide-K30 plus celecoxib capsules combination regimen.

[0097] [Example 7] In CT26 tumor-carrying mice, we will identify the optimal therapeutic response dose of tidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 antibody, and evaluate tidamide-H2SO4 plus celecoxib-Na in combination with an anti-PD-1 antibody. To test the optimal therapeutic response dose of tidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 antibody in CT26 tumor-carrying mice, tumor size approximately 300 mm was used. 3Mice with the specified characteristic were treated with different doses of thidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 antibody. As shown in Figure 11A, thidamide-HCl plus amorphous celecoxib-Na in combination with anti-PD-1 antibody (2.5 mg / kg; lot #717918D1), at a dose of 50 mg / kg, was superior to doses of 25 mg / kg and 12.5 mg / kg. This result also indicated that thidamide-HCl plus celecoxib-Na in combination with anti-PD-1 antibody (2.5 mg / kg), at a dose of 25 mg / kg, had a similar therapeutic response compared to thidamide-K30 plus celecoxib-capsules in combination with anti-PD-1 antibody (2.5 mg / kg), at a dose of 50 mg / kg. In CT26 tumor-carrying mice, thidamide-HCl plus celecoxib-Na, when combined with an anti-PD-1 antibody, suggested that it possessed potent anticancer activity at the same dose as thidamide-K30 plus celecoxib capsules. In addition, as shown in Figure 11B, in CT26 tumor-carrying mice, thidamide-HCl plus crystalline celecoxib-Na, when combined with an anti-PD-1 antibody, exhibited lower anticancer activity at the same dose compared to thidamide-HCl plus amorphous celecoxib-Na. On the other hand, as shown in Figure 11B, thidamide-H2SO4 plus celecoxib-Na, when combined with an anti-PD-1 antibody, possessed potent anticancer activity similar to that of thidamide-HCl plus celecoxib-Na, when combined with an anti-PD-1 antibody. In this experiment, the tumors had an average volume of approximately 300 mm³ before the different treatments. 3 The anti-PD-1 antibody protein activity was lower compared to previous studies, indicating that the anti-cancer therapeutic effect of anti-PD-1 antibody treatment was very weak in this study. As shown in Figure 11B, the optimal dose of thidamide-HCl salt 50 mg / kg or thidamide-H2SO4 salt 50 mg / kg plus celecoxib-Na salt, dose 50 mg / kg, combined with anti-PD-1 antibody (2.5 mg / kg), exhibited the best therapeutic response in this study. Furthermore, amorphous celecoxib-Na salt achieved a better response rate than crystalline celecoxib-Na salt in the combination regimen. Before treatment, the average tumor size was approximately 300 mm.3 In this case, thidamide-K30 plus celecoxib capsules combined with an anti-PD-1 antibody achieved a response rate of only about 33%. However, thidamide-HCl salt or thidamide-H2SO4 salt plus celecoxib-Na salt combined with an anti-PD-1 antibody significantly improved the response rate to up to 62.5% and 55.5%, respectively. These results demonstrate that in CT26 tumor-carrying mice, the salt forms of thidamide and celecoxib are more potent in enhancing the immune response rate than thidamide-K30 and celecoxib capsules. As shown in Figure 11C, none of the treated mice lost any body weight.

[0098] After discontinuing treatment on day 26, tumors in CT26-retaining tumor mice grew faster in the IgG control group. However, regimens of thidamide-HCl or thidamide-H2SO4 plus celecoxib-Na combined with an immune checkpoint inhibitor were very potent in inhibiting tumor growth, and thus significantly increased survival (Figure 11D). As shown in Figure 11D, in this study, the group receiving thidamide-K30 50 mg / kg plus celecoxib capsules 50 mg / kg combined with an anti-PD-1 antibody showed only about a 22% increase in survival, as the anti-PD-1 antibody's anticancer activity was lower compared to previous results. On the other hand, in a CT26-retaining tumor mouse model, the survival rates of the groups receiving 50 mg / kg of tidamid-HCl or tidamid-H2SO4 plus 50 mg / kg of celecoxib-Na in combination with an anti-PD-1 antibody were significantly increased to approximately 37.5% and 44.4%, respectively. This result suggests that tidamid-HCl or tidamid-H2SO4 plus celecoxib-Na in combination with an anti-PD-1 antibody is more potent in controlling and regulating the tumor microenvironment and enhancing the immune response to some extent than tidamid-K30 plus celecoxib capsules in combination with an anti-PD-1 antibody. This study also demonstrated that tidamid-HCl or tidamid-H2SO4 plus celecoxib-Na in combination with an immune checkpoint inhibitor is more potent in enhancing the anti-cancer immune response than tidamid-K30 plus celecoxib capsules in combination with an immune checkpoint inhibitor. On the other hand, when combined with anti-PD-1 Ab, a one-to-one comparison between thidamide-HCl plus celecoxib-Na and thidamide-K30 plus celecoxib capsules demonstrated that the anticancer activity of the thidamide-HCl plus celecoxib-Na combination regimen was better than that of the thidamide-K30 plus celecoxib capsules combination regimen.

[0099] [Example 8] In CT26-retaining mice, resistance to first-line anti-PD-1 Ab treatment was overcome by second-line treatment with thidamide-HCl plus celecoxib-Na, combined with anti-PD-1 / anti-CTLA-4 Ab. In this study, mice were treated with a second-line therapy to mimic the treatment for first-line drug resistance that occurs in human first-line cancer therapy. The majority of human cancer patients receiving first-line anti-PD-1 antibody therapy were induced to develop resistance to a second-line therapy using tidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 / anti-CTLA-4 antibody in case of first-line anti-PD-1 antibody failure. The study evaluated whether tidamide-HCl plus celecoxib-Na could improve immune checkpoint inhibitor sensitivity by modulating the tumor microenvironment. Tumors were allowed to grow for 8 days before first-line treatment with anti-PD-1 antibody (2.5 mg / kg; lot #717918D1), administered in two doses (with a 3-day interval between doses) (average tumor size approximately 120 mm). 3 ). After the second dose of the first-line anti-PD-1 antibody treatment, the tumor tripled in size over three consecutive days (average tumor size 360 ​​mm). 3 The following criteria for treatment failure are met, and the tumor volume is <600 mm 3If this was the case, the mice were re-registered. These mice with resistance to anti-PD-1 Ab were further randomized. Ten different treatment regimens were used, as shown (n=9-11 mice / group). These mice were randomized to different second-line treatment groups, including: anti-IgG Ab (2.5 mg / kg; lot #65481701), anti-PD-1 Ab (2.5 mg / kg; lot #717918D1), and as positive controls, entinostat (20 mg / kg) in combination with anti-PD-1 Ab (2.5 mg / kg), thidamide-K30 plus celecoxib capsules, thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), thidamide-K30 plus celecoxib capsules in combination with anti-PD-1 Ab, thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg) in combination with anti-PD-1 Ab (2.5 mg / kg), and anti-CTLA-4 The mice were treated with Ab (2.5 mg / kg; lot #702418A2B), tidamide-K30 plus celecoxib capsules in combination with anti-CTLA-4 Ab (2.5 mg / kg), or tidamide-HCl salt (50 mg / kg) plus celecoxib-Na salt (50 mg / kg) in combination with anti-CTLA-4 Ab (2.5 mg / kg). Antibodies were administered intraperitoneally (ip) six times (with a three-day interval between two injections). Entinostat was administered orally eight times (every two days). Tidamide-K30 or tidamide-HCl salt and celecoxib capsules or celecoxib-Na salt were administered orally 16 times (daily). As shown in Figures 12A and 12B, no mice achieved a PR (0% response rate) in the anti-PD-1 Ab group, and eight mice had PD with rapid tumor growth. Treatment with thidamide-HCl plus celecoxib-Na was more potent in inhibiting tumor growth compared to thidamide-K30 plus celecoxib capsules. Treatment with thidamide-HCl plus celecoxib-Na resulted in complete response (CR) in 3 mice and disease progression (PD) in 4 mice with rapid tumor growth (response rate 33.3%).However, treatment with tidamide-K30 plus celecoxib capsules resulted in only one mouse achieving a partial response (PR), while eight mice progressed to progressive disease (PD) with rapid tumor growth (10% response rate). When tidamide-HCl salt plus celecoxib-Na salt was combined with anti-PD-1 Ab, the results showed that four mice achieved complete response (CR) (36.3% response rate), while six mice progressed to PD with much slower tumor growth. However, treatment with tidamide-K30 plus celecoxib capsules combined with anti-PD-1 Ab resulted in only one mouse achieving a PR, while nine mice progressed to PD with moderate tumor growth (10% response rate). These results suggest that anti-PD-1 Ab does not possess anticancer activity in mice resistant to anti-PD-1 Ab. Furthermore, in mice resistant to anti-PD-1 Ab, the tidamamide-HCl salt plus celecoxib-Na salt regimen was very potent in controlling the tumor microenvironment and increasing anti-PD-1 Ab sensitivity. Additionally, treatment with tidamamide-K30 plus celecoxib capsules showed significantly lower anticancer activity compared to the salt combination of tidamamide-HCl salt plus celecoxib-Na salt. As shown in Figure 12B, the anti-CTLA-4 Ab group showed less inhibition of tumor growth compared to the anti-PD-1 Ab group, but so far no mice have achieved CR or PR, with only 7 mice. Mice reached disease progression (PD) with moderate tumor growth. However, in the tidamide-HCl plus celecoxib-Na regimen combined with anti-CTLA-4 Ab, the results showed that 4 mice achieved complete response (CR), 2 mice achieved partial response (PR) (60% response rate), and no mice reached PD. Furthermore, in the tidamide-K30 plus celecoxib-capsules regimen combined with anti-CTLA-4 Ab, the results showed that 2 mice achieved CR, 1 mouse achieved PR (25% response rate), and 5 mice reached PD with moderate tumor growth. Finally, in the positive control group entinostat combined with anti-PD-1 Ab, 1 mouse achieved PR (9% response rate), and 8 mice reached PD with rapid tumor growth. In summary, the tidamide-HCl plus celecoxib-Na regimen was potent in enhancing the response rate in mice resistant to anti-PD-1 Ab. Furthermore, in mice resistant to anti-PD-1 Ab, thidamide-HCl plus celecoxib-Na was more potent in enhancing response rates when combined with anti-CTLA-4 Ab than when combined with anti-PD-1 Ab alone.

[0100] After discontinuing treatment on day 31, tumors in CT26 tumor-carrying mice grew faster in the anti-PD-1 and anti-CTLA-4 groups (Figure 12D). Survival rates were assessed on day 60. Treatment with tidamide-K30 plus celecoxib capsules, without the combination of anti-PD-1 Ab, showed better survival rates than the combination with anti-PD-1 Ab, reaching 11.1% and 0%, respectively. Furthermore, treatment with tidamide-HCl salt plus celecoxib-Na salt, without the combination of anti-PD-1 Ab, also showed better survival rates than the combination with anti-PD-1 Ab, reaching 44% and 40%, respectively. After discontinuing treatment, the results showed that thidamide-K30 plus celecoxib capsules or thidamide-HCl plus celecoxib sodium combined with anti-PD-1 Ab unexpectedly demonstrated faster tumor growth than thidamide-K30 plus celecoxib capsules or thidamide-HCl plus celecoxib sodium combined with anti-PD-1 Ab. This study also demonstrated that thidamide-HCl plus celecoxib sodium combined with anti-CTLA-4 Ab was more potent in enhancing the anti-cancer immune response than thidamide-HCl plus celecoxib sodium combined with anti-PD-1 Ab. However, thidamide-HCl plus celecoxib-Na, combined with anti-CTLA-4 Ab, was more potent in inhibiting tumor growth than thidamide-K30 plus celecoxib capsules, achieving survival rates of 77.8% and 41.6%, respectively (Figure 12D). On the other hand, a one-to-one comparison between thidamide-HCl plus celecoxib-Na and MS-275, combined with anti-PD-1 Ab, demonstrated that under anti-PD-1 resistance conditions, the anticancer activity of the thidamide-HCl plus celecoxib-Na combination regimen was better than that of the MS-275 combination regimen.

[0101] [Example 9] In CT26-retaining mice, resistance to first-line anti-PD-L1 Ab treatment was overcome by second-line treatment with anti-PD-1 / anti-CTLA-4 Ab combined with thidamide-HCl plus celecoxib-Na. In this study, the inventors further tested a second-line combination treatment to assess the incidence of drug resistance after treatment with first-line anti-PD-L1 antibody therapy. They evaluated the anticancer efficacy of a second-line treatment using tidamide-HCl plus celecoxib-Na in combination with an anti-PD-1 / anti-CTLA-4 antibody when first-line anti-PD-L1 antibody therapy failed. They also investigated whether tidamide-HCl plus celecoxib-Na could improve sensitivity to immune checkpoint inhibitors by modifying the tumor microenvironment after drug resistance to first-line anti-PD-L1 antibody treatment. (CT-26 tumor-carrying mice, average tumor size approximately 160 mm) 3 The tumor was treated with first-line therapy using anti-PD-L1 antibody (2.5 mg / kg; lot #720619F1) twice (with a 3-day interval between injections). The tumor tripled in size over 3 days after the second dose of first-line anti-PD-L1 antibody therapy (mean tumor size 320 mm). 3 The following criteria for treatment failure are met, and the tumor volume is <600 mm 3If this was the case, the mice were re-registered. These mice with resistance to anti-PD-L1 Ab were further randomized. Ten different treatment regimens were available, as shown (n=9-11 mice / group). These mice were randomized to different second-line treatment groups, including: anti-IgG Ab (2.5 mg / kg; lot #65481701), anti-PD-1 Ab (2.5 mg / kg; lot #717918D1), entinostat (20 mg / kg) plus celecoxib capsules in combination with anti-PD-1 Ab (2.5 mg / kg) as a positive control, thidamide-K30 plus celecoxib capsules, thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg), thidamide-K30 plus celecoxib capsules in combination with anti-PD-1 Ab, thidamide-HCl (50 mg / kg) plus celecoxib-Na (50 mg / kg) in combination with anti-PD-1 Ab (2.5 mg / kg), and anti-CTLA-4 The groups were treated with Ab (2.5 mg / kg; lot #702418A2B), tidamide-K30 plus celecoxib capsules in combination with anti-CTLA-4 Ab (2.5 mg / kg), and tidamide-HCl salt (50 mg / kg) plus celecoxib-Na salt (50 mg / kg) in combination with anti-CTLA-4 Ab (2.5 mg / kg). Antibodies were administered intraperitoneally (ip) 6 times (every 3 days). Entinostat was administered orally 8 times (every 2 days). Tidamide-K30 or tidamide-HCl salt and celecoxib capsules or celecoxib-Na salt were administered orally 16 times (daily). As shown in Figures 13A and 13B, in the control group (anti-IgG), two mice achieved partial response (PR) and three mice progressed to disease progression (PD) with rapid tumor growth (response rate 28.6%). This was because mice that responded to the first-line anti-PD-L1 treatment were mistakenly identified as resistant to anti-PD-L1 Ab treatment due to their slower response to the first-line treatment. However, in the anti-PD-1 Ab group, one mouse achieved PR, two mice achieved complete response (CR), and three mice progressed to disease progression with rapid tumor growth (response rate 33.3%).Treatment with thidamide-HCl plus celecoxib-Na was more potent in inhibiting tumor growth compared to thidamide-K30 plus celecoxib capsules. Treatment with thidamide-HCl plus celecoxib-Na showed that 6 mice achieved complete response (CR), 1 mouse achieved partial response (PR), and no mice developed disease (PD) (70% response rate). However, treatment with thidamide-K30 plus celecoxib capsules showed that 2 mice achieved CR, 4 mice achieved PR, and 3 mice developed PD with rapid tumor growth (54.5% response rate). When thidamide-HCl plus celecoxib-Na was combined with anti-PD-1 Ab, the results demonstrated that 6 mice achieved CR (66.6% response rate) and 1 mouse developed PD with slow tumor growth. However, treatment with tidamide-K30 plus celecoxib capsules combined with anti-PD-1 Ab resulted in complete response (CR) in 4 mice, partial response (PR) in 1 mouse (response rate 62.5%), and progression (PD) in 1 mouse with rapid tumor growth. The data suggest that in anti-PD-L1 resistant mice, the tidamide-HCl salt plus celecoxib-Na salt regimen is more potent than the tidamide-K30 plus celecoxib capsule regimen in controlling the tumor microenvironment and increasing anti-PD-1 Ab sensitivity.

[0102] In Figure 13B, the data showed that the second-line treatment with anti-CTLA-4 Ab significantly inhibited tumor growth, with 2 mice achieving complete response (CR), 3 mice achieving partial response (PR), and 3 mice developing progressive disease (PD) with rapid tumor growth (response rate 55.5%). However, in the group treated with tidamide-HCl salt plus celecoxib-Na salt in combination with anti-CTLA-4 Ab, the results demonstrated that 4 mice achieved CR, 3 mice achieved PR, and no mice developed PD (response rate 77.7%). Furthermore, in the group treated with tidamide-K30 plus celecoxib capsules in combination with anti-CTLA-4 Ab, the results demonstrated that 2 mice achieved CR, 3 mice achieved PR, and 1 mouse developed PD with rapid tumor growth (response rate 55.5%). Finally, in the group treated with entinostat plus celecoxib capsules in combination with anti-PD-1 Ab as a positive control, the results showed that two mice achieved complete response (CR), one mouse achieved partial response (PR), and three mice reached disease progression (PD) with rapid tumor growth (50% response rate). In summary, the tidamide-HCl plus celecoxib-Na regimen was potent in enhancing the response rate in PD-L1-resistant mice. Furthermore, in PD-L1-resistant mice, tidamide-HCl plus celecoxib-Na in combination with an immune checkpoint inhibitor was more potent in enhancing the response rate than tidamide-K30 plus celecoxib capsules in combination with an immune checkpoint inhibitor.

[0103] After discontinuation of treatment on day 31, tumors in CT26 tumor-carrying mice grew more rapidly in the anti-PD-1 and anti-CTLA-4 groups (Figure 13D). Survival rates were assessed on day 62. Treatment with thidamide-K30 plus celecoxib capsules in combination with anti-PD-1 Ab showed better survival rates than in the absence of anti-PD-1 Ab, reaching 62.5% and 27.2%, respectively. Furthermore, treatment with thidamide-HCl salt plus celecoxib-Na salt in combination with anti-PD-1 Ab showed better survival rates than in the absence of anti-PD-1 Ab, reaching 77% and 44%, respectively. The results showed that, after discontinuation of treatment, thidamide-K30 plus celecoxib capsules or thidamide-HCl plus celecoxib sodium unexpectedly demonstrated faster tumor growth than thidamide-K30 plus celecoxib capsules or thidamide-HCl plus celecoxib sodium combined with anti-PD-1 Ab. This study also demonstrated that thidamide-HCl plus celecoxib sodium combined with anti-CTLA-4 Ab was potent in enhancing the anti-cancer immune response. However, thidamide-HCl plus celecoxib sodium combined with anti-CTLA-4 Ab was more potent in inhibiting tumor growth than thidamide-K30 plus celecoxib capsules combined with anti-CTLA-4 Ab, achieving survival rates of 66.6% and 44.4%, respectively (Figure 13D). On the other hand, a one-to-one comparison between thidamide-HCl plus celecoxib-Na and MS-275 plus celecoxib capsules, when combined with anti-PD-1 Ab, demonstrated that the anticancer activity of the thidamide-HCl plus celecoxib-Na combination regimen was better than that of the MS-275 plus celecoxib capsules combination regimen under anti-PD-L1 resistance conditions.

[0104] [Example 10] In male Wistar rats, the pharmacokinetic profile of thidamide-HCl salt in combination with celecoxib-sodium salt will be investigated. Tidamid-HCl salt, alone or in combination with an anti-PD-1 antibody, plus amorphous celecoxib-Na salt, possessed very potent anti-cancer immune activity. Therefore, we tested the PK profiles of tidamid-HCl salt in combination with celecoxib-Na salt versus tidamid-K30 in combination with celecoxib-capsules in Wistar rats. As shown in Figure 14A, the blood concentration-time profiles of tidamid for orally administered tidamid-HCl salt (50 mg / kg) and tidamid-K30 (50 mg / kg) were analyzed in Wistar rats. In Table 4, the results demonstrated a significant change in the Cmax and Tmax of tidamid for the salt form. In the tidamid-HCl salt group, the Cmax was 2065.2 (ng / mL) and the Tmax was 0.14 hours. However, in the thidamide-K30 group, the Cmax was 786.3 ng / mL and the Tmax was 0.39 hours. Compared to the absorption rate of thidamide-K30, the absorption rate of thidamide-HCl salt increased significantly. However, as shown in Table 4, AUC, MRT, and Tmax were... 1 / 2 The values ​​did not change significantly. These results suggest that in Wistar rats, tidamid-HCl salt possessed faster absorption characteristics and achieved a higher Cmax, but the overall amount of tidamid in the circulating system did not increase compared to tidamid-K30. As shown in Figure 14B, the celecoxib blood concentration-time profiles of 50 mg / kg celecoxib-Na salt and 50 mg / kg celecoxib-capsules administered orally were analyzed in Wistar rats. As shown in Table 5, the results showed that in Wistar rats, Tmax, Cmax, AUC, AUMC, MRT, and Tmax increased after oral administration. 1 / 2 The values ​​were not significantly different between celecoxib sodium salt and celecoxib capsules.

[0105] Next, in Wistar rats, we analyzed the comparison of the PK profiles of tidamide between tidamide-HCl salt plus celecoxib-Na salt and tidamide-K30 plus celecoxib capsules at an orally administered dose of 50 mg / kg. As shown in Figure 14C and Table 4, the Cmax value of tidamide was significantly increased in the tidamide-HCl salt plus celecoxib-Na salt group compared with the tidamide-K30 plus celecoxib capsule group, with values ​​of approximately 2244.5 and 862.3 ng / mL, respectively. As shown in Table 4, the Tmax value of tidamide was significantly decreased in the tidamide-HCl salt plus celecoxib-Na salt group compared with the tidamide-K30 plus celecoxib capsule group, with values ​​of approximately 0.14 and 0.25 hours, respectively. The AUC value of thidamide was slightly increased in the thidamide-HCl salt plus celecoxib-Na salt group compared to the thidamide-K30 plus celecoxib-capsule group, with values ​​of approximately 5977 and 4201 ng* hours / mL, respectively. A comparison of similar AUMC values ​​between the two combinations is shown in Table 4. As shown in Table 4, MRT and T 1 / 2 The values ​​did not show any difference between the two groups. As shown in Figure 14E and Table 4, the comparison of PK profiles between thidamide-HCl salt and thidamide-HCl salt plus celecoxib-Na salt showed only slight changes. Treatment with thidamide-HCl salt plus celecoxib-Na salt suggested that the thidamide PK profile was not significantly affected by the presence of celecoxib-Na salt with respect to ADME (absorption, distribution, metabolism, and excretion). However, the AUC value of thidamide was mildly affected, with the AUC values ​​for thidamide-HCl and thidamide-HCl salt plus celecoxib-Na salt groups being approximately 4113 and 5977 ng / mL, respectively, as shown in Table 4.

[0106] On the other hand, as shown in Figure 14D and Table 5, in Wistar rats, the celecoxib PK profile did not change significantly when comparing tidamid-K30 plus celecoxib capsules with tidamid-HCl salt plus celecoxib-Na salt administered orally at 50 mg / kg. However, as shown in Figure 14F and Table 5, celecoxib-Na salt or celecoxib capsules alone had significantly lower Cmax and AUC than tidamid-K30 plus celecoxib capsules or tidamid-HCl salt plus celecoxib-Na salt. These results suggest that the presence of tidamid-K30 or tidamid-HCl salt significantly altered the celecoxib ADME profile and therefore significantly increased the Cmax and AUC values ​​of celecoxib. However, the tidamide PK profile was not significantly affected by the presence of celecoxib-Na salt or celecoxib-Capsules. In conclusion, tidamide-HCl salt plus celecoxib-Na salt possessed a significantly altered ADME profile and, therefore, demonstrated to achieve effective tumor inhibition and increased survival when combined with an immune checkpoint inhibitor. This suggests that the salt form possesses better anticancer potential than tidamide-K30 plus celecoxib-Capsules in addressing the challenges faced by second-line treatments against drug resistance.

[0107] [Table 5]

[0108] [Table 6] The following are examples of the forms of this disclosure: [1] A combination including the acidic salt of thidamide and the basic salt of celecoxib. [2] The combination according to Embodiment 1, wherein the amounts of the acidic salt of thidamide and the basic salt of celecoxib are in the range of approximately 5% (w / w) to approximately 80% (w / w) and approximately 95% (w / w) to approximately 20% (w / w), respectively. [3] The combination according to Embodiment 1, wherein the amounts of the acidic salt of thidamide and the basic salt of celecoxib are in a weight ratio of approximately 8:1, approximately 4:1, approximately 2:1, approximately 1:1, approximately 1:2, approximately 1:4, or approximately 1:8. [4] The combination according to Embodiment 1, wherein the acidic salt of thidamide and the basic salt of celecoxib are contained in the same dosage form or independently in separate dosage forms. [5] The combination according to embodiment 4, wherein the dosage form is a tablet or a capsule. [6] The combination according to Embodiment 1, wherein the acidic salt of thidamide is the hydrochloride or sulfate of thidamide. [7] The combination according to embodiment 6, wherein the salt form of thidamide is in crystalline form. [8] The combination according to embodiment 6, wherein the hydrochloride salt of thidamide has a crystalline form (form A) having a powder X-ray diffraction (XRPD) pattern with peaks including 2-theta values ​​of approximately 16.12 degrees, approximately 19.02 degrees, approximately 21.62 degrees, approximately 23.38 degrees, and approximately 30.16 degrees. [9] The combination according to embodiment 8, wherein the XRPD pattern of form A further has peaks including 2-theta values ​​of approximately 21.08 degrees, approximately 23.76 degrees, approximately 25.58 degrees, approximately 27.82 degrees, and approximately 28.18 degrees.

[10] The hydrochloride of thidamide is approximately 3162 cm³. -1 , approx. 3059cm -1 , about 3036cm -1 , approx. 2751cm -1 , approx. 2588cm -1 , approx. 2359cm -1 , approx. 2341cm -1 , approx. 1667cm -1 , approx. 1658cm -1 , approx. 1639cm -1 , about 1620cm -1 , about 1610cm -1 , approx. 1562cm -1 , approx. 1517cm -1 , about 1508cm -1 , approx. 1485cm -1 , approx. 1468cm -1 , approx. 1444cm -1 , approx. 1431cm -1 , about 1307cm -1 Approximately 1282cm -1 , approx. 1265cm -1 , approx. 1243cm -1 , about 1220cm -1 , approx. 1182cm -1 , approx. 1145cm -1 , approx. 1074cm -1 , approx. 1046cm -1 The combination according to embodiment 6, which has a crystal morphology (morphology A) having a Fourier transform infrared spectroscopy (FTIR) pattern with a peak.

[11] The combination according to embodiment 8 or 10, further characterized in that embodiment A exhibits substantially the same XRPD pattern as shown in Figure 3(B) or substantially the same FTIR pattern as shown in Figure 4(B).

[12] The combination according to embodiment 6, wherein the sulfate of thidamide has a crystalline form (form B) having a powder X-ray diffraction (XRPD) pattern with peaks including two theta values ​​of approximately 21.15°, approximately 24.65°, approximately 17.00°, approximately 18.49°, and approximately 26.69°.

[13] The combination according to embodiment 12, wherein the XRPD pattern of form B further has peaks including 2-theta values ​​of approximately 14.74 degrees, approximately 19.45 degrees, approximately 22.00 degrees, approximately 23.55 degrees, and approximately 27.94 degrees.

[14] The sulfate of thydamide is approximately 3249 cm³. -1 , approx. 3067cm -1 , approx. 2578cm -1 , approx. 2360cm -1 , approx. 1689cm -1 , approx. 1664cm -1 , approx. 1647cm -1 , approx. 1614cm -1 , approx. 1568cm -1 , approx. 1521cm -1 , about 1510cm -1 , approx. 1486cm -1 , approx. 1467cm -1 , approx. 1434cm -1 , approx. 1412cm -1 , approx. 1388cm -1 , approx. 1354cm -1 , approx. 1328cm -1 , approx. 1283cm -1 , approx. 1266cm -1 , approx. 1252cm -1 , approx. 1226cm -1 , approx. 1184cm -1 Approximately 1099cm -1 , approx. 1059cm -1 , approx. 1034cm -1 and approximately 1022cm -1 The combination according to embodiment 6, which is a crystal form (form B) having an FTIR pattern with a peak.

[15] The combination according to embodiment 12 or 14, further characterized in that embodiment B exhibits substantially the same XRPD pattern as shown in Figure 3(C) or substantially the same FTIR pattern as shown in Figure 4(C).

[16] The combination according to Embodiment 1, wherein the basic salt of celecoxib is the sodium salt of celecoxib.

[17] The combination according to embodiment 16, wherein the sodium salt of celecoxib is in amorphous or crystalline form.

[18] The combination according to embodiment 17, wherein the amorphous morphology has substantially the same XRPD pattern as that shown in Figure 7(B).

[19] The combination according to embodiment 16, wherein the crystalline form (form I) has a powder X-ray diffraction (XRPD) pattern having peaks with two theta values ​​of approximately 19.85 degrees, approximately 20.51 degrees, approximately 21.51 degrees, approximately 22.55 degrees, and approximately 18.25 degrees.

[20] The combination according to embodiment 19, wherein the XRPD pattern of form I further has peaks including 2-theta values ​​of approximately 10.95 degrees, approximately 14.05 degrees, approximately 14.601 degrees, approximately 17.2 degrees, approximately 25.80 degrees, and approximately 27.30 degrees.

[21] The combination according to embodiment 19, further characterized in that form I exhibits substantially the same XRPD pattern as that shown in Figure 7(C).

[22] The combination according to embodiment 1, further comprising an immune checkpoint inhibitor and / or a chemotherapeutic agent. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[23] The combination according to embodiment 22, wherein the immune checkpoint inhibitor is pembrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, atezolizumab, tripalimab, cintilimab, camrelizumab, or MIHI.

[24] A method for modifying the tumor microenvironment in cancer immunotherapy, comprising administering an effective amount of any one of the combinations described in Embodiments 1 to 22.

[25] The method according to embodiment 24, wherein the acidic salt of thidamide and the basic salt of celecoxib are administered simultaneously, separately, or sequentially.

[26] A method for treating cancer, comprising administering an effective amount of any one of the combinations described in any one of embodiments 1 to 22 to the target.

[27] The method according to embodiment 24 or 26, further comprising administering an immune checkpoint inhibitor.

[28] The method according to embodiment 24 or 26, wherein administration of an acidic salt of tidamide and a basic salt of celecoxib improves the pharmacokinetic profile compared to administration of free tidamide and free celecoxib.

[29] The method according to embodiment 27, wherein the combination and immune checkpoint inhibitor described in any one of embodiments 1 to 22 are administered simultaneously, separately, or sequentially.

[30] The method according to aspect 24 or 26, wherein the cancer is glioblastoma, liver cancer, colorectal cancer, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, benign prostatic hyperplasia, prostate cancer, ovarian cancer, melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), gallbladder cancer, bile duct cancer, bladder cancer, or uterine cancer.

[31] A method for treating cancer by modulating the tumor microenvironment and improving the immune response, comprising administering an effective amount of thidamide in combination with an effective amount of celecoxib.

[32] The method according to embodiment 31, wherein tidamide and celecoxib are administered simultaneously, separately, or sequentially.

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