RU2811733C1 - Antitumor combination of chidamide and celecoxib - Google Patents

Abstract

FIELD: medicine, oncology.

SUBSTANCE: group of inventions discloses an antitumor combination containing the acid salt of chidamide and the basic salt of celecoxib, in which the acid salt of chidamide is a hydrochloride or sulfate salt. Also the following is disclosed: a method of regulating the tumor microenvironment during cancer immunotherapy, a method of treating cancer, and a method of treating cancer by regulating the tumor microenvironment and increasing the rate of formation of the immune response.

EFFECT: increase in the rate of formation of the immune response and antitumor activity.

31 cl, 5 tbl, 14 dwg, 10 ex

Description

The present invention relates to the field of cancer therapy. In particular, the present invention provides a combination containing chidamide and celecoxib in salt forms and its use in tumor microenvironment regulation and cancer immunotherapy.

State of the art

Cancer immunotherapy is a rapidly developing field in which exciting and promising discoveries have been made. The discovery of the existence of tumor-associated antigens now provides the opportunity to use the host immune system to interfere with tumor growth. Currently, research is underway on various mechanisms of using both the humoral and cellular components of the immune system for cancer immunotherapy.

Several strategies to break immune tolerance have been proposed, including adaptive transfer of immune effectors, immunomodulatory therapy, and vaccination. However, these strategies still fail to prevent immune escape. The main immune evasion pathways used by cancer cells are mechanisms related to the activation of antiapoptotic signaling pathways, mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate (cAMP). The tumor microenvironment is an important area of research because it is dynamic and interconnected with the process of tumor progression. Tumors develop mechanisms to evade the immune response through a process called “immune editing,” which provides selective pressure in the tumor microenvironment that can lead to malignant progression. During the tumor-promoting phase, called “immune escape,” the immune system can promote further tumor progression by selecting cancer cells that are better able to survive host immunologic activity or by altering the tumor microenvironment so that it promoted tumor growth. The distinctive properties of the tumor microenvironment are associated with the influence of various factors, such as hypoxia, acidic pH, vascular structure, metabolic state, immunosuppression of immune cells, and cytokines or chemokines. These factors control the process of evading the immune response and also help reduce the rate of formation of the immune response. Thus, control of the tumor microenvironment is one of the most important strategies for antitumor treatment, especially immunotherapy.

Immune system homeostasis includes the presence of both stimulatory and inhibitory mechanisms designed to control the balance of the immune response process. Inhibitory mechanisms include cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD28 homologue) and programmed cell death protein-1 (PD-1) or its ligand (PD-L1), TIM-3 (T-cell immunoglobulin- 3), BTLA (B- and T-lymphocyte attenuator), VISTA (V-domain Ig suppressor of T-cell activation) and LAG-3 (lymphocyte activation gene-3). Promoting mechanisms include cluster differentiation 28 (CD28), tumor necrosis factor receptor superfamily member 4 (TNFRSF4), also known as CD134 or OX40, glucocorticoid-induced TNFR-related protein (GITR), tumor necrosis factor receptor (TNF) family member (CD137 ; 4-1BB), a member of the tumor necrosis factor receptor superfamily (CD27), a herpesvirus entry mediator (HVEM). Currently, many immune checkpoint inhibitor monoclonal antibodies, including anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies, have been registered by the US Food and Drug Administration (FDA). , the European Medicines Agency (EMA), the Pharmaceuticals and Medical Devices Agency (PMDA) and the National Medical Products Authority (NMPA) for therapeutic use in several oncology indications. However, when using these immune checkpoint inhibitors, tumor response to monotherapy is observed in about 20% - 30% of cancer patients. Efficiency remains unsatisfactory. New drug combination strategies with immune checkpoint inhibitors represent the most recent approaches to increasing the rate of response to appropriate immune checkpoint inhibitors. They will provide an opportunity to evaluate the benefits of immunotherapy for patients with different types of common malignant tumors. On the other hand, resistance to immune checkpoint inhibitors has resulted in treatment benefits that are less than expected. A large number of promising combination approaches are in preclinical studies and clinical trials. Advances in these promising combination regimens offer hope of solving the problem of drug resistance by increasing the rate of immune response and activity.

Patent applications US 20180355042 and US 20190211103 propose combinations that contain an HDACi and a PD-1 inhibitor useful for treating cancer, including reducing and/or preventing the occurrence of cancer metastases. However, there is still a need to develop a therapeutic solution to control the tumor microenvironment and achieve more pronounced antitumor activity.

Disclosure of the Invention

The present invention provides a combination containing a chidamide salt and a celecoxib salt, as well as methods for regulating the tumor microenvironment, significantly increasing the rate of formation of the immune response and antitumor activity under the influence of the combination of a chidamide salt and a celecoxib salt.

According to a first aspect of the present invention, there is provided a combination comprising a chidamide acid salt and a celecoxib basic salt.

In one embodiment, the amounts of chidamide acid salt and celecoxib base salt range from about 5% by weight to about 80% by weight and from about 95% by weight to about 20% by weight, respectively. In one embodiment, the amounts of chidamide acid salt and celecoxib base salt are in a weight ratio of 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.

In one embodiment, the acid salt of chidamide and the basic salt of celecoxib are contained in the same dosage form or independently distributed among different dosage forms. In another embodiment of the invention, the dosage form is a tablet or capsule.

In one embodiment of the invention, the chidamide acid salt is a hydrochloride or sulfate salt. In one embodiment of the invention, the chidamide acid salt is in crystalline or amorphous form.

In one embodiment, the chidamide hydrochloride salt is in crystalline form (Form A) and exhibits peaks with 2θ values of about 16.12°, about 19.02° in the X-ray powder diffraction (XRPD) pattern. , about 21.62°, about 23.38° and about 30.16°. In another embodiment of the invention, additional peaks are observed in the X-ray diffraction pattern of Form A with 2θ values of about 21.08°, about 23.76°, about 25.58°, about 27.82°, and about 28.18°.

In yet another embodiment of the invention, the chidamide hydrochloride salt is in crystalline form (Form A), with peaks observed in the spectrum obtained by Fourier transform infrared spectroscopy (FTIR) at a level of 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 .

In yet another embodiment, it is further shown that the radiograph of Form A is substantially the same as that shown in FIG. 3, fragment B, or the IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), largely repeats the spectrum presented in FIG. 4, fragment B.

In one embodiment, the chidamide sulfate salt is in crystalline form (Form B) and exhibits peaks with 2θ values of about 21.15°, about 24.65° in the X-ray powder diffraction (XRPD) pattern. , about 17.00°, about 18.49° and about 26.69°. In another embodiment of the invention, additional peaks are observed in the X-ray diffraction pattern of Form B with 2θ values at about 14.74°, about 19.45°, about 22.00°, about 23.55°, and about 27.94°.

In one embodiment of the invention, the chidamide sulfate salt is in crystalline form (Form B), with peaks observed in the spectrum obtained by Fourier transform infrared spectroscopy (FTIR) at a level of about 3249 cm ^-1 , about 3067 cm ^{- 1} , about 2578 cm ^-1 , about 2360 cm ^-1 , about 1689 cm ^-1 , about 1664 cm ^-1 , about 1647 cm ^-1 , about 1614 cm -1, about 1568 ^{cm -1 ,} about 1521 cm ^-1 , about 1510 cm ^-1 , about 1486 cm ^-1 , about 1467 cm ^-1 , about 1434 cm ^-1 , about 1412 cm ^-1 , about 1388 cm ^-1 , about 1354 cm ^-1 , about 1328 cm ^-1 , about 1283 cm ^-1 , about 1266 cm ^-1 , about 1252 cm ^-1 , about 1226 cm ^-1 , about 1184 cm ^-1 , about 1099 cm -1 , about 1059 cm ^-1 , about 1034 ^{cm -1 and} about 1022 cm ^{- 1} .

In yet another embodiment of the invention, it is further shown that the radiograph of Form B is substantially the same as that shown in FIG. 3, fragment C, or the IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), largely repeats the spectrum presented in FIG. 4, fragment C.

In one embodiment, the base salt of celecoxib is a sodium salt. In another embodiment of the invention, the sodium salt of celecoxib is in amorphous or crystalline form. In another embodiment of the invention, it is further shown that the x-ray diffraction pattern of the amorphous form of celecoxib sodium salt is substantially similar to the x-ray diffraction pattern shown in FIG. 7, fragment B.

In one embodiment, the sodium salt of celecoxib is in crystalline form (Form I) and exhibits peaks with 2θ values of about 19.85°, about 20.51° in the X-ray powder diffraction (XRPD) pattern. , about 21.51°, about 22.55° and about 18.25°. In another embodiment, the X-ray diffraction pattern of Form I exhibits additional peaks with 2θ values of about 10.95°, about 14.05°, about 14.60°, about 17.2°, about 25.80°, and about 27. 30°. In yet another embodiment of the invention, it is further shown that the radiograph of Form I is substantially the same as that shown in FIG. 7, fragment C.

In one embodiment, the combination further comprises an immune checkpoint inhibitor and/or a chemotherapeutic agent. In some embodiments, the immune checkpoint inhibitors are anti-CTLA-4 antibodies, anti-PD-1 antibodies, or anti-PD-L1 antibodies. In some embodiments, immune checkpoint inhibitors include pembrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, atezolizumab, toripalimab, sintilimab, camrelizumab, and MIH1.

According to a first aspect of the present invention, there is provided a method of treating cancer by regulating the tumor microenvironment and increasing the rate of immune response, comprising administering a combination of effective amounts of chidamide and celecoxib. In yet another embodiment, chidamide and celecoxib are administered simultaneously, separately, or sequentially.

In accordance with a first aspect of the present invention, there is provided a method of regulating the tumor microenvironment in cancer immunotherapy, comprising administering to a subject an effective amount of a combination described herein. In one embodiment, the chidamide acid salt and the celecoxib basic salt are administered simultaneously, separately, or sequentially.

In accordance with a second aspect of the present invention, there is provided a method of treating cancer, comprising administering to a subject an effective amount of a combination described herein. In one embodiment, cancer is treated by regulating the tumor microenvironment and increasing the rate of immune response. In one embodiment of the invention, the method further comprises administering an immune checkpoint inhibitor. In another embodiment of the invention, a combination comprising the invention and an immune checkpoint inhibitor is administered simultaneously, separately or sequentially. Examples of immune checkpoint inhibitors are described herein.

In one embodiment, administration of chidamide acid salt and celecoxib base salt, as opposed to a method using chidamide free base and celecoxib free acid, improves the pharmacokinetic profile.

In some embodiments, the types of cancer include glioblastoma, liver cancer, colorectal carcinoma, glioblastoma, stomach cancer, colorectal cancer, esophageal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, benign prostatic hyperplasia, prostate cancer, cancer ovarian, melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), gallbladder cancer, cholangiocarcinoma, bladder cancer and uterine cancer.

Brief description of drawings

Figure 1, fragments A – G shows ¹ H-NMR and ¹³ C-NMR spectra of the substance chidamide-API, chidamide-HCl salt and chidamide-H ₂ SO ₄ salt . Shown are ¹ H-NMR spectrum of chidamide-API (active pharmaceutical ingredient) (A), ¹ H-NMR spectrum of chidamide-HCl salt (B), and ¹ H- _NMR spectrum of chidamide- _H2SO4 salt (C) . Shown are the ¹³ C-NMR spectrum of the chidamide-API substance (D), ^{the 13} C-NMR spectrum of the chidamide-HCl salt (E), and ^{the 13} C-NMR spectrum of the chidamide-H ₂ SO ₄ salt (F). A comparison of ¹³ C-NMR spectra of different forms of chidamide (G) is presented.

In Figure 2, fragments A – D, the spectra of the chidamide-HCl salt and the chidamide-H 2 SO ₄ salt _obtained by mass spectrometry (MS) with electrospray ionization (ESI) in the positive and negative ion recording mode are presented . Shown are MS spectra (ESI) of chidamide-HCl salt obtained in positive ion mode (A) and negative ion mode (B). Shown are MS spectra (ESI) of chidamide-H ₂ SO ₄ salt obtained in positive ion mode (C) and negative ion mode (D).

In Figure 3, fragments A – D are powder X-ray diffraction (XRD) X-ray diffraction patterns of the chidamide-API substance, the chidamide-HCl salt, and the chidamide-H ₂ SO ₄ salt . According to the results of comparison of the X-ray diffraction patterns of the chidamide-API substance (A), the chidamide-HCl salt (B) and the chidamide-H ₂ SO ₄ salt (C), it is shown that the 2θ values of the chidamide-API substance, the chidamide-HCl salt and the chidamide-H ₂ salt SO ₄ are different (D).

In Figure 4, fragments A – D, the spectra of chidamide-API, chidamide-HCl salt and chidamide-H ₂ SO ₄ salt obtained by Fourier transform infrared spectroscopy (FTIR) are presented . To assess the characteristics of the chidamide-API substance, chidamide-HCl salt and chidamide-H ₂ SO ₄ salt (D), the IR spectra of the chidamide-API substance (A), chidamide-HCl salt (B) and chidamide-H ₂ SO salt were analyzed ₄ (C), obtained by Fourier transform infrared spectroscopy (FTIR).

Figure 5, fragments A – E shows ¹ H-NMR and ¹³ C-NMR spectra of the celecoxib-API substance and celecoxib-Na salt. ¹ H-NMR spectra (400 MHz, CDCl ₃ ) of the celecoxib-API substance (active pharmaceutical ingredient) and celecoxib-Na salt are presented in Figure 5, fragments A and B, respectively. ¹³ C-NMR spectra of the celecoxib-API substance and celecoxib-Na salt are presented in Figure 5, fragments C and D, respectively. A comparison of the ¹³ C-NMR spectra (100 MHz, DMSO-d ₆ ) of celecoxib-API and celecoxib-Na salt is presented in Figure 5, fragment E. Celecoxib-Na salt can be obtained in amorphous or crystalline form through various processes . The characteristics of the ¹ H-NMR and ¹³ C-NMR spectra of the amorphous celecoxib-Na salt and the spectra of the crystalline form of the salt are similar.

Figure 6 shows the fast atom bombardment (FAB) mass spectrometry (MS) spectrum of celecoxib-Na salt. The parameters of the MS spectrum (FAB) of the amorphous celecoxib-Na salt are similar to the parameters of the spectrum of the crystalline form of the salt.

Figure 7, panels A–D, shows powder X-ray diffraction (XRD) X-ray diffraction patterns of celecoxib-API, as well as the amorphous and crystalline forms of celecoxib-Na salt. X-ray diffraction patterns of the celecoxib-API substance, as well as the amorphous and crystalline forms of the celecoxib-Na salt, are presented in Figure 7, fragments A, B and C, respectively. Significant differences in diffraction peaks are observed between the amorphous and crystalline forms.

Figure 8, fragments A – D, shows the spectra of celecoxib-API, as well as the amorphous and crystalline forms of celecoxib-Na salt, obtained by Fourier transform infrared spectroscopy (FTIR). FTIR spectra of the celecoxib API and the amorphous and crystalline forms of the celecoxib Na salt are shown in Figure 8, sections A, B, and C, respectively. A comparison was made of the IR spectra of celecoxib-API and celecoxib-Na salts obtained by Fourier transform infrared spectroscopy (FTIR) (Figure 8, fragment D).

Figure 9, panels A–D, shows the therapeutic response to the combination of chidamide-HCl salt and celecoxib capsules in combination with anti-PD-1 antibodies in CT26 tumor-bearing mice. BALB/c mice bearing CT26 colon tumors were treated with various therapeutic treatments as outlined below. IgG, IgG control antibodies (vehicle carrier, 2.5 mg/kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); CD-HCl, chidamide-HCl salt (12.5, 25, 50 mg/kg); CD-K30, chidamide-K30 (chidamide supported on polyvinylpyrrolidone K30, 50 mg/kg); C-cap 50, celecoxib capsules (50 mg/kg, Celebrex®). Total values and fold changes in tumor volume (A), individual tumor volume values (B), body weight of mice bearing the CT26 tumor (C), and animal survival (D) were recorded. Mice bearing the CT26 tumor were subjected to the indicated therapy and sacrificed when the tumor volume reached 3000 mm ³ after tumor implantation. Means and SDs are indicated. The number of animals used in each experimental group and P values are also indicated. * P < 0.05 (compared with IgG); ^# P < 0.05 (vs. PD-1). P values were calculated using Student's t test, which was used to compare tumor sizes in the indication group with the IgG group. Differences in survival rates between different treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

Figure 10, panels A–D, shows the therapeutic response to the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies in CT26 tumor-bearing mice. BALB/c mice bearing CT26 colon tumors were treated with various therapeutic treatments as outlined below. IgG, IgG control antibodies (vehicle carrier, 2.5 mg/kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); CD-HCl, chidamide-HCl salt (50 mg/kg); C-Na, amorphous celecoxib-Na salt (12.5, 25 and 50 mg/kg); CD-K30, chidamide-K30 (chidamide supported on polyvinylpyrrolidone K30, 50 mg/kg); C-cap 50, celecoxib capsules (50 mg/kg, Celebrex®). Total values and fold changes in tumor volume (A), individual values of tumor volume (B), the proportion of tumor-free mice (C), body weight of mice bearing the CT26 tumor (D), and animal survival (E) were recorded. Mice bearing the CT26 tumor were subjected to the indicated therapy and sacrificed when the tumor volume reached 3000 mm ³ after tumor implantation. Means and SDs are indicated. The number of animals used in each experimental group and P values are also indicated. * P < 0.05 (compared with IgG); ^# P < 0.05 (vs. PD-1). P values were calculated using Student's t test, which was used to compare tumor sizes in the indication group with the IgG group. Differences in survival rates between different treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

The data presented in Figure 11, panels A–D, confirm the optimal dosages corresponding to the therapeutic response to the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1, and allow the assessment of the therapeutic response to the combination of chidamide-H salt ₂ SO ₄ and celecoxib-Na salts in combination with antibodies to PD-1, occurring in mice bearing the CT26 tumor. BALB/c mice bearing a CT26 colon tumor with a volume of approximately 300 mm ³ received various therapeutic treatments as indicated below. IgG, IgG control antibodies (vehicle carrier, 2.5 mg/kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); CD-HCl, chidamide-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-H ₂ SO ₄ , chidamide-H ₂ SO ₄ salt (50 mg/kg); CD-K30, chidamide-K30 (chidamide supported on polyvinylpyrrolidone K30, 50 mg/kg); C-cap, celecoxib capsules (50 mg/kg, Celebrex®). Total values and fold changes in tumor volume (A), individual tumor volume values (B), body weight of mice bearing the CT26 tumor (C), and animal survival (D) were recorded. Mice bearing the CT26 tumor were subjected to the indicated therapy and sacrificed when the tumor volume reached 3000 mm ³ after tumor implantation. Means and SDs are indicated. The number of animals used in each experimental group and P values are also indicated. * P < 0.05 (compared with IgG); ^# P < 0.05 (vs. PD-1). P values were calculated using Student's t test, which was used to compare tumor sizes in the indication group with the IgG group . Differences in survival rates between different treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

Figure 12, panels A–D shows that in mice bearing CT26 tumor, resistance to PD-1 immune checkpoint blockade therapy is overcome by anti-PD-1 antibodies or anti-CTLA-4 antibodies in combination with a chidamide salt combination -HCl and celecoxib-Na salts. Mice bearing CT26 tumors (average tumor volume approximately 120 mm ³ ) received first-line therapy with anti-PD-1 antibodies (2.5 mg/kg), administered twice (twice weekly). When tumors met criteria for first-line treatment failure, which was a threefold increase in tumor size (to a mean of approximately 360 mm ³ ) and tumor volume was <600 mm ³ , mice were re-enrolled in the second-line treatment study. Mice exhibiting anti-PD-1 antibody resistance were treated with seven different regimens (n=9 – 11 mice/group): IgG, control IgG antibodies (vehicle vehicle, 2.5 mg/kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); CTLA-4, monoclonal antibodies to CTLA-4 (2.5 mg/kg); CD-HCl, chidamide-HCl salt (50 mg/kg); C-Na, amorphous celecoxib-Na salt (50 mg/kg); MS275, entinostat (20 mg/kg). Total values and fold changes in tumor volume (A), individual tumor volume values (B), body weight of mice bearing the CT26 tumor (C), and animal survival (D) were recorded. Mice bearing the CT26 tumor were subjected to the indicated therapy and sacrificed when the tumor volume reached 3000 mm ³ after tumor implantation. Means and SDs are indicated. The number of animals used in each experimental group and P values are also indicated. * P < 0.05 (compared with IgG); ^# P < 0.05 (vs. PD-1). P values were calculated using Student's t test, which was used to compare tumor sizes in the indication group with the IgG group. Differences in survival rates between different treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

Figure 13, panels A–D shows that in mice bearing CT26 tumor, resistance to PD-L1 immune checkpoint blockade therapy is overcome by anti-PD-1 antibodies or anti-CTLA-4 antibodies in combination with a chidamide salt combination -HCl and celecoxib-Na salts. Mice bearing CT26 tumors (average tumor volume approximately 160 mm ³ ) received first-line therapy with anti-PD-L1 antibodies (2.5 mg/kg), administered twice (twice weekly). When tumors met criteria for first-line treatment failure, which was a threefold increase in tumor size (to a mean of approximately 320 mm ³ ) and tumor volume was <600 mm ³ , mice were re-enrolled in the second-line treatment study. Mice exhibiting anti-PD-L1 antibody resistance were treated with seven different regimens (n=9 – 11 mice/group). IgG, IgG control antibodies (vehicle carrier, 2.5 mg/kg); PD-1, anti-PD-1 monoclonal antibody (2.5 mg/kg); CTLA-4, monoclonal antibodies to CTLA-4 (2.5 mg/kg); CD-HCl, chidamide-HCl salt (50 mg/kg); C-Na, amorphous celecoxib-Na salt (50 mg/kg); MS275, entinostat (20 mg/kg). Total values and fold changes in tumor volume (A), individual tumor volume values (B), body weight of mice bearing the CT26 tumor (C), and animal survival (D) were recorded. Mice bearing the CT26 tumor were subjected to the indicated therapy and sacrificed when the tumor volume reached 3000 mm ³ after tumor implantation. Means and SDs are indicated. The number of animals used in each experimental group and P values are also indicated. * P < 0.05 (compared with IgG); ^# P < 0.05 (vs. PD-1). P values were calculated using Student's t test, which was used to compare tumor sizes in the indication group with the IgG group . Differences in survival rates between different treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

Figure 14, panels A–F presents the PK profiles of chidamide-HCl salt and celecoxib-Na salt administered alone or in combination to male Wistar rats. Rats were orally administered chidamide-K30, chidamide-HCl salt, celecoxib capsules (Celebrex®, celecoxib/caps.) or amorphous celecoxib-Na salt at a dosage of 50 mg/kg. Differences between the PK profiles of chidamide-K30 and chidamide-HCl salt (A) were analyzed. Differences between the PK profiles of celecoxib capsules and amorphous celecoxib-Na salt (B) were analyzed. Differences between the PK profiles of chidamide using the combination of chidamide-K30 and celecoxib capsules and the combination of chidamide-HCl salt and amorphous celecoxib-Na salt are shown in Figure 14, panel C. Differences between the PK profiles of celecoxib using the combination of chidamide-K30 and celecoxib capsules and the combination of chidamide-HCl salt and celecoxib-Na salt are shown in Figure 14, fragment D. Differences between the PK profiles of chidamide using the example of chidamide-K30 and chidamide-HCl salt, as well as the combination of chidamide-K30 and celecoxib capsules and combinations of chidamide-HCl salt and celecoxib-Na salt are shown in Figure 14, fragment E. Differences between the PK profiles of celecoxib using the example of celecoxib capsules and celecoxib-Na salt, as well as the combination of chidamide-K30 and celecoxib capsules and the combination of chidamide-HCl salt. HCl and celecoxib-Na salts are shown in Figure 14, fragment F.

Carrying out the invention

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods and materials are described below. All publications referred to herein are incorporated herein by reference.

The use of "about" when specifying a value or parameter in this document includes (and describes) options that are directed directly at that value or parameter. For example, a description referring to "about X" includes a description of "X". For example, a 2θ value at "about X°" on a radiograph corresponds to +/-0.2 degrees of a 2θ value.

The use of a term in the singular means one or more than one (i.e., at least one) grammatical object in question. For example, "element" means one element or more than one element. The use of “or” means “and/or” unless specifically stated otherwise.

The term "polymorph" refers to the crystalline form of a compound (eg, compound 1) or its hydrate or solvate with a particular crystal lattice type. All polymorphs of a particular compound have the same elemental composition. The term "crystalline", as used herein, means a solid-phase form with an ordered arrangement of structural units. Different crystalline forms of the same compound, its hydrate or solvate, arise due to different packing of molecules in the solid state, which leads to differences in crystal symmetry and/or unit cell parameters. Different crystalline forms typically exhibit different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, optical and electrical properties, stability and/or solubility.

The term "substantially replicates" when referring to, for example, radiographs, refers to a pattern that is not necessarily identical to that shown herein, but is within the range of experimental error or variation when examined by one of ordinary skill in the art.

As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably and refer to a vertebrate organism, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sporting animals and pets. Also included are tissues, cells of a biological organism and their progeny obtained in vitro or cultured in vitro.

As used herein, the term “therapeutically effective amount” means an amount sufficient to treat a subject affected by a disease (eg, a neurodegenerative disease) or to reduce symptoms or complications associated with the disease.

As used herein, the terms “treat,” “cure,” “therapy,” and the like mean the reduction or elimination of a disorder and/or symptoms associated with a disease. It should be kept in mind that, although not excluded, treatment of a disorder or condition does not require complete elimination of the disorder, condition or symptoms associated with it.

As used herein, the term “immunotherapy” means treating a subject affected by, or at risk of developing or relapsing, a disease using a method involving enhancing, suppressing, or otherwise modifying an immune response.

As used herein, the term “programmed cell death protein-1 (PD-1)” refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly in previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1", as used herein, includes human PD-1 (hPD-1), variants, isoforms and homologues of hPD-1 belonging to certain biological species, as well as analogues having at least one common epitope with hPD-1. The complete sequence of hPD-1 is in the GenBank database, accession number U64863.

As used herein, the term “programmed cell death protein ligand 1 (PD-L1)” refers to one of two cell surface glycoprotein ligands of the PD-1 protein (the other being PD-L2) that has a down-regulating effect on T cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1", as used herein, includes human PD-L1 (hPD-L1), variants, isoforms and homologs of hPD-L1 belonging to certain biological species, as well as analogues having at least one common epitope with hPD-L1. The complete sequence of hPD-L1 is in the GenBank database, accession number Q9NZQ7.

As used herein, the term “antibodies” and “antigen-binding fragment of the latter” includes naturally occurring immunoglobulins (e.g., IgM, IgG, IgD, IgA, IgE, etc.) as well as immunoglobulins not found in nature, including, for example, single chain antibodies, chimeric antibodies (eg, humanized mouse antibodies), heteroconjugate antibodies (eg, bispecific antibodies), Fab', F(ab').sub.2, Fab, Fv and rIgG. As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody molecule that retains the ability to specifically recognize an antigen, as well as various combinations of such portions.

As used herein, the term “cancer” refers to a broad group of different diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth lead to the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic or circulatory system. The term "cancer", as used herein, refers to primary, metastatic and recurrent cancer.

The tumor microenvironment is an important aspect of cancer biology and contributes to tumor initiation, tumor progression, and response to therapy. The tumor microenvironment consists of a heterogeneous cell population that includes malignant cells and cells that support tumor proliferation, invasion, and metastatic potential through a variety of interactions. Tumor cells often induce the formation of an immunosuppressive microenvironment that promotes the development of immunosuppressive immune cell populations, such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and T regulatory cells (Tregs). Therefore, targets have been discovered in the tumor microenvironment that can help guide and improve the effects of various anticancer therapies, particularly immunotherapies, which work by potentiating the host's antitumor immune responses.

In the context of the present invention, it has been unexpectedly discovered that a combination containing a histone deacetylase (HDAC) inhibitor (for example, chidamide or its acid salt) and non-steroidal anti-inflammatory drugs (NSAIDs) (for example, celecoxib or its basic salt) can significantly increase the rate of formation of the immune response , influence the regulation of the tumor microenvironment and thus significantly increase antitumor activity. These two active pharmaceutical ingredients are preferably in salt form (crystalline or amorphous).

Chidamide (Epidase®) is known as a histone deacetylase (HDAC) inhibitor, which has relevant effects on HDAC1, HDAC2, HDAC3 class I, as well as HDAC10 class IIb. Chemical name of chidamide: 4-(((E)-3-(pyridin-3-yl)acrylamido)methyl)-N-(2-amino-4-fluorophenyl)benzamide; the structure is shown below

Celecoxib, sold under the brand name Celebrex®, is a nonsteroidal anti-inflammatory drug (NSAID) that is a selective COX-2 inhibitor. Chemical name of celecoxib: 4-[5-(4-methylphenyl)-3-(trifluoromethyl)pyrazol-1-yl]benzenesulfonamide; the structure is shown below

The present invention uses an acid salt of chidamide (chidamide-HCl or chidamide-H ₂ SO ₄ salts) and a base form of celecoxib (such as celecoxib-Na salt). Preferably, the chidamide salt form is in crystalline form and the celecoxib salt form is in amorphous form.

Specifically, the chidamide-HCl salt crystalline form (crystal form A) and the chidamide-H2SO4 salt crystalline form (form B).

X-ray diffraction patterns and Fourier transform infrared spectroscopy (FTIR) spectra are shown and described herein for Form A and Form B. As used herein, the term “highest peak” refers to the peak with the highest intensity in the x-ray diffraction pattern. . As used herein, the term “high intensity peak” includes any peak whose intensity corresponds to the top 20% of the peaks in a particular X-ray powder diffraction pattern.

The X-ray diffraction pattern of Crystal Form A exhibits peaks with the 2θ values reported herein. In this case, peaks are observed in the spectrum of the crystalline form of the hydrochloride salt of chidamide, obtained by Fourier transform infrared spectroscopy (FTIR), the data for which is given below. In addition, it is further shown that the X-ray pattern of Form A is substantially similar to the X-ray pattern shown in FIG. 3, fragment B, or the IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), largely repeats the spectrum presented in FIG. 4, fragment B.

The X-ray diffraction pattern of Crystal Form B exhibits peaks with the 2θ values reported herein. In this case, peaks are observed in the IR spectrum of the crystalline form of the chidamide sulfate salt (form B), the data for which is given below. In addition, it is further shown that the X-ray pattern of Form B is substantially the same as the X-ray pattern shown in FIG. 3, fragment C, or the IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), largely repeats the spectrum presented in FIG. 4, fragment C.

The main salt of celecoxib is a sodium salt that is in amorphous or crystalline form. In one embodiment, the invention further shows that the x-ray diffraction pattern of the amorphous form of celecoxib sodium salt is substantially similar to the x-ray diffraction pattern shown in FIG. 7, fragment B.

The X-ray powder diffraction (XRPD) X-ray diffraction pattern of the crystalline form (Form I) of celecoxib sodium salt exhibits peaks as described below. In yet another embodiment of the invention, it is further shown that the radiograph of Form I is substantially the same as that shown in FIG. 7, fragment C.

Chidamide acid salt is produced under strongly acidic conditions (Arrhenius acid, pKa < 3) as part of the manufacturing process, as well as during a specific synthesis process of new crystalline forms of chidamide-HCl and chidamide-H ₂ SO ₄ salts. These salts significantly improve aqueous solubility and pharmacokinetic profile, significantly increasing the effectiveness of immunotherapy when combined with celecoxib-Na salt and an immune checkpoint inhibitor. The processes for obtaining crystalline forms of chidamide-HCl and chidamide-H ₂ SO ₄ salts are illustrated by the examples given herein.

The base salt of celecoxib is obtained from a metal hydride, such as NaH, as part of the manufacturing process, as well as through specific processes for the synthesis of “anhydrous” amorphous and crystalline forms of celecoxib-Na salts. The amorphous celecoxib-Na salt has good aqueous solubility and a novel pharmacokinetic profile, and has been shown to improve the efficacy of immunotherapy when used in combination with chidamide acid salt and immune checkpoint inhibitors. Similar results were also observed using the crystalline form of celecoxib-Na salt. The processes for obtaining amorphous and crystalline forms of celecoxib-Na salt are illustrated by the examples provided herein.

In some embodiments, the amount of chidamide-HCl or chidamide-H ₂ SO ₄ salts in combination ranges from about 5% by weight to about 80% by weight, from about 30% to about 80% by weight, from about 40 % to about 80% by weight, from about 20% to about 60% by weight, from about 30% to about 60% by weight, from about 40% to about 60% by weight, or from about 35% to about 60% by weight weight

In some embodiments, the amount of celecoxib-Na salt in the combination ranges from about 5% by weight to about 80% by weight, from about 30% to about 80% by weight, from about 40% to about 80% by weight , from about 20% to about 60% by weight, from about 30% to about 60% by weight, from about 40% to about 60% by weight, or from about 35% to about 60% by weight.

In one embodiment, the combination of the present invention is produced using different ratios of chidamide-HCl or chidamide-H ₂ SO ₄ salts (also called chidamide salt) and celecoxib-Na salt (called celecoxib salt). The pharmacokinetic properties of chidamide and celecoxib salts were improved compared to chidamide-K30 (the original formulation of Epidaza®, manufactured with chidamide) and celecoxib capsules (the original formulation of Celebrex®, manufactured with celecoxib).

Additionally, when combined with an immune checkpoint inhibitor, the combination (chidamide salt and celecoxib salt) significantly increased antitumor activity compared to the combination of chidamide-K30 and celecoxib capsules. Treatment with the combination of the present invention in combination with immune checkpoint inhibitors significantly improves the effectiveness of tumor growth inhibition compared to the use of immune checkpoint inhibitors alone, the combination of chidamide-K30 and celecoxib capsules, and even their combined use. In addition, the combination of combos and immune checkpoint inhibitors has a significant impact on the tumor killing process and increases survival rates to about 80 – 100%.

In one embodiment, an immune checkpoint inhibitor can be used in combination with a pharmaceutical combination described herein to stimulate immune system activity against cancer cells and treat cancer. Immune checkpoint inhibitors suitable for use in the practice of the present invention include inhibitory receptor antagonists that inhibit PD-1, PD-L1, CTLA-4, T-cell immunoglobulin-3 (TIM-3), B- and T-attenuator lymphocyte activation gene (BTLA), V domain Ig suppressor of T cell activation (VISTA) or lymphocyte activation gene-3 (LAG-3) 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 include, but are not limited to, humanized antibodies that block human PD-1 such as pembrolizumab (anti-PD-1, trade name Keytruda®), nivolumab (anti-PD-1, trade name Opdivo ®) or pidilizumab (anti-PD-1 antibodies, CT-011), toripalimab (anti-PD-1 antibodies, trade name To I®), sintilimab (anti-PD-1 antibodies, trade name Tivit®), camrelizumab (anti-PD antibodies -1), Bavencio® (anti-PD-L1, avelumab), Imfinzi® (anti-PD-L1, durvalumab) and Tecentriq® (anti-PD-L1, atezolizumab), as well as fully human antibodies such as nivolumab ( anti-PD-1 antibodies, trade name Opdivo®) and cemiplimab (anti-PD-1 antibodies, trade name Libtayo®). Other PD-1 inhibitors may include soluble PD-1 ligand preparations, including but not limited to PD-L2 Fc fusion protein, also known as B7-DC-Ig and AMP-244, and other PD-1 inhibitors currently in development under research and/or development as agents intended for use in therapy. In addition, immune checkpoint inhibitors may include, but are not limited to, humanized or fully human PD-L1 blocking antibodies such as durvalumab and MIH1 and other PD-L1 inhibitors currently in research. In some embodiments, the amount of immune checkpoint inhibitor is in the range of about 0.5% by weight to about 15% by weight, from 0.5% by weight to about 10% by weight, from 0.5% by weight by weight up to about 5% by weight, from 1.0% by weight to about 20% by weight, from 1.0% by weight to about 15% by weight, from 1.0% by weight to about 10% by weight, or from 1.0% by weight to about 5% by weight.

In some embodiments of the present invention, the chidamide-HCl or chidamide-H ₂ SO ₄ salts, the celecoxib-Na salt, and the immune checkpoint inhibitor are administered simultaneously. In some embodiments, the chidamide-HCl or chidamide-H ₂ SO ₄ salts, the celecoxib-Na salt, and the immune checkpoint inhibitor are administered sequentially in any order or alternately.

The pharmaceutical combination of the present invention may include a "carrier". As used herein, the term "carrier" includes any solvent, dispersion medium, carrier base, coating, diluent, antibacterial and/or antifungal agent, isotonic agent, absorption retarding agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is known to those skilled in the relevant field. For example, the pharmaceutical combination may be formulated specifically for administration in solid or liquid form, including forms adapted for the following modes of administration: (1) oral administration, such as liquid dosage forms (aqueous or non-aqueous solutions or suspensions) , lozenges, dragees, capsules, pills, tablets (for example, tablets intended for buccal, sublingual or systemic use), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, for example, as a sterile solution or suspension, or a sustained release preparation; (3) topical application, such as a cream, lotion, gel, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal administration, for example, in the form of a pessary, cream, suppository or foam; (5) sublingual administration; (6) ocular administration; (7) transdermal administration; (8) transmucosal administration; or (9) intranasal administration.

The combination proposed in the present invention can be used for the regulation of the tumor microenvironment and cancer immunotherapy. Types of cancer include, but are not limited to, glioblastoma, liver cancer (eg, hepatocellular carcinoma), colorectal carcinoma, glioblastoma, stomach cancer, colorectal cancer, esophageal cancer, lung cancer (eg, non-small cell lung cancer (NSCLC) and small cell lung cancer), cancer pancreas, renal cell carcinoma, benign prostatic hyperplasia, prostate cancer, ovarian cancer, melanoma, breast cancer, chronic lymphocytic leukemia (CLL), Merkel cell carcinoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), gallbladder cancer, cholangiocarcinoma , bladder cancer and uterine cancer.

The pharmaceutical combination proposed in the present invention can be released in the form of a single preparation. In other embodiments, the pharmaceutical combination provided by the present invention may be provided in separate formulations. The pharmaceutical combination may be formulated in different and/or multiple forms designed for one or more preferred routes of administration. Thus, the pharmaceutical combination may be administered using one or more known routes, including, for example, oral administration, parenteral administration (for example, intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.) or topical administration (for example, intranasal , intrapulmonary, intramammary, vaginal, intrauterine, intradermal, percutaneous, rectal, etc.). The pharmaceutical combination, or part thereof, can be administered through the surface of the mucous membrane, by application, for example, to the mucous membrane of the nose or respiratory tract (for example, in the form of a spray or aerosol). The pharmaceutical combination, or part thereof, may also be administered in a sustained or delayed release form.

The pharmaceutical combination of the present invention may conveniently be provided in unit dosage form or may be prepared using methods known to those skilled in the art of pharmaceuticals. Methods for preparing a combination using a pharmaceutically acceptable carrier include the step of combining the pharmaceutical combination of the present invention with a carrier that consists of one or more auxiliary ingredients. In general, the pharmaceutical combination of the present invention can be prepared by uniformly and/or uniformly combining the active ingredient with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, forming the preparation into the desired form.

In some embodiments, the method may include administering a sufficient amount of the pharmaceutical combination to ensure that the subject receives a dose, for example, from about 10 mg/kg to about 1000 mg/kg.

The present invention is illustrated by the following examples. It should be understood that the specific examples, materials, quantities and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention set forth herein.

EXAMPLES

Materials and methods

Materials and equipment. Chidamide-API, chidamide-K30, chidamide-HCl salt, chidamide-H ₂ SO ₄ salt and celecoxib-Na salt were provided by GNT Biotech & Medicals Co. Ltd (Taiwan). Celecoxib API was purchased from Aarti Drugs Ltd (India). Celecoxib capsules (Celebrex®, 200 mg) were purchased from (Pfizer, Taiwan). The following antibodies and reagents were used in animal experiments: mouse monoclonal antibodies to PD-L1 (B7-H1) (10F.9G2; Bio X Cell), mouse monoclonal antibodies to PD-1 (CD279) (RMP1-14); Bio X Cell), mouse monoclonal antibody to CTLA-4 (CD152) (BE0164; Bio X Cell), and isotype control rat monoclonal IgG2a antibody (2A3; Bio X Cell). LC/MS grade methanol, HPLC grade acetonitrile, sodium 1-heptanesulfonic acid, talc and ethylenediaminetetraacetic acid were purchased from JTBaker® (USA). Formic acid, sodium chloride, lactose, magnesium stearate, polyvinylpyrrolidone and tribasic sodium phosphate 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), 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 H-NMR and ¹³ C-NMR spectra were recorded on a Bruker AVANCE 400MHz PLUS instrument. FTIR spectra were recorded on a Perkin Elmer Spotlight 200i Sp2 instrument with an AutoATR System (Perkin Elmer IR spectrometer). Measurements by powder X-ray diffraction analysis were carried out on a PANalytical EMPYREAN X-ray diffractometer. The masses obtained by electrospray ionization were recorded on a Bruker microTOF instrument. The masses obtained by bombardment with fast atoms were recorded on a JEOL JMS-700 instrument. Gibco RPMI 1640 and DMEM culture media with L-glutamine were purchased from Invitrogen Life Technologies. HyClone fetal bovine serum (FBS) was purchased from Thermo Scientific.

Preparation of chidamide-HCl salt. 1 gram of chidamide-API substance (active pharmaceutical ingredient) was placed in a flask and 3~5 ml of 6~8 N HCl (aq) was added, then stirred until completely dissolved; the process was monitored visually. Without stirring, a solid precipitate formed. The solid precipitate was separated from the solution by suction filtration and further purified by slurrying four times to remove impurities using diethyl ether. Condensed and concentrated (to dryness) the pure solid. The solid product was then dried at 50 – 60°C for 16 hours in an oven and ground into powder (100 mesh). After preparation, the characteristics of the chidamide-HCl salt were determined by HPLC, ¹ H-NMR, ¹³ C-NMR, XRD, MS, FTIR, solubility at saturation was assessed, etc. Other methods for preparing the chidamide-HCl salt are presented below.

65 mg of chidamide-API substance was suspended in 50~150 ml of EtOH, MeOH, DCM, THF or _H2O , then 2~6 drops of 37% HCl were added and stirred until completely dissolved. The mixture was concentrated to remove the solvent until 1 ml of liquid remained, which was then added dropwise to 50 ml of ether and the solid salt precipitated.

500 mg of chidamide-API substance was added to 4~10 ml of 4~8 N HCl (aq) and stirred until completely dissolved. 10~20 ml EtOH was added followed by 10~20 ml ether until a mist formed. The crystallization process was continued at 4°C for 12 hours. The salt was collected by filtration and washed with ether and then dried in an oven at 60°C for 5 hours.

Preparation of chidamide salt-H ₂ SO ₄ . 1 gram of chidamide-API substance was placed in a flask and 3~5 ml of 3~5 MH ₂ SO ₄ (aq) was added, then stirred until completely dissolved; the process was monitored visually. The solution was slowly added dropwise to 150 – 200 ml of ethanol and the solid precipitated. The solid was separated from the solution by suction filtration and washed three times with ethanol. The solid was purified by slurrying with ethanol three times and then further removing excess moisture with diethyl ether. Condensed and concentrated (to dryness) the pure solid. The solid product was then dried at 50 – 60°C for 16 hours in an oven and ground into powder (100 mesh). After preparation, the characteristics of the chidamide-H ₂ SO ₄ salt were determined by HPLC, ¹ H-NMR, ¹³ C-NMR, XRD, MS, FTIR, solubility at saturation, etc. was assessed.

Preparation of celecoxib-Na salt. 5 grams of celecoxib-API substance was placed in a round-bottom flask and 150 – 200 ml of THF was added in the absence of air in a nitrogen gas environment. The compound dissolved completely; the process was monitored visually. 450 – 500 mg of NaH (sodium hydride) was added to the solution and stirred vigorously. A solid precipitate formed after approximately 70 – 90 minutes. THF was removed by suction filtration and the solid was washed three times with 20 ml THF. The solid was then dissolved in 300 ml of dichloromethane (DCM) and any undissolved components were removed by suction filtration. The filtrate was collected, then the solid was condensed and concentrated (to dryness) on a rotary evaporator at a pressure of 30 - 50 mbar and a rotation speed of 140 rpm. The pure solid was dried at 60°C for 16 hours and ground into powder (100 mesh). After preparation, the characteristics of the anhydrous amorphous celecoxib-Na salt were determined according to the spectra obtained by ¹ H-NMR, ¹³ C-NMR, XRD, MS, FTIR, etc.

Another method for preparing anhydrous amorphous celecoxib-Na salt is presented below. 1 gram of celecoxib-API was placed in a round bottom flask and 6 ml of THF was added in the absence of air under nitrogen gas. The compound was completely dissolved; the process was monitored visually. 75~100 mg NaH (sodium hydride) was added to the solution and stirred vigorously. A solid precipitate formed after about 40~80 minutes. The THF was removed by suction filtration and the solid was washed three times with diethyl ether. The solid was purified by slurrying with diethyl ether three times. The solid was then dissolved in 150~200 ml of dichloromethane (DCM), and any undissolved components were removed by suction filtration. The filtrate was collected, then condensed and concentrated (to dryness). During the condensation process, the initial pressure was set at 400~430 mbar until there was no distillate. The pressure was then adjusted to 10~30 mbar until the solid salt precipitation process was completed. The pure solid was dried at 60°C for 16 hours and ground into powder (100 mesh). After preparation, the characteristics of the amorphous celecoxib-Na salt were determined by HPLC, ¹ H-NMR, ¹³ C-NMR, XRD, MS, FTIR, solubility at saturation, etc. were assessed.

Anhydrous crystalline celecoxib-Na salt was also prepared by the method described above, except that during condensation, the pressure was set at 10~30 mbar until the solid salt precipitation process was completed.

Determination of the solubility of chidamide-HCl, chidamide-H ₂ SO ₄ and celecoxide-Na salts at saturation. A 5 mg sample of chidamide-HCl, chidamide-H ₂ SO ₄ or celecoxib-Na salts was placed in 5 ml volumetric flasks containing distilled demineralized water and shaken at 100 rpm in an incubator at 25°C for 90 minutes . The resulting suspension was passed through a 0.22 μm filter. The concentrations of chidamide-HCl, chidamide-chidamide-H ₂ SO ₄ and celecoxib-Na salts were determined spectrophotometrically at 256 nm, 256 nm and 253 nm, respectively. The solubility of each sample at saturation was determined in triplicate, and the mean and standard deviation were recorded. Data on the construction of calibration curves are presented below. Chidamide and celecoxib stock solutions were prepared in 99.99% MeOH. The λ _max values were 256 nm and 253 nm, respectively. The calibration curve demonstrated an adequate level of linearity with a correlation coefficient ( ^R2 ) of 0.9998 over the Beer concentration range of 0 – 20 µg/mL.

Cell lines. CT26 (CRL-2638; murine colorectal adenocarcinoma cells) were obtained from the American Type Culture Collection (ATCC). Cells of the CT26 tumor cell lines were grown in McCoy 5A medium supplemented with 10% (v/v) FBS at a temperature of 37°C, 5% CO ₂ .

Antitumor activity in experimental animal models. The animal study was approved and reviewed by the Laboratory Animal Care and Use Committee of Taipei Medical University (TMU IACUC, No: LAC-2018-0340). Six to eight weeks old male BALB/C mice (BioLASCO, Taiwan) were used for all animal experiments. Inoculation of CT26 cancer cells (5 × 10 ⁶ ) was performed by subcutaneous injection into the right flank of each mouse. Tumors grew for 10–11 days (tumor size approximately 200–300 mm ³ ) before randomization and therapy. CT26 tumor-bearing mice received 2.5 mg/kg IgG antibodies (lot no. 65481701), anti-PD-1 antibodies (lot no. 640517M1 and 717918D1), anti-PD-L1 antibodies (lot no. 720619F1) or anti-CTLA-4 antibodies (lot no. 702418A2B) intraperitoneally on days 11, 14, 17, 20, 23 and 26 after tumor implantation, all antibodies were diluted to appropriate concentrations in 100 μl of sterile phosphate buffered saline (PBS) (pH 7.4) (Invitrogen Life Technologies ). Chidamide-K30, chidamide-HCl salt, chidamide-H ₂ SO ₄ salt, celecoxib capsules (Celebrex®, 200 mg) and celecoxib-Na salt (amorphous or crystalline form) were administered orally on day 11 after tumor implantation. Chidamide-K30, chidamide-HCl salt and chidamide-H ₂ SO ₄ salt were administered orally to treat tumor-bearing mice at a dosage of 12.5, 25 and 50 mg/kg daily from 11 to 26 days. Daily treatment with celecoxib (capsule/Celebrex®, 200 mg) or celecoxib-Na salt at dosages of 12.5, 25.0 and 50 mg/kg was carried out from days 11 to 26. Antitumor activity was measured from the start of therapy aimed at reducing tumor growth until the tumor reached a volume of 3000 mm ³ . Tumor volume was calculated using the formula: length × width ² × 0.5.

Survival among experimental animal models. The administration of antibodies or drugs was carried out from 11 to 25 or 26 days. The tumor continued to grow in the bodies of the tumor-bearing mice. Tumor volume in mice was measured every three or four days (twice a week). Tumor-bearing mice were considered dead once the tumor volume reached 3000 mm ³ . Data for all treatment groups were recorded and analyzed.

Overcoming resistance to first-line therapy through PD-1 immune checkpoint blockade. The animal study was approved and reviewed by the Laboratory Animal Care and Use Committee of Taipei Medical University (TMU IACUC, No: LAC-2018-0340). Six to eight weeks old male BALB/C mice (BioLASCO, Taiwan) were used for all animal experiments. Inoculation of CT26 cancer cells (5 × 10 ⁶ ) was performed by subcutaneous injection into the right flank of each mouse. Tumors grew for 8 days (mean tumor size approximately 120 mm ³ ) before starting first-line therapy with anti-PD-1 antibodies (2.5 mg/kg), which were administered twice (the interval between two doses was 3 days). When tumors met failure criteria, which was a gradual threefold increase in size over 3 days (mean tumor size 360 mm ³ ) after the second dose of anti-PD-1 antibody as part of first-line therapy, and tumor volumes were < 600 mm ³ , mice were re-treated. included in the study. Mice exhibiting anti-PD-1 antibody resistance were further randomized. Mice exhibiting anti-PD-1 resistance were treated with seven different regimens, including IgG (2.5 mg/kg; lot no. 65481701), anti-PD-1 (2.5 mg/kg; lot no. 640517M1 ), anti-PD-1 antibodies (2.5 mg/kg) in combination with entinostat (20 mg/kg), anti-PD-1 antibodies (2.5 mg/kg) in combination with chidamide-HCl salt combination (50 mg /kg) and celecoxib-Na salt (50 mg/kg), combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg), antibodies to CTLA-4 (2.5 mg/kg ; lot no. 702418A2B) alone or in combination with a combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg). Antibodies were administered intraperitoneally on days 14, 17, 20, 23, 26 and 29 (six treatments, interval between two injections was 3 days), all antibodies were diluted to appropriate concentrations in 100 μl of sterile PBS (pH 7.4) (Invitrogen Life Technologies). Celecoxib-Na salt, chidamide-HCl salt, and entinostat were administered orally from days 14 to 29. Celecoxib-Na salt (50 mg/kg), chidamide-HCl salt (50 mg/kg) were given daily, and entinostat (20 mg/kg) every two days. Antitumor activity was measured from the start of therapy aimed at reducing tumor growth until the tumor reached a volume of 3000 mm ³ . Tumor volume was calculated using the formula: length × width ² × 0.5. An animal study was developed to demonstrate a potential treatment option for failure of first-line anti-PD-1 therapy in human cancer patients who have developed primary/secondary resistance to anti-PD-1 therapy.

Overcoming resistance to first-line therapy through PD-L1 immune checkpoint blockade. The animal study was approved and reviewed by the Laboratory Animal Care and Use Committee of Taipei Medical University (TMU IACUC, No: LAC-2018-0340). Six to eight weeks old male BALB/C mice (BioLASCO, Taiwan) were used for all animal experiments. Inoculation of CT26 cancer cells (5 × 10 ⁶ ) was performed by subcutaneous injection into the right flank of each mouse. Tumors grew for 8 days (mean tumor size approximately 160 mm ³ ) before starting first-line therapy with anti-PD-L1 antibodies (2.5 mg/kg), which were administered twice (the interval between two doses was 3 days). When tumors met failure criteria, which was a gradual threefold increase in size over 3 days (mean tumor size 320 mm ³ ) after the second dose of anti-PD-L1 antibody (Lot #720619F1), and tumor volumes were <600 mm ³ , mice were re-included in the study. Mice exhibiting anti-PD-L1 antibody resistance were further randomized. Mice exhibiting anti-PD-L1 resistance were treated with seven different regimens, including IgG (2.5 mg/kg; lot no. 65481701), anti-PD-1 (2.5 mg/kg; lot no. 717918D1 ), anti-PD-1 antibodies (2.5 mg/kg) in combination with entinostat (20 mg/kg), anti-PD-1 antibodies (2.5 mg/kg) in combination with chidamide-HCl salt combination (50 mg /kg) and celecoxib-Na salt (50 mg/kg), combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg), antibodies to CTLA-4 (2.5 mg/kg ; lot no. 702418A2B) alone or in combination with a combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg). Antibodies were administered intraperitoneally on days 14, 17, 20, 23, 26 and 29 (six treatments, interval between two injections was 3 days), all antibodies were diluted to appropriate concentrations in 100 μl of sterile PBS (pH 7.4) (Invitrogen Life Technologies). Celecoxib-Na salt, chidamide-HCl salt, and entinostat were administered orally from days 14 to 29. Celecoxib-Na salt (50 mg/kg), chidamide-HCl salt (50 mg/kg) were given daily, and entinostat (20 mg/kg) every two days. Antitumor activity was measured from the start of therapy aimed at reducing tumor growth until the tumor reached a volume of 3000 mm ³ . Tumor volume was calculated using the formula: length × width ² × 0.5. An animal study was developed to demonstrate a potential treatment option for failure of first-line anti-PD-L1 therapy in human cancer patients who have developed primary/secondary resistance to anti-PD-L1 therapy.

Analysis of pharmacokinetic (PK) profiles of chidamide-HCl salt and celecoxib-Na salt in Wistar rats. Pharmacokinetic studies of chidamide, celecoxib and their salt forms (chidamide-HCl salt and celecoxib-Na salt) were carried out on male Wistar rats at the age of 7 weeks by oral administration of the compounds at a dosage of 50 mg/kg in water. Male Wistar rats were purchased from BioLasco (Taiwan). Before the start of pharmacokinetic studies, animals were fasted for 12 hours without restricting access to water. Blood samples were collected (n > 5/time point) at 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours after dosing. At each time point, approximately 250 μl of blood was collected from the jugular vein into a labeled Microtainer™ tube containing ethylenediaminetetraacetic acid. Blood samples were processed to obtain plasma samples within 30 minutes of the scheduled sampling time. All plasma samples were stored at temperatures below -80°C until analysis. Plasma samples were analyzed for traces of treatment with chidamide-K30, chidamide-HCl salt, celecoxib (capsule/Celebrex®, 200 mg) and the amorphous form of celecoxib-Na salt by liquid chromatography-mass spectrometry (LC-MS/MS, 6470 Agilent Tech., USA) with limits of quantitation of 14.2 ng/ml (chidamide) and 45.5 ng/ml (celecoxib). The PK parameters of chidamide-K30, chidamide-HCl salt, celecoxib/Celebrex® and celecoxib-Na salt were calculated using the trapezoidal rule, as well as using the non-compartmental analysis tool of validated Phoenix WinNonlin software (version 6.3). Pharmacokinetic studies were conducted at Taipei Medical University and approved by the Laboratory Animal Care and Use Commission (IACUC No.: LAC-2017-0331). Sample preparation and analysis were performed as described below. To 50 μL of calibration standards or plasma samples, 150 μL of acetonitrile (containing 10% methanol) was added, then the samples were vortexed for 1 min to precipitate the protein. After centrifugation at 4°C and 21130 g for 15 min, 5 μL of supernatant was injected directly into LC-MS/MS for analysis. The analysis was performed on a 6470 series liquid chromatograph (Agilent Tech., USA), equipped with a pump for quaternary mixtures (LC system 1260 Infinity II Quaternary Pump), a degasser, an automatic sampler, a thermostated compartment for the column and LC-MS/MS-mass. spectrometer 6470 (Agilent Tech., USA). Chromatographic separation was carried out on a LiChrospher® 60 RP-select B column (5 µm, 125 × 4.6 mm, Merck, Germany) at 40°C, in gradient elution mode, as described in the table below. The flow rate was 0.5 ml/min. The total analysis time is 10 minutes. The drying gas and atomizing gas flow rates were 6 and 1.5 L/min. The dry gas temperature and system capillary voltage were 250°C and 3000 V, respectively. LC-MS/MS analysis was carried out in the selective detection mode of selected reactions of the decay of several ions using target ions with m/z values of 391.1 and 265.1 for chidamide during electrospray ionization in positive ion detection mode, and m/z values 380 and m/z 316 for celecoxib using electrospray ionization in negative ion detection mode.

LC/MS Gradient Elution Mode Datasheet

Time (minutes) 2.5% formic acid water acetonitrile 0 – 3 2% 68% thirty% 3.01 – 5 2% 48% 50% 5.01 – 9 2% 38% 60% 9.01 – 12 2% 68% thirty%

Statistical indicators. Means and standard errors were calculated for all data points examined in at least four independent experiments. Pairwise comparisons of tumor size were made between each experimental condition and the IgG control group using a two-sample Student's t test (Systat Software, San Jose, CA, USA). Student's t test or one-way analysis of variance (ANOVA) was used to evaluate efficacy data from animal studies. Kaplan-Meier curves and log-rank tests were generated using sigma stat 3.5 software. All P values <0.05 were considered statistically significant.

Example 1. Characterization of a new crystalline form of chidamide-HCl salt.

Chidamide was approved by the China Food and Drug Administration (CFDA) and the National Medical Products Administration (NMPA) for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL) in 2014. Chidamide (trade name Eipdaza®) is available as oral tablets containing 5 mg chidamide, and the recommended dose is 30 mg twice a week at intervals greater than 3 days. The tablet contains the chidamide API coated with polyvinylpyrrolidone K30 (PVP-K30) to improve its aqueous solubility and oral bioavailability. In the present invention, formulations for chidamide-API substance were developed to obtain chidamide-HCl and chidamide-H ₂ SO ₄ salts in new crystalline forms. The properties of chidamide-HCl and chidamide-H ₂ SO ₄ salts can significantly improve aqueous solubility and oral bioavailability. The structure of chidamide salts was identified by ¹ H-NMR and ¹³ C-NMR spectroscopy, the results are presented in Figure 1. ¹ H-NMR spectrum was recorded on a Bruker AVANCE 400MHz PLUS instrument using dimethyl sulfoxide (DMSO-d ₆ ) as a solvent. ¹³ C-NMR spectrum was recorded at 100 MHz. ^{The 1} H-NMR spectrum data demonstrated that the chemical shift _δH 5,20 of the NH ₂ group of aniline in the chidamide-HCl salt disappears, in contrast to the substance chidamide-API, as shown in Figure 1, fragments A and B. According to the results obtained , the salt form is formed at the C ₂₁ -NH ₂ position. It can be designated as C ₂₁ -NH ₃ ⁺ Cl ^- salt or chidamide-HCl salt. The ^13C -NMR data of the chidamide API and chidamide-HCl salt are shown in Figure 1, fragments D and E. Details of the chemical shift of the chidamide API and chidamide-HCl salt are presented in Table 1 and Figure 1, fragment G. In addition, MS (ESI) method was used to determine the molecular weight. Mass spectra of the chidamide-HCl salt were recorded on a Bruker microTOF instrument with an ESI source in positive/negative ion recording mode. MS spectra (ESI) of the chidamide-HCl salt obtained in positive ion mode are shown in Figure 2, fragment A. The most common peak is characterized by m/z 391.158 [M+H] ⁺ . MS spectra (ESI) of the chidamide-HCl salt obtained in negative ion mode are shown in Figure 2, fragment B. The most common peak is characterized by m/z 425.118 [M+Cl] ^- . Then the parameters of the crystalline form of the chidamide-HCl salt were determined by XRD. The differences between the X-ray diffraction patterns of the chidamide-API substance and the chidamide-HCl salt were analyzed. XRD measurements were carried out on a PANalytical EMPYREAN X-ray diffractometer. A copper (Cu) (λ=45 kV, 40 mA) anode was used as an X-ray source, 2θ values ranged from 3° to 40°, scanning speed was 1/min. The results of XRD analysis demonstrated that the XRD patterns of the chidamide-API substance and the chidamide-HCl salt were different, as shown in Figure 3, fragments A (chidamide-API) and B (chidamide-HCl salt). The 2θ values for chidamide-API and chidamide-HCl salt were different, as shown in Figure 3, fragment D. The data obtained showed that the chidamide-HCl salt had a new crystalline form that was different from that of chidamide-API. Two different crystalline forms of the chidamide-API substance and the chidamide-HCl salt were examined as part of the saturation solubility assessment. The solubility of the chidamide-HCl salt in water significantly exceeded the solubility of the substances chidamide-API and chidamide-K30, as shown in Table 2. The substance chidamide-API was not soluble in water, but chidamide-K30, which is part of the formulation of the drug chidamide tablets (Epidaza® ), showed low solubility in water (about 26.03 μg/ml). Three independent series of chidamide-HCl salt were tested and showed saturation solubility of about 554.83, 566.90, and 536.06 μg/mL, respectively. The results obtained demonstrated that chidamide-HCl salt contributed to a significant improvement in water solubility (more than 20 times) compared to chidamide-K30, as shown in Table 2. Improved water solubility of chidamide-HCl salt may lead to increased oral bioavailability , which will subsequently lead to an improvement in the PK profile and increased antitumor activity. The structure of the chidamide-HCl salt was further confirmed by Fourier transform infrared (FTIR) spectroscopy, the results are presented in Figure 4. FTIR spectra were recorded on a Perkin Elmer Spotlight 200i Sp2 instrument with an AutoATR System (Perkin Elmer IR spectrometer). . IR spectra (FTIR) were recorded in the range 4000 – 700 cm ^-1 . In the spectrum of the chidamide-HCl salt there was no signal of stretching vibrations of the NH bond of aniline with a wave number of 3275 and 3309 cm ^-1 , as shown in Figure 4, fragment B. Differences between the data of the BR spectra of the substance chidamide-API (Figure 4, fragment A) and chidamide-HCl salts obtained by Fourier transform infrared spectroscopy (FTIR) are shown in Figure 4, fragment D.

Example 2 Characterization of a New Crystalline Form of Chidamide-H Salt ₂ SO ₄ .

The second salt form of chidamide was prepared using H ₂ SO ₄ . The structure of the chidamide-H ₂ SO ₄ salt was identified by ¹ H-NMR and ¹³ C-NMR spectroscopy, the results are presented in Figure 1, fragments C and F. ¹ H-NMR spectrum was recorded on a Bruker AVANCE 400MHz PLUS instrument using dimethyl sulfoxide (DMSO-d ₆ ) as a solvent. ¹³ C-NMR spectrum was recorded at 100 MHz. ¹ H-NMR spectrum data demonstrated that the chemical shift _δH 5,20 of the NH ₂ group of aniline in the chidamide-H ₂ SO ₄ salt disappears, in contrast to the chidamide-API substance, as shown in Figure 1, fragments C and A. According to the results obtained, the salt form is formed at the C ₂₁ -NH ₂ position. It can be designated as C ₂₁ -NH ₃ ⁺ HSO ₄ ^- salt or chidamide-H ₂ SO ₄ salt. Details of the chemical shift of the chidamide-API substance and the chidamide-H ₂ SO ₄ salt are presented in Table 1 and Figure 1, fragment G. In addition, MS (ESI) was used to determine the molecular weight. Mass spectra of chidamide-H ₂ SO ₄ salt were recorded on a Bruker microTOF instrument with an ESI source in positive/negative ion recording mode. MS spectra (ESI) of chidamide-H ₂ SO ₄ salt, obtained in negative ion mode, are shown in Figure 2, fragment C. The most common peak is characterized by m/z 391.16 [M+H] ⁺ . MS spectra (ESI) of chidamide-H ₂ SO ₄ salt obtained in negative ion mode are shown in Figure 2, fragment D. The most common peak is characterized by m/z 487.12 [M+ HSO ₄ ] ^- . Then the parameters of the crystalline form of the chidamide-H ₂ SO ₄ salt were determined by XRD. The differences between the X-ray diffraction patterns of the chidamide-API substance and the chidamide-H ₂ SO ₄ salt were analyzed. XRD measurements were carried out on a PANalytical EMPYREAN X-ray diffractometer. A copper (Cu) (λ=45 kV, 40 mA) anode was used as an X-ray source, 2θ values ranged from 3° to 40°, scanning speed was 1/min. The results of XRD analysis demonstrated that the XRD patterns of the chidamide-API substance and the chidamide-H ₂ SO ₄ salt are different, as shown in Figure 3, fragments A (chidamide-API) and B (chidamide-H ₂ SO ₄ salt). The 2θ values for the chidamide-API substance and the chidamide-H ₂ SO ₄ salt were quite different, as shown in Figure 3, fragment D. The data obtained showed that the chidamide-H ₂ SO ₄ salt had a new crystalline form, different from the form of the chidamide substance -AFI. Two different crystalline forms of the substance chidamide-API and the salt chidamide-H ₂ SO ₄ were studied as part of the evaluation of solubility at saturation. The solubility of the chidamide-H ₂ SO ₄ salt in water significantly exceeded the solubility of the substances chidamide-API and chidamide-K30, as shown in Table 2. The substance chidamide-API was not soluble in water, but chidamide-K30, which is part of the formulation of the drug chidamide in tablets (Epidase®), showed low solubility in water (about 26.03 μg/ml). Three independent series of chidamide-H ₂ SO ₄ salt were studied and showed saturation solubility of about 597.39, 652.90 and 561.5 μg/ml, respectively. The results obtained demonstrated that chidamide-H ₂ SO ₄ salt contributed to a significant improvement in water solubility (more than 20 times) compared to chidamide-K30, as shown in Table 2. Improvement in water solubility of chidamide-H ₂ SO ₄ salt may lead to increased oral bioavailability, which will subsequently lead to an improved PK profile and increased antitumor activity. The structure of the chidamide-H ₂ SO ₄ salt was further confirmed by Fourier transform infrared spectroscopy (FTIR), the results are presented in Figure 4, fragment C. FTIR spectra were recorded on a Perkin Elmer Spotlight 200i Sp2 instrument with an AutoATR System ( Perkin Elmer IR spectrometer). IR spectra (FTIR) were recorded in the range 4000 – 700 cm ^-1 . In the spectrum of the chidamide-H ₂ SO ₄ salt there was no signal of stretching vibrations of the NH bond of aniline with a wave number of 3412 and 3309 cm ^-1 , as shown in Figure 4, fragment C. Differences between the data of the IR spectra of the substance chidamide-API (Figure 4, fragment A) and chidamide-H ₂ SO ₄ salts are shown in Figure 4, fragment D.

Example 3. Characterization of a new amorphous form of celecoxib-Na salt.

Amorphous forms are characterized by the presence of short-range molecular order, in contrast to crystalline forms with long-range molecular packing order. Celecoxib has been classified in BCS (biopharmaceutical classification system) class II. It was characterized by low solubility and high permeability. Most commercial drug products have acceptable levels of permeability; dissolution is the rate-limiting step in the absorption of these drugs. On the other hand, in drug development, solubility has been one of the important issues; preparation of the amorphous form provides an effective solution to the problem of low solubility. A unique method for obtaining an amorphous form of celecoxib-Na salt was developed and tested. Under conditions of exposure to the strong base NaH, the hydrogen in the sulfonamide group of the celecoxib-API substance was replaced by Na, and through several stages, including purification and condensation, a new amorphous celecoxib-Na salt was obtained. First, ^{the 1} H-NMR spectra of the celecoxib-API substance and the celecoxib-Na salt, which are shown in Figure 5, fragments A and 5B, were compared. It was clearly shown that the two signals corresponding to the hydrogen in the sulfonamide group disappeared, see Figure 5, fragment B. It was suggested that two Na atoms replaced the two hydrogen atoms of the sulfonamide group to participate in the formation of the new celecoxib-Na salt. Data from ^{the 1} H-NMR spectrum (400 MHz, CDCl ₃ ) of the celecoxib-API substance: 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). ¹ H-NMR spectrum data (400 MHz, CDCl ₃ ) of celecoxib-Na salt: δ 2.09 (3H, s), 6.59 (1H, s), 6.87 (4H, s), 6.94 (2H, d), 7.61 (2H, d). ^{The 1} H-NMR spectrum data demonstrated that the chemical shift δ _H 4,86 of the NH ₂ sulfonamide group in celecoxib-Na salt disappears, unlike the celecoxib-API substance, as shown in Figure 5, fragments B and A. In addition, ¹³ C-NMR data are shown in Figure 5, fragments C, D and E. MS (FAB) was then used to estimate the molecular weight of celecoxib-Na salt, the data is presented in Figure 6. Mass spectra of celecoxib-Na salt were recorded on JEOL JMS-700 instrument with FAB source in positive ion detection mode. According to the data obtained, the experimental m/z value was 426.1 [M+H ⁺ ]. It has been suggested that the formula of celecoxib-Na salt is C ₁₇ H ₁₂ F ₃ N ₃ Na ₂ O ₂ S and the molecular weight is 425.04. The calculated m/z value for C ₁₇ H ₁₂ F ₃ N ₃ Na ₂ O ₂ S was 425.04, the experimental value was 426.1 (M+H) ⁺ (MS method (FAB)). The data reconfirmed that celecoxib-Na salt contains two sodium atoms instead of two hydrogen atoms. The solubility in water of the amorphous celecoxib-Na salt was then assessed. As shown in Table 3, celecoxib API was insoluble in water, and celecoxib capsules (Celebrex®) were slightly soluble in water (about 1.19 μg/mL). Three independent batches of the amorphous form of celecoxib-Na salt were tested and demonstrated saturation solubilities of approximately 54.72, 54.45, and 56.72 μg/mL, respectively. The solubility of celecoxib-Na salt in water, in contrast to the solubility of celecoxib-API and celecoxib capsules, has improved significantly. The results obtained suggest that improved water solubility of the amorphous form of celecoxib-Na salt may contribute to increased oral bioavailability and, consequently, therapeutic efficacy. In addition, as shown in Figure 7, according to the results of XRD analysis, the celecoxib-API substance has a specific X-ray pattern (Figure 7, fragment A), and the amorphous celecoxib-Na salt has an amorphous X-ray pattern as shown in Figure 7, fragment B. The obtained The results indicate that the amorphous celecoxib-Na salt is characterized by a specific shape combined with a significant improvement in water solubility at saturation. Many studies have been devoted to the preparation of an amorphous form of celecoxib using various polymers as carriers. The structure of the amorphous celecoxib-Na salt was reconfirmed by Fourier transform infrared (FTIR) spectroscopy, the results are presented in Figure 8. The spectrum of the amorphous celecoxib-Na salt did not show the signal of stretching vibrations of the NH sulfonamide bond with a wavenumber of 3234 and 3341 cm ^-1 . as shown in Figure 8, fragments A and B. The differences between the FTIR spectra of celecoxib-API and amorphous celecoxib-Na salt are shown in Figure 8, fragment D.

Table 3. Solubility of celecoxib-API, celecoxib capsules and celecoxib-Na salt (amorphous or crystalline form) at saturation

Substances Solubility at saturation (µg/ml) Celecoxib-API NGO Celecoxib capsules (Celebrex®) 1.19±0.05 Celecoxib-Na salt No. 1903261 (amorphous) 54.72±1.0 Celecoxib-Na salt No. 1903262 (amorphous) 54.45±1.8 Celecoxib-Na salt No. 1903263 (amorphous) 56.72±0.8 Celecoxib-Na salt No. 190307 (crystal) 111.5±5.7 Celecoxib-Na salt No. 1903282 (crystal) 133.63±1.8 Celecoxib-Na salt No. 1903283 (crystal) 95.34±5.7

*NGO: below detection limit

amorphous: amorphous form; crystal: crystalline form

Example 4. Determination of the characteristics of the crystalline form of celecoxib-Na salt.

After preparation, the characteristics of the crystalline celecoxib-Na salt were determined by ¹ H-NMR, ¹³ C-NMR, XRD, MS, FTIR. As shown in Table 3, the water solubility of the crystalline form of celecoxib-Na salt from three different batches is about 111.5, 133.63 and 95.34 μg/ml. As shown in Figure 7, panels C and D, the x-ray diffraction pattern of the crystalline celecoxib-Na salt is different from the x-ray diffraction pattern of the celecoxib-API substance. The results obtained indicate that crystalline celecoxib-Na salt is characterized by a specific crystalline form, which contributes to a significant improvement in solubility in water. The crystalline structure of celecoxib-Na salt was reconfirmed by Fourier transform infrared spectroscopy (FTIR), the results are presented in Figure 8, fragment C. In the spectrum of celecoxib-Na salt, in contrast to the spectrum of the celecoxib-API substance, there was no signal of stretching vibrations of the bond. NH sulfonamide with wavenumber 3234 and 3341 cm ^-1 as shown in Figure 8, fragment C and D.

Example 5. Comparison of the antitumor activity of the chidamide-K30 substance and the chidamide-HCl salt in combination with celecoxib capsules and antibodies to PD-1 in mice bearing the CT26 tumor

To determine whether the use of the chidamide salt form would improve the effectiveness of tumor growth inhibition, the therapeutic effect of the combination of chidamide-K30 and celecoxib capsules was assessed in comparison with the combination of chidamide-HCl salt and celecoxib capsules in combination with antibodies to PD- 1 (2.5 mg/kg; lot no. 640517M1) in mice bearing the CT26 tumor. As shown in Figure 9, on day 11, tumor size in mice bearing the CT26 tumor increased to about 200 – 250 mm ³ . The mice were then treated with 6 different regimens as follows. As shown in Figure 9, fragment A, the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with anti-PD-1 antibodies contributed to a significant increase in tumor growth inhibition in mice carrying CT26 tumor versus anti-PD-1 antibody group. Results from the use of a combination of chidamide-HCl salt at a dosage of 12.5, 25 or 50 mg/kg and celecoxib capsules (50 mg/kg) in combination with antibodies to PD-1 also showed a significant increase in the effectiveness of tumor growth inhibition in tumor-bearing mice CT26, compared with the anti-PD-1 antibody group. As part of the comparison of the antitumor activity of the salt form of chidamide and the substance chidamide-K30, the effectiveness was assessed according to the classification system presented below. The present study determined complete response (CR, ≤0.5-fold increase in tumor size in tumor-bearing mice at the end of treatment); partial response (PR, increase in tumor size in tumor-bearing mice ranging from >0.5 to ≤2-fold at the end of treatment); stable disease (SD, increase in tumor size in tumor-bearing mice ranging from 2 to 5 times the end of treatment); progressive disease (PD, a 5-fold or greater increase in tumor size in tumor-bearing mice at the end of treatment).

As shown in Figure 9, fragment B, the combination of chidamide-HCl salt (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with anti-PD-1 antibodies (2.5 mg/kg) is even more effective in regarding the effect on the process of inhibiting tumor growth in mice bearing the CT26 tumor, in comparison with the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with antibodies to PD-1. Treatment with a combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with antibodies to PD-1 resulted in the following results: 6 mice with CR (60%) and 4 mice with PD and moderate tumor growth; treatment with a combination of chidamide-HCl salt (50 mg/kg) and celecoxib-HCl salt (50 mg/kg) in combination with antibodies to PD-1 achieved an 89% positive clinical response in 5 mice with PR and 3 mice with CR (PD mice were absent). The results obtained indicate that the effectiveness of the chidamide-HCl salt was higher than the effectiveness of the chidamide-K30 substance due to better solubility in water and oral bioavailability and, as a result, therapeutic efficacy. As shown in Figure 9, fragment A and B, in combination with anti-PD-1 antibodies, the combination of chidamide-HCl salt (12.5 mg/kg) and celecoxib capsules (50 mg/kg) was sufficient to produce an effect on tumor microenvironment and reactivate cytotoxic T lymphocytes to kill the tumor. As shown in Figure 9, panel C, none of the mice in the treatment groups experienced weight loss. After stopping treatment on day 26, tumors in mice bearing the CT26 tumor grew faster in the IgG control group. However, a regimen using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with an immune checkpoint inhibitor showed high efficacy in inhibiting tumor growth and thus contributed to a significant increase in survival rates (Figure 9, fragment D). As shown in Figure 9, fragment D, the combination of chidamide-HCl salt (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with anti-PD-1 antibodies contributed to a significant increase in survival rates to about 77.7%, however, the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) combined with anti-PD-1 antibodies achieved only 60% survival in a CT26 tumor-bearing mouse model. Notably, the combination of chidamide-HCl salt (25 mg/kg) and celecoxib capsules (50 mg/kg) combined with PD-1 antibodies contributed to a significant increase in survival rates to about 66.6%. The results indicate that the combination of chidamide-HCl salt and celecoxib capsules was more effective than the combination of chidamide-K30 and celecoxib capsules in controlling and regulating the tumor microenvironment and, to some extent, increasing the effectiveness of immunotherapy.

The present study also demonstrated that the combination of chidamide-HCl and celecoxib capsules combined with an immune checkpoint inhibitor was more effective than the combination of chidamide-K30 and celecoxib capsules in increasing the rate of antitumor immune response. On the other hand, a direct comparison between the combination of chidamide-HCl salt and celecoxib capsules and the combination of chidamide-K30 and celecoxib capsules in combination with anti-PD-1 antibodies demonstrated that the antitumor activity of the combination regimen using the chidamide salt-HCl combination and celecoxib-Na salts have higher antitumor activity when implementing a combination regimen using a combination of the substance chidamide-K30 and celecoxib in capsules.

Example 6. Comparison of the antitumor activity of the combination of chidamide-K30 and celecoxib in capsules and the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1 in mice bearing the CT26 tumor

To demonstrate increased tumor growth inhibition, the therapeutic effect of the combination of chidamide-K30 and celecoxib capsules was evaluated compared with the combination of chidamide-HCl salt and celecoxib capsules in combination with anti-PD-1 antibodies (2.5 mg/kg ; batch no. 640517M1) in mice bearing the CT26 tumor. As shown in Figure 10, treatment in each group began on day 10, when the tumor size in mice bearing the CT26 tumor increased to about 200 – 250 mm ³ . First of all, the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with antibodies to PD-1 significantly increases the effectiveness of tumor growth inhibition in mice bearing the CT26 tumor, compared with group of antibodies to PD-1 (Figure 10, fragment A). The results of using a combination of chidamide-HCl salt (50 mg/kg) and amorphous celecoxib-Na salt at a dosage of 12.5, 25 and 50 mg/kg in combination with antibodies to PD-1 showed a significant increase in the effectiveness of tumor growth inhibition in mice carrying CT26 tumor compared with the anti-PD-1 antibody group (Figure 10, panel A). As shown in Figure 10, fragment B, the combination of chidamide-HCl salt (50 mg/kg) and amorphous celecoxib-Na salt at different dosages in combination with anti-PD-1 antibodies (2.5 mg/kg) is even more effective against effects on the process of inhibiting tumor growth in mice bearing the CT26 tumor, in comparison with the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with antibodies to PD-1 (2.5 mg /kg). The results obtained indicate that chidamide-HCl salt and celecoxib-Na salt were more effective than chidamide-K30 and celecoxib capsules due to better water solubility and oral bioavailability, as well as, as a consequence, the therapeutic effectiveness of the salt forms.

As shown in Figure 10, fragments A and B, chidamide-HCl salt (50 mg/kg) in combination with celecoxib-Na salt (12.5 mg/kg) was sufficient to affect the tumor microenvironment and reactivate cytotoxic T- lymphocytes to destroy the tumor. Direct comparison of antitumor activity between identical doses (50 mg/kg) of the combination of chidamide-K30 and celecoxib capsules (4 mice with CR, 50%) and the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies (2.5 mg/kg) showed that the latter regimen using a combination of salt forms was more effective in inhibiting tumor growth in mice bearing the CT26 tumor, with 7 CR mice present (100%), as shown in Figure 10, fragment B. Additionally, as shown in Figure 10, panel C, the proportion of tumor-free (CR) animals was assessed in different treatment groups. When combined with anti-PD-1 antibodies, all saline regimens were superior (higher proportion of tumor-free cases) to chidamide-K30 and celecoxib capsule regimens in inhibiting tumor growth. The results indicate that celecoxib-Na salt was more effective than celecoxib capsules in combination with an immune checkpoint inhibitor in inhibiting tumor growth in mice bearing the CT26 tumor. A similar result was demonstrated for chidamide-HCl when compared with the substance chidamide-K30. This finding also demonstrated that the dosage of the combination of chidamide-HCl salt and celecoxib-Na salt could be reduced when used in combination with an immune checkpoint inhibitor to reactivate cytotoxic T lymphocytes in the tumor microenvironment and subsequently inhibit tumor growth, as shown in Figure 10 , fragment A and B. As shown in Figure 10, fragment D, none of the mice in the treatment groups experienced weight loss.

After stopping treatment on day 25, tumors in mice bearing the CT26 tumor grew faster in the IgG control group (2.5 mg/kg; lot no. 65481701). As shown in Figure 10, fragment E, in combination with anti-PD-1 antibodies, the combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) contributed to a significant increase in survival rates to about 100% in compared with the combination of chidamide-K30 and celecoxib capsules (about 75%) in mice bearing the CT26 tumor. Survival in the anti-PD-1 antibody group was only 37.5%. The results obtained indicate that the combination of chidamide-HCl salt and celecoxib-Na salt was more effective than the combination of chidamide-K30 and celecoxib capsules in terms of controlling and regulating the tumor microenvironment and, to some extent, increasing the rate of formation of the immune response. Ultimately, the combination regimen of chidamide-HCl salt and celecoxib-Na salt in combination with an immune checkpoint inhibitor demonstrated high efficacy in inhibiting tumor growth and thus contributed to a significant increase in survival rates (Figure 10, detail E). The present study demonstrated that the combination of chidamide-HCl salt and celecoxib-Na salt in combination with an immune checkpoint inhibitor was more effective than the combination of chidamide-K30 and celecoxib capsules in increasing the rate of formation of the antitumor immune response. On the other hand, a direct comparison between the combination of chidamide-HCl and celecoxib-Na salt and the combination of chidamide-K30 and celecoxib capsules in combination with anti-PD-1 antibodies demonstrated that the antitumor activity of the combination regimen using the combination of chidamide-HCl-Na salt HCl and celecoxib-Na salts have higher antitumor activity when implementing a combination regimen using a combination of the substance chidamide-K30 and celecoxib in capsules.

Example 7 Confirmation of optimal dosages corresponding to therapeutic response to the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies, and evaluation of therapeutic response to the combination of chidamide-H salt ₂ SO ₄ and celecoxib-Na salts in combination with antibodies to PD-1, occurring in mice bearing the CT26 tumor

To test the optimal dosages corresponding to the therapeutic response to the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1 arising in mice bearing CT26 tumors, mice with tumors measuring approximately 300 mm³ were treated with different dosages of a combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies. As shown in Figure 11, fragment A, a combination of chidamide-HCl salt and celecoxib-Na salt at a dosage of 50 mg/kg in combination with antibodies to PD-1 (2.5 mg/kg; lot no. 717918D1) was more effective than similar combinations at a dosage of 25 mg/kg and 12.5 mg/kg. The results obtained indicate that the combination of chidamide-HCl salt and celecoxib-Na salt in dosages of 25 mg/kg in combination with antibodies to PD-1 (2.5 mg/kg) and the combination of chidamide-K30 substance and celecoxib capsules in dosages of 50 mg/kg in combination with anti-PD-1 antibodies (2.5 mg/kg) corresponded to a similar therapeutic response. It was suggested that the combination of chidamide-HCl salt and celecoxib-Na salt, compared with the combination of chidamide-K30 and celecoxib capsules at a similar dosage in combination with anti-PD-1 antibodies, had superior antitumor activity in mice bearing the CT26 tumor. Additionally, when combined with anti-PD-1 antibodies, the combination of chidamide-HCl salt and crystalline celecoxib-Na salt compared with the combination of chidamide-HCl salt and amorphous celecoxib-Na salt at a similar dosage had less antitumor activity in mice bearing the CT26 tumor. , as shown in Figure 11, fragment B. On the other hand, the chidamide-H salt combination₂SO₄and celecoxib-Na salt in combination with anti-PD-1 antibodies had very high antitumor activity similar to the antitumor activity of the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies, as shown in Figure 11, fragment B. In the present study, since the average tumor volume reached about 300 mm³ before various treatments, and the activity of antibodies to PD-1 proteins was lower compared with previous studies, the antitumor therapeutic effect of treatment with antibodies to PD-1 was significantly worse. As shown in Figure 11, fragment B, in the present study, the optimal dosage of the combination of chidamide-HCl salt (50 mg/kg) or chidamide-H salt₂SO₄ (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with anti-PD-1 antibodies (2.5 mg/kg) corresponded to the best therapeutic response. In addition, the use of amorphous celecoxib-Na salt compared to the use of crystalline celecoxib-Na salt in a combination regimen resulted in higher rates of positive clinical response. When the tumor size reached an average of about 300 mm³ Before treatment, the use of a combination of chidamide-K30 and celecoxib capsules in combination with antibodies to PD-1 allowed only about 33% of a positive clinical response to be achieved. However, the combination of chidamide-HCl salt or chidamide-H salt₂SO₄ and celecoxib-Na salts in combination with anti-PD-1 antibodies contributed to a significant increase in positive clinical response rates to 62.5% and 55.5%, respectively. The results obtained demonstrated that in mice bearing the CT26 tumor, the salt forms of chidamide and celecoxib, in contrast to the substance chidamide-K30 and celecoxib in capsules, were more effective in increasing the rate of formation of the immune response. As shown in Figure 11, panel C, none of the mice in the treatment groups experienced weight loss.

After stopping treatment on day 26, tumors in mice bearing the CT26 tumor grew faster in the IgG control group. However, a regimen using a combination of chidamide-HCl salt or chidamide-H ₂ SO ₄ salt and celecoxib-Na salt in combination with an immune checkpoint inhibitor showed high efficacy in affecting tumor growth inhibition and thus contributed significantly increasing survival rates (Figure 11, fragment D). As shown in Figure 11, panel D, in the present study, the combination of chidamide-K30 (50 mg/kg) and celecoxib capsules (50 mg/kg) in combination with anti-PD-1 antibodies increased survival rates to approximately 22% , since the antitumor activity of antibodies to PD-1 was lower compared to previous results. On the other hand, the combination of chidamide-HCl salt or chidamide-H ₂ SO ₄ salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with anti-PD-1 antibodies contributed to a significant increase in survival rates to about 37 .5% or 44.4%, respectively, in mice bearing the CT26 tumor. The results obtained indicate that the combination of chidamide-HCl salt or chidamide-H ₂ SO ₄ salt and celecoxib-Na salt in combination with anti-PD-1 antibodies was more effective than the combination of chidamide-K30 and celecoxib capsules in combination with anti-PD antibodies -1 in relation to the control and regulation of the tumor microenvironment and, to some extent, increasing the rate of formation of the immune response. The present study also proved that the combination of chidamide-HCl salt or chidamide-H ₂ SO ₄ salt and celecoxib-Na salt in combination with an immune checkpoint inhibitor was more effective than the combination of chidamide-K30 and celecoxib capsules in combination with an immune checkpoint inhibitor in regarding increasing the rate of formation of the antitumor immune response. On the other hand, a direct comparison between the combination of chidamide-HCl and celecoxib-Na salt and the combination of chidamide-K30 and celecoxib capsules in combination with anti-PD-1 antibodies demonstrated that the antitumor activity of the combination regimen using the combination of chidamide-HCl-Na salt HCl and celecoxib-Na salts have higher antitumor activity when implementing a combination regimen using a combination of the substance chidamide-K30 and celecoxib in capsules.

Example 8: Overcoming Resistance to First-Line Anti-PD-1 Antibody Therapy by Including Second-Line Anti-PD-1 Antibody/Anti-CTLA-4 Antibody Therapy in Combination with a Combination of Chidamide-HCl Salt and Celecoxib-Na Salt in mice bearing CT26 tumor

In the present study, mice received second-line therapy to mimic drug resistance that occurs in human cancer patients in first-line anti-PD-1 therapy to evaluate the antitumor activity of second-line anti-PD-1 therapy. combinations of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies/anti-CTLA-4 antibodies in cases where first-line therapy with anti-PD-L1 antibodies has failed. We assessed whether the combination of chidamide-HCl salt and celecoxib-Na salt could enhance the sensitivity of immune checkpoint inhibitors through regulation of the tumor microenvironment. Tumors grew for 8 days (mean tumor size approximately 120 mm ³ ) before starting first-line therapy with anti-PD-1 antibodies (2.5 mg/kg; lot no. 717918D1), which were administered twice (interval between two injections - 3 day). When tumors met the criteria for treatment failure, which was a gradual threefold increase in size over 3 days (mean tumor size 360 mm ³ ) after the second dose of anti-PD-1 antibodies as part of first-line therapy, and tumor volumes were < 600 mm ³ , mice were re-included in the study. Mice exhibiting anti-PD-1 antibody resistance were further randomized. Ten different treatment regimens were used (n=9 – 11 mice/group). Mice were randomized to different second-line treatment groups, including an IgG antibody group (2.5 mg/kg; lot no. 65481701), an anti-PD-1 antibody group (2.5 mg/kg; lot no. 717918D1), an entinostat positive control group ( 20 mg/kg) in combination with antibodies to PD-1 (2.5 mg/kg), group of combination of chidamide-K30 substance and celecoxib in capsules, group of combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg), group of combination of chidamide-K30 substance and celecoxib in capsules in combination with antibodies to PD-1, group of combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with antibodies to PD-1 (2.5 mg/kg), a group of antibodies to CTLA-4 (2.5 mg/kg; batch No. 702418A2B), a group of combinations of chidamide-K30 and celecoxib in capsules in combination with antibodies to CTLA-4 (2.5 mg/kg), combination group of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with antibodies to CTLA-4 (2.5 mg/kg ). Antibodies were administered intraperitoneally six times (the interval between two injections was 3 days). Entinostat was administered orally eight times (every 2 days). The combination of chidamide-K30 or chidamide-HCl salt and celecoxib capsules or celecoxib-Na salt was administered orally 16 times (daily). As shown in Figure 12, fragments A and B, there were no mice with PR (positive clinical response - 0%) in the anti-PD-1 antibody group, while there were 8 mice with PD and rapid tumor growth. Treatment with a combination of chidamide-HCl salt and celecoxib-Na salt was more effective than treatment with a combination of chidamide-K30 and celecoxib capsules in inhibiting tumor growth. In the treatment group using the combination of chidamide-HCl salt and celecoxib-Na salt, there were 3 mice with CR and 4 mice with PD and rapid tumor growth (positive clinical response - 33.3%). However, in the treatment group using the combination of chidamide-K30 and celecoxib capsules, there was only 1 mouse with PR and 8 mice with PD and rapid tumor growth (positive clinical response - 10%). In the treatment group using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1, there were 4 mice with CR (positive clinical response - 36.3%) and 6 mice with PD and much slower tumor growth. However, in the treatment group using the combination of chidamide-K30 and celecoxib capsules in combination with anti-PD-1 antibodies, there was only 1 mouse with PR and 9 mice with PD and moderate tumor growth (positive clinical response - 10%). The results obtained indicate the absence of antitumor activity of anti-PD-1 antibodies in mice exhibiting resistance to anti-PD-1 antibodies. Moreover, a combination regimen of chidamide-HCl salt and celecoxib-Na salt demonstrated high efficacy in controlling the tumor microenvironment and increasing sensitivity to anti-PD-1 antibodies in mice exhibiting anti-PD-1 antibody resistance. Treatment using a combination of chidamide-K30 and celecoxib capsules compared with treatment using a combination of salt forms (chidamide-HCl salt and celecoxib-Na salt) demonstrated much less antitumor activity. As shown in Figure 12, fragment B, the anti-CTLA-4 group compared with the anti-PD-1 group showed moderate rates of tumor growth inhibition; There were no mice with CR or PR, while there were 7 mice with PD and moderate tumor growth. However, in the treatment group using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to CTLA-4, there were 4 mice with CR, 2 mice with PR (positive clinical response - 60%), while mice with There were no PDs. In the treatment group using a combination of chidamide-K30 and celecoxib capsules in combination with antibodies to CTLA-4, there were 2 mice with CR, 1 mouse with PR (positive clinical response - 25%) and 5 mice with PD and moderate tumor growth. The positive control group of entinostat plus anti-PD-1 antibodies included 1 mouse with PR (positive clinical response - 9%) and 8 mice with PD and rapid tumor growth. It can be concluded that the treatment regimen using a combination of chidamide-HCl salt and celecoxib-Na salt was effective in increasing the rate of immune response in mice exhibiting resistance to PD-1 antibodies. In addition, the combination of chidamide-HCl salt and celecoxib-Na salt was more effective in combination with anti-CTLA-4 antibodies than in combination with anti-PD-1 antibodies in increasing the rate of immune response in mice exhibiting resistance to anti-PD antibodies. -1.

After stopping treatment on day 31, tumors in mice bearing the CT26 tumor grew faster in the anti-PD-1 and anti-CTLA-4 groups (Figure 12, panel D). Survival was assessed at 60 days. Treatment with a combination of chidamide-K30 and celecoxib capsules without anti-PD-1 antibodies compared with treatment in combination with anti-PD-1 antibodies demonstrated better survival rates; the values were 11.1% and 0%, respectively. Treatment with a combination of chidamide-HCl salt and celecoxib-Na salt without anti-PD-1 antibodies compared with treatment in combination with anti-PD-1 antibodies demonstrated better survival rates; the values were 44% and 40%, respectively. The results indicate that after treatment cessation, the combination of chidamide-K30 and celecoxib capsules or the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies unexpectedly corresponded to faster tumor growth than the combination of chidamide-K30 and celecoxib-Na salts. K30 and celecoxib capsules or a combination of chidamide-HCl salt and celecoxib-Na salt. The present study also proved that the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to CTLA-4 was more effective than the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1 in increasing the rate of formation of an antitumor immune response. However, the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to CTLA-4 was more effective than the combination of chidamide-K30 and celecoxib in capsules in combination with antibodies to CTLA-4 in terms of affecting the process of inhibiting tumor growth; while the survival rates were 77.8%% and 41.6%, respectively (Figure 12, fragment D). On the other hand, a direct comparison between the combination of chidamide-HCl salt and celecoxib-Na salt and MS-275 in combination with anti-PD-1 antibodies demonstrated that the antitumor activity of the combination regimen using the combination of chidamide-HCl salt and celecoxib-Na salt higher antitumor activity when implementing a combination regimen using MS-275 in conditions of resistance to antibodies to PD-1.

Example 9: Overcoming Resistance to First-Line Anti-PD-L1 Therapy by Including Second-Line Anti-PD-1/Anti-CTLA-4 Therapy in Combination with a Combination of Chidamide-HCl Salt and Celecoxib-Na Salt mice bearing CT26 tumor

The present study further examined second-line combination therapy regarding the incidence of drug resistance after first-line anti-PD-L1 therapy, and also assessed the antitumor activity of second-line therapy using a combination of chidamide-HCl salt and celecoxib. Na salts in combination with anti-PD-1 antibodies/anti-CTLA-4 antibodies in cases where first-line therapy with anti-PD-L1 antibodies has been ineffective. We tested whether the combination of chidamide-HCl salt and celecoxib-Na salt could enhance the sensitivity of immune checkpoint inhibitors by regulating the tumor microenvironment after the discovery of resistance to first-line anti-PD-L1 therapy. Mice bearing CT26 tumors (average tumor volume - approximately 160 mm ³ ) received first-line therapy using antibodies to PD-L1 (2.5 mg/kg; lot no. 720619F1), which were administered twice (the interval between two injections was 3 days ). When tumors met the criteria for treatment failure, which was a gradual threefold increase in size over 3 days (mean tumor size 320 mm ³ ) after the second dose of anti-PD-L1 antibodies as part of first-line therapy, and tumor volumes were < 600 mm ³ , mice were re-included in the study. Mice exhibiting anti-PD-L1 antibody resistance were further randomized. Ten different treatment regimens were used (n=9 – 11 mice/group). Mice were randomized to different second-line treatment groups, including an IgG antibody group (2.5 mg/kg; lot no. 65481701), an anti-PD-1 antibody group (2.5 mg/kg; lot no. 717918D1), an entinostat combination positive control group (20 mg/kg) and celecoxib in capsules in combination with antibodies to PD-1 (2.5 mg/kg), group of combination of chidamide-K30 substance and celecoxib in capsules, group of combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg), a group of combinations of the substance chidamide-K30 and celecoxib in capsules in combination with antibodies to PD-1, a group of combinations of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with antibodies to PD-1 (2.5 mg/kg), a group of antibodies to CTLA-4 (2.5 mg/kg; batch No. 702418A2B), a group of combinations of chidamide-K30 and celecoxib in capsules in combination with antibodies to CTLA-4 (2.5 mg/kg), group of combination of chidamide-HCl salt (50 mg/kg) and celecoxib-Na salt (50 mg/kg) in combination with antibodies to CTLA-4 (2 .5 mg/kg). Antibodies were administered intraperitoneally six times (every 3 days). Entinostat was administered orally eight times (every 2 days). The combination of chidamide-K30 or chidamide-HCl salt and celecoxib capsules or celecoxib-Na salt was administered orally 16 times (daily). As shown in Figure 13, fragments A and B, in the control group of IgG antibodies there were 2 mice with PR and 3 mice with PD and rapid tumor growth (positive clinical response - 28.6%), this was due to the fact that mice responders to first-line anti-PD-L1 therapy were misdiagnosed as anti-PD-L1 resistant due to delayed response to first-line therapy. However, in the anti-PD-1 antibody group there was 1 mouse with PR, 2 mice with CR and 3 mice with PD and rapid tumor growth (positive clinical response - 33.3%). Treatment with a combination of chidamide-HCl salt and celecoxib-Na salt was more effective than treatment with a combination of chidamide-K30 and celecoxib capsules in inhibiting tumor growth. In the treatment group using a combination of chidamide-HCl salt and celecoxib-Na salt, there were 6 CR mice, 1 PR mouse, and no PD mice (positive clinical response - 70%). However, in the treatment group using the combination of chidamide-K30 and celecoxib capsules, there were 2 mice with CR, 4 mice with PR and 3 mice with PD and rapid tumor growth (positive clinical response - 54.5%). In the treatment group using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to PD-1, there were 6 mice with CR (positive clinical response - 66.6%) and 1 mouse with PD and slow tumor growth. However, in the treatment group using a combination of chidamide-K30 and celecoxib capsules in combination with antibodies to PD-1, there were 4 mice with CR, 1 mouse with PR (positive clinical response - 62.5%) and 1 mouse with PD and rapid tumor growth. The findings suggest that the chidamide-HCl and celecoxib-Na salt combination regimen was superior to the chidamide-K30 and celecoxib capsule regimen in controlling the tumor microenvironment and increasing sensitivity to anti-PD-1 antibodies. in mice exhibiting anti-PD-L1 resistance.

As shown in Figure 13, panel B, second-line anti-CTLA-4 therapy produced a significant increase in tumor growth inhibition efficacy, with 2 CR mice, 3 PR mice, and 3 PD mice with rapid tumor growth (positive clinical response – 55.5%). However, in the treatment group using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to CTLA-4, there were 4 CR mice, 3 PR mice, and no PD mice (positive clinical response - 77 .7%). In the treatment group using a combination of chidamide-K30 and celecoxib capsules in combination with antibodies to CTLA-4, there were 2 mice with CR, 3 mice with PR and 1 mouse with PD and rapid tumor growth (positive clinical response - 55.5% ). The positive control group of treatment using a combination of entinostat and celecoxib capsules in combination with antibodies to PD-1 included 2 mice with CR, 1 mouse with PR and 3 mice with PD and rapid tumor growth (positive clinical response - 50%). It can be concluded that the treatment regimen using a combination of chidamide-HCl salt and celecoxib-Na salt was effective in increasing the rate of immune response in mice exhibiting resistance to PD-L1 antibodies. The present study proved that the combination of chidamide-HCl and celecoxib-Na salt plus an immune checkpoint inhibitor was more effective than the combination of chidamide-K30 and celecoxib capsules plus an immune checkpoint inhibitor in increasing the rate of immune response in mice. exhibiting resistance to anti-PD-L1 antibodies.

After stopping treatment on day 31, tumors in mice bearing the CT26 tumor grew faster in the anti-PD-1 and anti-CTLA-4 groups (Figure 13, panel D). Survival was assessed at 62 days. Treatment with a combination of chidamide-K30 and celecoxib capsules in combination with anti-PD-1 antibodies, compared with treatment without the use of anti-PD-1 antibodies, demonstrated better survival rates; the values were 62.5% and 27.2%, respectively. Treatment using a combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-PD-1 antibodies compared with treatment without using anti-PD-1 antibodies demonstrated better survival rates; the values were 77% and 44%, respectively. The results indicate that after treatment cessation, the combination of chidamide-K30 and celecoxib capsules or the combination of chidamide-HCl salt and celecoxib-Na salt unexpectedly corresponded to faster tumor growth than the combination of chidamide-K30 and celecoxib capsules or the salt combination chidamide-HCl and celecoxib-Na salts in combination with antibodies to PD-1. The present study also proved that the combination of chidamide-HCl salt and celecoxib-Na salt in combination with anti-CTLA-4 antibodies was effective in increasing the rate of antitumor immune response. However, the combination of chidamide-HCl salt and celecoxib-Na salt in combination with antibodies to CTLA-4 was more effective than the combination of chidamide-K30 and celecoxib in capsules in combination with antibodies to CTLA-4 in terms of affecting the process of inhibiting tumor growth; while the survival rates were 66.6%% and 44.4%, respectively (Figure 13, fragment D). On the other hand, a direct comparison between the combination of chidamide-HCl salt and celecoxib-Na salt and the combination of MS-275 and celecoxib capsules in combination with anti-PD-1 antibodies demonstrated that the antitumor activity of the combination regimen using the chidamide-HCl salt combination and celecoxib-Na salts have higher antitumor activity when implementing a combination regimen using a combination of MS-275 and celecoxib capsules in conditions of resistance against PD-L1 antibodies.

Example 10 Study of the pharmacokinetic (PK) profile of chidamide-HCl salt in combination with celecoxib-Na salt in male Wistar rats

The combination of chidamide-HCl salt and amorphous celecoxib-Na salt alone or in combination with anti-PD-1 antibodies had very high antitumor activity. Therefore, the PK profile of chidamide-HCl salt in combination with celecoxib-Na salt was studied in comparison with chidamide-K30 in combination with celecoxib capsules in Wistar rats. As shown in Figure 14, panel A, chidamide blood concentration-time profiles were analyzed for chidamide-HCl salt (50 mg/kg) and chidamide-K30 (50 mg/kg) when administered orally to Wistar rats. According to the results presented in Table 4, the C values_maxand T_max chidamide for the salt form have changed significantly. In the chidamide-HCl salt group, the C value is_max was 2065.2 ng/ml, and T_max – 0.14 hours. However, in the substance group chidamide-K30, the value is C_max was 786.3 ng/ml, and T_max – 0.39 hours. A significant increase in the rate of absorption of the chidamide-HCl salt was observed in comparison with the rate of adsorption of the chidamide-K30 substance. However, AUC, MRT and T values_1/2 did not change significantly as shown in Table 4. The results obtained indicate that chidamide-HCl salt was absorbed faster and achieved higher C values_max, however, did not contribute to an increase in the total amount of chidamide in the circulatory system compared to the application of the substance chidamide-K30 in Wistar rats. As shown in Figure 14, Panel B, the blood concentration-time profiles of celecoxib Na salt (50 mg/kg) and celecoxib capsules (50 mg/kg) were analyzed when administered orally to Wistar rats. The results obtained indicate that the values of T_max, C_max, AUC (area under the pharmacokinetic curve), AUMC (area under the first moment of the pharmacokinetic curve), MRT (mean drug retention time) and T_1/2, obtained in Wistar rats were not significantly different after oral administration of celecoxib-Na salt and celecoxib capsules, as shown in Table 5.

Differences between the PK profiles of chidamide were then analyzed using the combination of chidamide-HCl salt and celecoxib-Na salt and the combination of chidamide-K30 and celecoxib capsules at a dosage of 50 mg/kg administered orally to Wistar rats. As shown in Figure 14, panel C and Table 4, the chidamide C _max values were significantly increased in the chidamide-HCl salt and celecoxib-Na salt combination treatment group compared with the chidamide-K30 and celecoxib salt combination treatment group. in capsules; the values were about 2244.5 and 862.3 ng/ml, respectively. As shown in Table 4, chidamide _Tmax values were significantly reduced in the chidamide-HCl salt and celecoxib-Na salt combination treatment group compared with the chidamide-K30 and celecoxib capsule combination treatment group; the values were about 0.14 and 0.25 h, respectively. Chidamide AUC values were slightly increased in the chidamide-HCl and celecoxib-Na salt combination treatment group compared with the chidamide-K30 and celecoxib capsules treatment group; the values were about 5977 and 4201 ng·h/ml, respectively. Similar results comparing the AUMC values of the two combinations are shown in Table 4. The MRT and T _1/2 values showed no difference between the two groups, as shown in Table 4. Results of comparing the PK profiles of chidamide-HCl salt and the combination of chidamide-HCl salt and celecoxib-Na salts showed little change, see Figure 14, fragment E and Table 4. It was suggested that the presence of celecoxib-Na salt, when treated with a combination of chidamide-HCl salt and celecoxib-Na salt, did not have a significant effect on the PK profile of chidamide in terms of ADME (absorption, distribution, metabolism and excretion). However, there was some effect on chidamide AUC values; AUC values in the chidamide-HCl salt and the combination of chidamide-HCl salt and celecoxib-Na salt treatment groups were approximately 4113 and 5977 ng/mL, respectively, as shown in Table 4.

On the other hand, the PK profile of celecoxib did not change significantly when comparing the combination of chidamide-K30 and celecoxib capsules and the combination of chidamide-HCl salt and celecoxib-Na salt (50 mg/kg) when administered orally to Wistar rats, as shown in Figure 14 , fragment D and in Table 5. However, celecoxib-Na salt or celecoxib capsules alone had significantly lower _Cmax and AUC values than the combination of chidamide-K30 substance and celecoxib capsules or the combination of chidamide-HCl salt and celecoxib-Na salt , as shown in Figure 14, fragment F and in Table 5. The results obtained indicate that the presence of chidamide-K30 or chidamide-HCl salt has a significant effect on the ADME profile of celecoxib and, therefore, markedly increases the _Cmax and AUC values of celecoxib . However, the presence of celecoxib-Na salt or celecoxib capsules did not have a significant effect on the PK profile of chidamide. In conclusion, the ADME profile of the combination of chidamide-HCl salt and celecoxib-Na salt was demonstrated to be significantly altered to provide effective tumor growth inhibition and increased survival rates when used in combination with immune checkpoint inhibitors; In order to address the problem of drug resistance in second-line therapy, it can be assumed that the antitumor activity of the salt forms exceeds the antitumor activity of the combination of chidamide-K30 and celecoxib capsules.

Table 4. Pharmacokinetic parameters of chidamide in the treatment of male Wistar rats with chidamide-K30 substance, chidamide-HCl salt, a combination of chidamide-K30 substance and celecoxib in capsules and a combination of chidamide-HCl salt and celecoxib-Na salt.

Scroll Khidamid-K30
N=6 Chidamide salt
-HCl
N=6 Combination of the substance chidamide-K30 and
celecoxib capsules N=6 Combination of substance chidamide-HCl and
Celecoxib-Na salt
N=6 T _max, (h) 0.39±0.2 0.14± ^0.1a 0.25±0.01 ^a,b 0.14±0.09 ^a,c C _max (ng/ml) 786±243 2065± ^1136a 862± ^245a 2244±841 ^b AUC _0→t, (ng h/ml) 4422±1894 4113±1773 4201±848 5977±2161 AUMC, (ng h ² /ml) 57808±43710 46570±25207 38478±5671 53542±19097 MRT (h) 12.1±5.5 11.3±2.8 8.9±1.1 9.0±2.9 T _1/2 (h) 16.9±3.7 14.0±4.9 20.7±3.1 18.8±2.2

Data represent mean ± standard deviation (SD). ^a P < 0.05 compared with the substance chidamide-K30, ^b P < 0.05 compared with chidamide-HCl salt; ^c P < 0.05 compared with the combination of chidamide-K30 and celecoxib capsules. Differences between rats administered chidamide-K30, chidamide-HCl salt, the combination of chidamide-K30 and celecoxib capsules, and the combination of chidamide-HCl salt and celecoxib-Na salt were reported as the mean. ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

T _max : Time to reach C _max .

C _max : Peak plasma concentration of a drug after administration.

AUC _0→t : area under the pharmacokinetic curve.

MRT: mean drug retention time

T _1/2: The time required for the drug concentration to reach half the initial value.

Table 5. Pharmacokinetic parameters of chidamide in the treatment of male Wistar rats using celecoxib capsules, celecoxib-Na salt, a combination of celecoxib capsules and chidamide-K30 substance and a combination of celecoxib-Na salt and chidamide-HCl salt.

Scroll Celecoxib capsules
N=5 Celecoxib-Na salt
N=5 Combination of the substance chidamide-K30 and
celecoxib capsules
N=6 Salt combination
chidamide-HCl
And
celecoxib-Na
salt
N=6 T _max, (h) 4.8±1.0 5.2±1 4.3± ^0.8a 3.3±1.0 ^s C _max (ng/ml) 7104±2962 7631±2727 15021±1563 ^b 16576±1181 ^s AUC _0→t, (ng h/ml) 105511±34816 106723±35778 163453±11461 ^b 168033±14588 ^a,b AUMC, (ng h ² /ml) 1062627±301118 1124599±333210 1481893± ^243790a 1330378±158873 MRT (h) 9.3±0.4 9.4±0.5 9.0±1.1 7.9±0.6 ^a,b,c T _1/2 (h) 6.2±2 7.7±3.1 3.7±0.2 ^a,b 3.4±0.1 ^a,b

Data represent mean ± standard deviation (SD). ^a P < 0.05 compared with celecoxib capsules, ^b P<0.05 compared with celecoxib-Na salt; ^c P < 0.05 compared with the combination of chidamide-K30 and celecoxib capsules. Differences between rats administered chidamide-K30, chidamide-HCl salt, the combination of chidamide-K30 and celecoxib capsules, and the combination of chidamide-HCl salt and celecoxib-Na salt were reported as the mean. ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

T _max : Time to reach C _max .

C _max : Peak plasma concentration of a drug after administration.

AUC _0→t : area under the pharmacokinetic curve.

MRT: mean drug retention time.

T _1/2: The time required for the drug concentration to reach half the initial value.

Claims (31)

1. An antitumor combination containing an acid salt of chidamide and a basic salt of celecoxib, in which the acid salt of chidamide is a hydrochloride or sulfate salt. 2. The combination of claim 1, wherein the amounts of chidamide acid salt and celecoxib base salt are in the range of 5% by weight to 80% by weight and 95% by weight to 20% by weight, respectively. 3. The combination according to claim 1, in which the amounts of the acid salt of chidamide and the basic salt of celecoxib are in a mass ratio of 8:1, 4:1, 2:1, 1:1, 1:2, 1:4 or 1:8. 4. The combination of claim 1, wherein the amounts of chidamide acid salt and celecoxib base salt are contained in the same dosage form or independently distributed among different dosage forms. 5. The combination according to claim 4, in which the dosage form is a tablet or capsule. 6. The combination according to claim 1, in which the chidamide acid salt is in crystalline form. 7. The combination of claim 1, wherein the chidamide hydrochloride salt is in crystalline form (Form A) and exhibits peaks with 2θ values of about 16.12° in the X-ray powder diffraction (XRPD) pattern, about 19.02°, about 21.62°, about 23.38° and about 30.16°. 8. The combination of claim 7, wherein the X-ray diffraction pattern of Form A exhibits additional peaks with 2θ values of about 21.08°, about 23.76°, about 25.58°, about 27.82°, and about 28.18 ° 9. The combination according to claim 1, in which the hydrochloride salt of chidamide is in crystalline form (Form A), while the spectrum obtained by Fourier transform infrared spectroscopy (FTIR) shows peaks at a level of 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 . 10. The combination according to claim 7 or 9, in which the radiograph of form A repeats the profile of the radiograph presented in Fig. 3, fragment B, or IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), repeats the profile of the spectrum presented in FIG. 4, fragment B. 11. The combination according to claim 1, in which the chidamide sulfate salt is in crystalline form (Form B), and the X-ray powder diffraction (XRPD) pattern shows peaks with 2θ values at about 21.15°, about 24.65°, about 17.00°, about 18.49° and about 26.69°. 12. The combination of claim 11, wherein the X-ray diffraction pattern of Form B exhibits additional peaks with 2θ values of about 14.74°, about 19.45°, about 22.00°, about 23.55°, and about 27.94 °. 13. The combination according to claim 1, in which the chidamide sulfate salt is in crystalline form (Form B), while the spectrum obtained by Fourier transform infrared spectroscopy (FTIR) shows peaks at a level of about 3249 cm ^-1 , about 3067 cm ^-1 , about 2578 cm ^-1 , about 2360 cm ^-1 , about 1689 cm ^-1 , about 1664 cm ^-1 , about 1647 cm ^-1 , about 1614 cm ^-1 , about 1568 cm ^-1 , about 1521 cm ^-1 , about 1510 cm ^-1 , about 1486 cm ^-1 , about 1467 cm ^-1 , about 1434 cm ^-1 , about 1412 cm ^-1 , about 1388 cm ^-1 , about 1354 cm ^-1 , about 1328 cm ^-1 , about 1283 cm ^-1 , about 1266 cm ^-1 , about 1252 cm ^-1 , about 1226 cm ^-1 , about 1184 cm -1 , about 1099 cm ^-1 , about 1059 ^{cm -1 ,} about 1034 cm ^-1 and about 1022 cm ^-1 . 14. The combination of claim 11 or 13, wherein the radiograph of Form B follows the profile of the radiograph shown in FIG. 3, fragment C, or the IR spectrum obtained by Fourier transform infrared spectroscopy (FTIR), repeats the profile of the spectrum presented in FIG. 4, fragment C. 15. The combination according to claim 1, wherein the main salt of celecoxib is a sodium salt. 16. The combination according to claim 15, in which the sodium salt of celecoxib is in amorphous or crystalline form. 17. The combination according to claim 16, in which the X-ray pattern of the amorphous form of the sodium salt of celecoxib repeats the profile of the X-ray pattern shown in FIG. 7, fragment B. 18. The combination of claim 16, wherein the sodium salt of celecoxib is in crystalline form (Form I) and exhibits peaks with 2θ values at about 19.85° in the X-ray powder diffraction (XRPD) pattern, about 20.51°, about 21.51°, about 22.55° and about 18.25°. 19. The combination of claim 18, wherein the X-ray diffraction pattern of Form I exhibits additional peaks with 2θ values of about 10.95°, about 14.05°, about 14.601°, about 17.2°, about 25.80°, and about 27.30°. 20. The combination according to claim 18, in which the radiograph of Form I repeats the profile of the radiograph presented in FIG. 7, fragment C. 21. The combination of claim 1, further comprising an immune checkpoint inhibitor and/or a chemotherapeutic agent, wherein in some embodiments the immune checkpoint inhibitors are anti-CTLA-4 antibodies, anti-PD-1 antibodies, or anti-PD antibodies -L1. 22. The combination of claim 21, wherein the immune checkpoint inhibitors are pembrolizumab, pidilizumab, nivolumab, durvalumab, avelumab, atezolizumab, toripalimab, sintilimab, camrelizumab or MIH1. 23. A method for regulating the tumor microenvironment during cancer immunotherapy, including administering an effective amount of an antitumor combination, according to any one of claims. 1-22. 24. The method according to claim 23, in which the acid salt of chidamide and the basic salt of celecoxib are administered simultaneously, separately or sequentially. 25. A method of treating cancer, comprising administering to a subject an effective amount of an antitumor combination described in claims. 1-22. 26. The method of claim 23 or 25, which further comprises administering an immune checkpoint inhibitor. 27. The method of claim 23 or 25, wherein the administration of chidamide acid salt and celecoxib base salt, as opposed to the method using chidamide free base and celecoxib free acid, improves the pharmacokinetic profile. 28. The method according to claim 26, in which the antitumor combination according to any one of claims. 1-22 and an immune checkpoint inhibitor, administered simultaneously, separately or sequentially. 29. The method of claim 23 or 25, wherein the cancers include glioblastoma, liver cancer, colorectal carcinoma, glioblastoma, stomach 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's lymphoma, acute myeloid leukemia (AML), gallbladder cancer, cholangiocarcinoma, bladder cancer and uterine cancer. 30. A method of treating cancer by regulating the tumor microenvironment and increasing the rate of formation of the immune response, including the introduction of an antitumor combination according to any one of claims. 1-22. 31. The method of claim 30, wherein chidamide and celecoxib are administered simultaneously, separately or sequentially.