A pharmaceutical combination for the treatment of a cancer

Abstract

The use of ABTL0812 in the treatment of cancer in human patients, wherein the cancer treatment is related to chemotherapy, targeted therapy, immunotherapy or radiation therapy.

Description

[Technical Field]

[0001] This invention relates to the use of ABTL0812 in the treatment of cancer in human patients, wherein the cancer treatment is related to chemotherapy, targeted therapy, immunotherapy or radiation therapy. [Previous Technology]

[0002] EP2409963B1 (Lipopharma - applied in 2010) describes the use of a 1,2-derived polyunsaturated fatty acid compound (called D-PUFA) for cancer treatment.

[0003] The fatty acid derivative compound has the following formula:

[0003] COOR1-CHR2-(CH2)a-(CH=CH-CH2)b-(CH2)c-CH3

[0003] One example of a preferred compound is:

[0003] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(182A1)

[0004] The article “Erazo et al.; Clinical Cancer Research; 22(10) May 15, 2016” provides a more detailed description of the compound (182A1) mentioned above – which is referred to as “ABTL0812” in the article and is also used in this paper.

[0005] As is known in the art, the pharmacological treatment of cancer is generally based on four major classes of drugs, including chemotherapy, targeted therapy, hormone therapy and immunotherapy.

[0006] In addition, radiation therapy is also the foundation of cancer treatment, and it is often administered in conjunction with drug therapy.

[0007] WO2018 / 210830A1 (Ability Pharmaceuticals) describes the use of an ABTL0812 compound in combination with other chemotherapeutic agents in the treatment of cancer—such as the combination of ABTL0812 with chemotherapeutic agents docetaxel, paclitaxel, carboplatin, or cisplatin as a first-line therapy. [Summary of the Invention]

[0008] Originating from WO2018 / 210830A1 (Ability Pharmaceuticals), which is the most relevant prior art document (so-called closest prior art document), the problem to be solved by the present invention can be regarded as providing an alternative use of ABTL0812, which can provide an improved cancer treatment.

[0009] As discussed above, the compound COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 is referred to as ABTL0812 in this paper.

[0010] As discussed above, WO2018 / 210830A1 (Ability Pharmaceuticals) describes the use of an ABTL0812 compound in combination with other chemotherapeutic agents in the treatment of cancer—such as the combination of ABTL0812 with chemotherapeutic agents docetaxel, paclitaxel, carboplatin or cisplatin in the context of first-line therapy.

[0011] WO2018 / 210830A1 (Ability Pharmaceuticals) does not directly and explicitly describe the use of ABTL0812 as a second-line therapy for cancer treatment - for example, the terms "second-line" or "second line" associated with second-line therapy are not even mentioned in WO2018 / 210830A1.

[0012] The working examples described herein provide seemingly reasonable and detailed experimental data that demonstrate a significant synergistic effect in relation to the use of the ABTL0812 compound discussed above in combination with other chemotherapeutic agents for, for example, second-line treatment of cancer in human patients.

[0013] Compound ABTL0812 is structurally and functionally similar to other 1,2-derived polyunsaturated fatty acid (D-PUFA) compounds described in EP2409963B1 as discussed above.

[0014] Therefore, it seems reasonable at first glance that virtually all fatty acid derivative compounds of EP2409963B1, in combination with chemotherapeutic agents and / or other better cancer treatments as discussed herein, will have the synergistic effects relevant herein.

[0015] Therefore, the first aspect of the present invention relates to a pharmaceutical combination comprising:

[0015] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0015] (i)a can be any integer value between 0 and 7.

[0015] (ii) b can be any integer value between 2 and 7.

[0015] (iii) c can be any integer value between 0 and 7.

[0015] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0015] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0015] and

[0015] (B1): Chemotherapy compound,

[0015] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is a second-line therapy for cancer.

[0016] As known in the art, first-line therapy is one or more treatment options commonly accepted by medical institutions for the initial treatment of a given type and stage of cancer. It is also referred to as primary treatment or therapy. Where possible, the intent of first-line therapy is to cure the cancer. This primary treatment is also known as induction therapy, which is the first attack of chemotherapy drugs on the malignant tumor.

[0017] Consistent with common sense among those familiar with this technology, the first-state term "second-line therapy" refers to second-line treatment attempted when first-line therapy has not been sufficiently effective. Cancer case management requires regular evaluation and adjustment of treatment as needed. Discontinuing primary first-line therapy and adopting a new regimen signifies "second-line therapy."

[0018] As understood by those skilled in this art in this context, the term "second-line therapy" requires that the patient in the first-line therapy has been treated with one or more cancer agents or mixtures that are different from the profile / mixture of one or more cancer agents in the "second-line therapy".

[0019] Some might argue that if the profile / mixture of one or more first-line cancer drugs is satisfactory (i.e., it cures the patient’s cancer), then “second-line therapy” may not be necessary.

[0020] By way of example only - in this context, first-line therapy may involve, for example, the use of docetaxel, paclitaxel and (or) ABTL0812, and subsequent second-line therapy may be different profiles / mixtures of one or more cancer agents, such as a combination of ABTL0812 and temozolomide as discussed in working example 1.1 herein.

[0021] According to this technology, chemotherapy is a type of cancer treatment that uses one or more anticancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. The term chemotherapy has been used to describe the non-specific use of intracellular toxic compounds to inhibit mitosis, cell division—that is, chemotherapeutic agent compounds are understood as compounds that interfere with cell replication. Because DNA / cell replication is a common method used by all cells when they want to replicate themselves more, chemotherapy cannot distinguish between cancer cells and normal cells. Therefore, typical chemotherapy may have significant side effects.

[0022] As discussed above, WO2018 / 210830A1 (Ability Pharmaceuticals) describes the use of ABTL0812 in combination with other chemotherapeutic agents in cancer treatment - therefore, this document does not directly and explicitly describe the use of ABTL0812 in targeted therapy, immunotherapy and / or radiotherapy for cancer treatment.

[0023] The working examples described herein provide seemingly reasonable and detailed experimental data that demonstrate significant positive effects related to the use of the ABTL0812 compound discussed above in targeted therapy, immunotherapy or radiotherapy for the treatment of cancer in human patients.

[0024] Therefore, the second aspect of the present invention relates to a pharmaceutical combination comprising:

[0024] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0024] (i)a can be any integer value between 0 and 7.

[0024] (ii) b can be any integer value between 2 and 7.

[0024] (iii) c can be any integer value between 0 and 7.

[0024] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0024] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0024] and

[0024] (B2): Targeted therapeutic compound,

[0024] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is a targeted therapy for cancer.

[0025] The term "targeted therapy" in the second state should be understood in accordance with this technology.

[0026] As known in the art, targeted therapy or molecular targeted therapy is one of the main modalities of drug treatment (pharmacological therapy) for cancer, others being, for example, cytotoxic chemotherapy. As a form of molecular medicine, targeted therapy inhibits the growth of cancer cells by interfering with specific target molecules required for cancer development and tumor growth, rather than simply interfering with all dividing cells (e.g., with conventional chemotherapy).

[0027] The third aspect of the present invention relates to a pharmaceutical combination, comprising:

[0027] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0027] (i)a can be any integer value between 0 and 7.

[0027] (ii) b can be any integer value between 2 and 7.

[0027] (iii) c can be any integer value between 0 and 7.

[0027] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0027] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0027] and

[0027] (B3): Immunotherapy compound,

[0027] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is an immunotherapy for cancer.

[0028] The term "immunotherapy" in the third state should be understood in accordance with this technology.

[0029] As known in the art, immunotherapy treats disease by activating or suppressing the immune system. Immunotherapy designed to elicit or enhance an immune response is classified as activating immunotherapy, while immunotherapy that reduces or suppresses an immune response is classified as suppressive immunotherapy. In recent years, immunotherapy has attracted great interest from researchers, clinicians, and pharmaceutical companies, particularly in its prospects for treating various forms of cancer.

[0030] The fourth aspect of the present invention relates to a pharmaceutical composition comprising:

[0030] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0030] (i)a can be any integer value between 0 and 7.

[0030] (ii) b can be any integer value between 2 and 7.

[0030] (iii) c can be any integer value between 0 and 7.

[0030] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0030] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0030] This pharmaceutical composition is used for the treatment of cancer in human patients, wherein the treatment is radiotherapy for cancer.

[0031] The term "radiotherapy" in the fourth category should be understood in accordance with this technique.

[0032] As is known in the art, radiation therapy (also known as radiotherapy) is a cancer treatment that uses high doses of radiation to kill cancer cells and shrink tumors.

[0033] The fifth aspect of the present invention relates to a pharmaceutical combination, comprising:

[0033] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0033] (i)a can be any integer value between 0 and 7.

[0033] (ii) b can be any integer value between 2 and 7.

[0033] (iii) c can be any integer value between 0 and 7.

[0033] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0033] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0033] and

[0033] (B1): Chemotherapy compound,

[0033] This pharmaceutical combination is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0033] Temozolomide;

[0033] Topotecan;

[0033] Irinotecan;

[0033] Cyclophosphamide;

[0033] Fluorouracil;

[0033] Oxaliplatin;

[0033] Tetrahydrofolate; and

[0033] Cranberries.

[0034] When compound (B) is referred to in this document, it should be understood as referring to any one of compound (B1), compound (B2) and / or compound (B3).

[0035] As understood by those skilled in this art in this context, the pharmaceutical preparation of the above-mentioned related state compound (B) is certainly not a compound within the scope of the above-mentioned related state compound (A).

[0036] As understood by those skilled in the art in this context—regarding the combination therapy discussed herein, if the two compounds (A) and (B) are administered simultaneously, for example, as a single composition, or sequentially, for example, as two separate compositions, then the combination therapy is not necessary. It is important that, when the second compound is administered, an effective amount of that compound / drug is first administered in the patient and / or has already exerted its effect in the patient.

[0037] As will be understood by those skilled in the art in this context, the present invention relates to combinations of compound (A) with at least one compound (B), such as combinations of compound (A) + compound (B1), compound (A) + compound (B2), compound (A) + compound (B3), or compound (A) + compound (B1) + compound (B3). It should also be understood that, since compound (A) can be administered in combination with radiotherapy according to the fourth embodiment of the present invention, radiotherapy can also be administered in combination with any of the combinations mentioned above with compound (B).

[0038] Therefore, for example in a single pharmaceutical composition, in a combined mixture such as a "tank-mix" consisting of an independent pharmaceutical formulation / composition of a single active compound, and in the combined use of a single active ingredient when administered sequentially (i.e., one after another over a reasonable short period of time such as hours or days) or simultaneously, the term "combination" in the relevant context above refers herein to various combinations of compounds (A) and (B). The order in which compounds (A) and (B) are administered is not required.

[0039] The combination of compounds (A) and (B) can be formulated for simultaneous, separate, or sequential administration. Specifically, if not administered simultaneously, the compounds are administered at relatively close time intervals. Furthermore, the compounds can be administered in the same or different dosage forms or via the same or different routes of administration, for example, one compound may be administered intravenously and the other orally. The combination of two compounds can be administered, for example, in the following manner:

[0039] As a combination, this combination is part of the same pharmaceutical formulation, and the two compounds are subsequently always administered simultaneously;

[0039] As a combination of two units / two compositions, each unit / each composition has one of the substances that can be produced simultaneously, sequentially or independently;

[0039] For example, compound (A) and compound (B) are administered independently (i.e., in two units) but simultaneously.

[0040] In another suitable example, compound (A) is first administered, and then compound (B) is administered independently or sequentially thereafter – or, compound (B) is first administered, and then compound (A) is administered independently or sequentially thereafter.

[0041] In another suitable example, when two compounds (B) are administered, compound (A) is administered first, followed by the first compound (B) administered independently or sequentially, and then the second compound (B) is administered last, independently or sequentially. Alternatively, the first compound (B) is administered first, followed by the second compound (B) administered independently or sequentially, and then compound (A) is administered last, independently or sequentially. Alternatively, the first compound (B) is administered first, followed by compound (A) administered independently or sequentially, and then the second compound (B) is administered last, independently or sequentially.

[0042] For example, the term "pharmaceutical" in relation to "pharmaceutical composition" should be understood in accordance with this technology—that is, it refers to a formulation / composition in a form that enables the biological activity of the active ingredient to be effective and is physiologically tolerable, i.e., a formulation / composition that does not contain any additional components that would have unacceptable toxicity to the individual to whom the composition will be administered. Specifically, the term "pharmaceuticalally acceptable" means that it is approved by a state or federal regulatory agency or included in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and more specifically, for use in humans.

[0043] Embodiments of the present invention are described below by way of example only.

[0044] The combination of the preferred embodiments described herein with another preferred embodiment described herein constitutes a more preferred embodiment. [Simplified Explanation of the Diagram]

[0045] ABTL0812 and ABTL are used interchangeably in this instruction manual.

[0045] 〔Figure 1〕: Cytotoxicity of ABTL0812 (ABTL) and temozolomide (TMZ) in LA1-5S and SK-N-BE(2) cells. See the working examples in this paper for further details.

[0045] [Figure 2]: Cytotoxicity of ABTL0812 and topotecan in LA1-5S. See the working examples in this article for further details.

[0045] [Figure 3]: Cytotoxicity of ABTL0812 and irinotecan in LA1-5S. See the working examples in this article for further details.

[0045] [Figure 4]: Cytotoxicity of ABTL0812 and cyclophosphamide in LA1-5S. See the working examples in this article for further details.

[0045] [Figure 5]: ABTL0812 and bortezomib exhibit a strong synergistic effect in vitro in multiple myeloma cell lines JJN-3 and OPM2. See the working examples in this article for further details.

[0045] [Figure 6]: ABTL0812 significantly enhanced the anticancer effect of Folfirinox without increasing toxicity in a human pancreatic cancer xenograft model using MiaPaca2 cells from nude mice. The results indicate that the combination treatment may have clinical significance for the treatment of pancreatic cancer. See the working examples in this article for further details.

[0045] [Figure 7]: ABTL0812 significantly enhanced the anticancer effect of cranberry without increasing toxicity in a human endometrial cancer xenograft model using Ishikawa cells implanted in nude mice. The results indicate that the combination therapy of ABTL0812 and cranberry may have clinical significance in the treatment of endometrial cancer. See the working examples in this article for further details.

[0045] [Figure 8]: ABTL0812 increased disease-free survival in mice with glioblastoma tumors and enhanced the antitumor activity of temozolomide. See the working examples in this article for further details.

[0045] [Figure 9]: ABTL0812 reduces the growth of glioblastoma tumors (U87MG, T98G cells) and enhances the antitumor activity of radiotherapy. See the working examples in this article for further details.

[0045] [Figure 10]: ABTL0812 increased disease-free survival in mice with glioblastoma tumors and enhanced the antitumor activity of radiotherapy. See the working examples in this article for further details.

[0045] [Figure 11]: ABTL0812 significantly enhanced the anticancer effect of olaparib without increasing toxicity in a human endometrial cancer xenograft model using Ishikawa cells implanted in nude mice. The results indicate that the combination therapy of ABTL0812 and olaparib may have clinical significance in the treatment of endometrial cancer. See the working examples in this article for further details.

[0045] [Figure 12]: ABTL0812 significantly enhanced the anticancer effect of bevacizumab without increasing toxicity in a human endometrial cancer xenograft model using Ishikawa cells implanted in nude mice. The results indicate that the combination therapy of ABTL0812 and bevacizumab may have clinical significance in the treatment of endometrial cancer. See the working examples in this article for further details.

[0045] [Figure 13]: ABTL0812 enhances macrophage polarization toward the M1 pro-inflammatory antitumor phenotype by significantly increasing the expression of IL-1B and TNF-α genes. Importantly, ABTL0812 inhibits M2 polarization toward the anti-inflammatory protumor phenotype by significantly inhibiting the expression of the IL10 gene (one of the major regulatory genes for immunosuppression). TBP is a TATA box-binding protein. NT refers to non-polarized macrophages. ABTL50uM refers to non-polarized macrophages treated with 50μM ABTL0812. M1 refers to macrophages polarized to the M1 phenotype. M1+ABTL50 refers to macrophages polarized to the M1 phenotype treated with 50μM ABTL0812. ABTL100uM refers to non-polarized macrophages treated with 100μM ABTL0812. M1+ABTL100 refers to macrophages polarized to the M1 phenotype after treatment with 100 μM ABTL0812. M2 refers to macrophages polarized to the M2 phenotype. M2+ABTL50 refers to macrophages polarized to the M2 phenotype after treatment with 50 μM ABTL0812. See the working examples in this article for further details.

[0045] [Figure 14]: ABTL0812 induces PDL1 expression in endometrial and pancreatic cancer cell lines. ABTL0812-mediated PDL1 expression is significantly lower than that induced by IFNγ (the main regulator of PDL1 expression). These results highlight the potential combination of ABTL0812 with immune checkpoint inhibitors, as the mediated induction of PDL1 levels would allow immune checkpoint inhibitors to target cancer cells. See the working examples in this article for further details.

[0045] [Figure 15]: ABTL0812 promotes immunogenic cancer cell death in human pancreatic MiaPaca2 cancer cells by significantly inducing sustained gene expression of IL-1β and TNF-α, thereby inducing sustained activation of macrophages and the release of immunogenic factors polarizing them toward the M1 pro-inflammatory and anti-tumor phenotype. These data, along with Figure 13, indicate that ABTL0812 can immunomodulate the tumor microenvironment through its anticancer effects on cancer cells, in addition to its direct effects on human macrophages, thus highlighting its potential for enhanced anticancer efficacy in combination with immune checkpoint inhibitors. RPMI or NT refers to the initial conditioned medium (control). MiaPACA-2 NT or MiaPACA-2 CM NT refers to the conditioned medium of untreated MiaPaca2 cells. MiaPACA-2 40μM ABTL refers to the conditioned medium of MiaPaca2 cells treated with ABTL0812 (40μM ABTL0812). MiaPACA-2 CM 70uM is a conditioned medium for MiaPaca2 cells treated with ABTL0812 (70μM ABTL0812). TBP is a TATA box-binding protein. See the working examples in this article for further details.

[0045] [Figure 16]: Administration of ABTL0812 alone increased survival in a genotype-specific lung cancer mouse model by subcutaneous implantation of LLC1 cells into C57BL6 mice. The combination of ABTL0812 and anti-PD1 showed a greater increase in survival compared to anti-PD1, ABTL0812, and mediator treatment. The data suggest that ABTL0812 synergistically enhances anti-PD1 treatment, thereby increasing mouse survival. This result indicates a potential benefit of combining anti-PD1 with ABT10812 in human patients. See the working examples in this article for further details.

[0045] [Figure 17]: In a genotype-dependent lung cancer mouse model using subcutaneous implantation of LLC1 cells into C57BL6 mice, administration of ABTL0812 alone showed tumor volume reduction similar to that of anti-PD1 plus carboplatin / paclitaxel treatment. Both treatments significantly reduced tumor volume compared to the mediator group. The triple combination treatment of ABTL0812 plus anti-PD1 plus carboplatin / paclitaxel induced a reduction in maximum tumor volume, thereby significantly improving the other treatments. This higher anticancer efficacy was associated with increased CD8 / CD4 gene expression in the tumor, thus validating previous in vitro observations in vivo (Figures 15 and 13). The CD8 / CD4 ratio is typically assessed to analyze cytotoxic antitumor T lymphocytes after drug treatment. The results indicate that the combination therapy of ABTL0812 plus anti-PD1 + paclitaxel / carboplatin (the standard treatment for lung cancer patients) may have clinical significance in the treatment of lung cancer. See the working examples in this article for further details.

[0045] [Figure 18]: The combination of ABTL0812 with anti-PD1 plus carboplatin / paclitaxel treatment induced a reduction in maximum tumor volume in a genotype-specific lung cancer mouse model of C57BL6 mice by intraperitoneal implantation of LLC1 cells. The results indicate that the combination therapy of ABTL0812 plus anti-PD1 plus paclitaxel / carboplatin (the standard treatment for lung cancer patients) may have clinical significance in the treatment of lung cancer. See the working examples in this article for further details.

[0045] [Figure 19]: ABTL0812 induces PDL1 expression in in vitro endometrial and pancreatic cancer cell lines. These results highlight the potential combination of ABTL0812 with immune checkpoint inhibitors, as the induction of PDL1 levels would allow the immune checkpoint inhibitors to target cancer cells. See the working examples in this article for further details.

[0045] [Figure 20]: ABTL0812 inhibits PD1 expression in activated and inactivated human primary T cells. PD1 mediates inhibitory signals for T cell activity; therefore, reducing it with ABTL0812 may contribute to T cell activation and induce their anticancer activity. RFU stands for Relative Fluorescent Unit. WB stands for Western Blot. See the working examples in this paper for further details.

[0045] [Figure 21]: ABTL0812 promotes the inhibition of the release of immunosuppressive chemokines in human cancer cells, thereby promoting a pro-inflammatory environment. See the working examples in this article for further details.

[0045] [Figure 22]: ABTL0812 induces ICD in human pancreatic cancer cells by inducing dose-dependent increases in extracellular Hmgb1 and ATP, surface calreticulin, and activated apoptotic proteases 3 and 8. See the working examples in this article for further details.

[0045] [Figure 23]: ABTL0812 promotes the infiltration of CD3 T cells into tumor lesions in PTEN-KO mice that have undergone endometrial carcinogenesis, accompanied by inhibition of carcinogenesis and reduction of tumor lesions (EIN, endometrial intraepithelial neoplasia). UN represents untreated cells.

[0045] [Figure 24]: ABTL0812 enhances the polarization of immortalized THP-1 and human primary macrophages toward the M1 pro-inflammatory antitumor phenotype by significantly increasing the expression of IL-1β and TNF-α genes. Importantly, ABTL0812 inhibits M2 polarization toward the anti-inflammatory protumor phenotype by significantly inhibiting the expression of the IL10 gene (one of the major regulatory genes of immunosuppression). These data demonstrate the immunomodulatory role of ABTL0812 in macrophages polarized toward the antitumor phenotype, which may potentially synergize with immunotherapy. (Note: The last sentence appears to be a separate, unrelated statement about a mediator.) See the working examples in this article for further details.

[0045] [Figure 25]: ABTL0812 promotes the release of pro-inflammatory cytokines and the inhibition of immunosuppressive cytokine secretion in human primary macrophages and immortalized THP1 macrophages, thereby promoting a pro-inflammatory environment. These results support potential synergistic effects with immunotherapy. CTRL stands for control. See the working examples in this article for further details.

[0045] [Figure 26]: When the two cell types are co-cultured in vitro, ABTL0812 enhances the cytotoxic effect of activated T cells against cancer cells.

[0045] [Figure 27]: In a syngeneic mouse model of C57BL6 mice with subcutaneous implantation of MT5 pancreatic cancer cells, ABTL0812 monotherapy demonstrated tumor volume reduction similar to anti-PD1 therapy. Both treatments significantly reduced tumor volume compared to the mediator group. ABTL0812 more effectively promoted a pro-inflammatory anti-tumor environment than anti-PD1 treatment, resulting in an increase in intratumoral bone marrow cells and NK anti-cancer cells, as well as an increase in the Th1 / Th2 ratio in the spleen.

[0045] [Figure 28]: The combination of anti-PD1 and FOLFIRINOX with ABTL0812 showed a reduction in maximum tumor volume compared with the other groups (including anti-PD1 alone and ABTL0812 + FOLFIRINOX), thereby promoting a pro-inflammatory anti-tumor environment that increased the infiltration of bone marrow cells and CD8 anti-cancer cells within the tumor.

Implementation Method

[0045] Compound of the relevant state (A)

[0046] A preferred embodiment is wherein...

[0046] (i)a can be any integer value between 5 and 7.

[0046] (ii) b can be any integer value between 2 and 4.

[0046] (iii)c can be any integer value between 1 and 5.

[0047] Preferably, R1 can be H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2.

[0048] Preferably, R2 can be OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2.

[0049] In a preferred embodiment, R1 is H and R2 is OH.

[0050] In another preferred embodiment, R1 is Na and R2 is OH.

[0051] Preferably, compound (A) is at least one compound selected from the group consisting of:

[0051] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0051] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0051] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0051] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0051] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0051] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0052] Optimally, compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0053] A pharmaceutically acceptable salt of compound (A) means any pharmaceutically acceptable salt of compound (A). As is known in the art, there are many known pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include, but are not limited to, sodium (Na) salts, potassium salts, acetates, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propynates, oxalates, malonates, succinates, octanoates, sebates, transbutenedioates, and cis-butenedioates. Salts, butyne-1,4-diacidates, hexyne-1,6-diacidates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, alkyl sulfonates (e.g., methane-sulfonates or methanesulfonates), propane sulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and amygdalinates. In a particular embodiment, the salt of compound (A) is a sodium salt.

[0054] As will be understood in this context by those skilled in the art, when a preferred formula of compound (A) (such as ABTL0812) is mentioned herein, it should be understood that it is also included as a salt of it. For example, when compound (A) is mentioned herein as COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812), it is also referred to as a salt of ABTL0812.

[0055] Preferably, compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0055] Chemotherapy Agent - First-State Compound (B1)

[0056] In some embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0056] Temozolomide;

[0056] Topotecan;

[0056] Irinotecan;

[0056] Cyclophosphamide;

[0056] Fluorouracil (5-fluorouracil, 5-FU);

[0056] Cisplatin;

[0056] Carboplatin;

[0056] Oxaliplatin;

[0056] Tetrahydrofolate;

[0056] Cranberries;

[0056] Bleomycin;

[0056] Capecitabine;

[0056] Mitomycin B;

[0056] Paclitaxel;

[0056] Albumin-bound paclitaxel;

[0056] Dorcetaxel;

[0056] Gemcitabine;

[0056] Methotrexate;

[0056] Pemetrexed;

[0056] Cytarabine;

[0056] Mercaptopurine;

[0056] Glucophosphamidon;

[0056] Ixabepilone;

[0056] Nimustine;

[0056] Carmustine;

[0056] Lomustine;

[0056] Mitoxantrone;

[0056] Etoposide;

[0056] Changchun Xinjian;

[0056] Vinpocetine; and

[0056] Tamoxifen.

[0057] As understood in this context - for any of the preferred enumerated examples of compound (B1), preferably compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0058] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0058] Temozolomide;

[0058] Topotecan;

[0058] Irinotecan;

[0058] Cyclophosphamide;

[0058] Fluorouracil;

[0058] Oxaliplatin;

[0058] Tetrahydrofolate; and

[0058] Cranberries.

[0059] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0059] Temozolomide;

[0059] Topotecan;

[0059] Fluorouracil;

[0059] Oxaliplatin; and

[0059] Formyltetrahydrofolate.

[0060] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0060] Irinotecan;

[0060] Fluorouracil;

[0060] Oxaliplatin; and

[0060] Formyltetrahydrofolate.

[0061] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0061] Carboplatin; and

[0061] Paclitaxel.

[0062] Preferably, the first-state compound (B1) comprises two or more different chemotherapeutic agents (especially when compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812)) - such preferably, wherein the first-state compound (B1) comprises:

[0062] Irinotecan, leucovorin, oxaliplatin, and fluorouracil; or

[0062] Irinotecan, Topotecan and Cyclophosphamide.

[0063] As will be understood in this context by those skilled in the art, the compound (B1) may be used in combination with other cancer treatment-related agents / compounds, such as one or more targeted therapeutic agents (B2).

[0064] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is temozolomide - specifically, wherein the cancer is neuroblastoma (see Example 1.1 for an example of this preferred embodiment).

[0065] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is topotecan—specifically, the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.2 herein). In other embodiments, compound (A) is ABTL0812 and compound (B1) is topotecan—specifically, the cancer is pancreatic cancer or glioblastoma.

[0066] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is irinotecan—specifically, the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.3 herein). In other embodiments, compound (A) is ABTL0812 and compound (B1) is irinotecan—specifically, the cancer is pancreatic cancer or glioblastoma.

[0067] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is cyclophosphamide - specifically, wherein the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.4 herein).

[0068] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is irinotecan, leucovorin, oxaliplatin and fluorouracil - specifically, wherein the cancer is pancreatic cancer. (For an example of this preferred embodiment, see Example 3.1 herein).

[0069] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is cranberry - specifically, the cancer is endometrial cell carcinoma. (For an example of this preferred embodiment, see Example 3.2 herein).

[0070] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is temozolomide – specifically, the cancer is glioblastoma. (For an example of this preferred embodiment, see Example 3.3 herein).

[0071] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is pemetrexed - specifically, the cancer is lung cancer.

[0072] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is methamidophos - specifically, the cancer is lung cancer.

[0073] Preferably, the pharmaceutical combination discussed herein is wherein compound (A) is ABTL0812 and wherein:

[0073] - Compound (B1) is temozolomide and the cancer is neuroblastoma;

[0073] - Compound (B1) is topotecan and the cancer is neuroblastoma;

[0073] - Compound (B1) is irinotecan and the cancer is neuroblastoma;

[0073] - Compound (B1) is cyclophosphamide and the cancer is neuroblastoma;

[0073] - Compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil, and the cancer is pancreatic cancer;

[0073] - Compound (B1) is a cranberry and the cancer is endometrial cancer; or

[0073] - Compound (B1) is temozolomide and the cancer is glioblastoma;

[0073] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0074] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0075] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0075] Targeted Therapeutic Agent - Second-State Compound (B2)

[0076] Preferably, compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0076] Imatinib;

[0076] Gefitinib;

[0076] Erlotinib;

[0076] Sorafenib;

[0076] Sunitinib;

[0076] Dasatinib;

[0076] Lapatinib;

[0076] Nilotinib;

[0076] Proteasome inhibitors (preferably carfilzomib, ixazomib, or bortezomib);

[0076] Tamoxifen;

[0076] Janus kinase inhibitor (preferably tofacitinib);

[0076] ALK inhibitors (better than crizotinib);

[0076] Bcl-2 inhibitors (preferably obatoclax, navitoclax, or gossypol);

[0076] PARP inhibitors (better than Iniparib or Olaparib);

[0076] PI3K inhibitor (better than Perifosine)

[0076] Apatinib;

[0076] Braf inhibitors (preferably vemurafenib or dabrafenib);

[0076] MEK inhibitor (better than trametinib);

[0076] CDK inhibitor;

[0076] Hsp90 inhibitor;

[0076] Salinomycin;

[0076] VAL-083 (Dehydrated Euonymus alcohol);

[0076] Vintafolide;

[0076] Serine / threonine kinase inhibitors (preferably sirolimus, everolimus, vemurafenib, trametinib, or dabrafenib); and

[0076] Monoclonal antibody (preferably anti-VEGF mAb; rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab or bevacizumab)).

[0077] As understood in this context - for any of the preferred enumerated examples of compound (B2), preferably compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0078] In other embodiments, compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0078] Proteasome inhibitors (preferably carfilzomib, isazomib, or bortezomib);

[0078] PARP inhibitors (preferably enoxaparin or olaparib); and

[0078] Monoclonal antibody (preferred anti-VEGF mAb, rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab or bevacizumab).

[0079] In other embodiments, compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0079] Bortezomib;

[0079] Olapani; and

[0079] Bevacizumab.

[0080] Preferably, the second-state compound (B2) includes two or more different targeted therapeutic agents (especially when compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812)).

[0081] As will be understood in this context by those skilled in the art, compound (B2) may be used in combination with other cancer treatment-related agents / compounds, such as one or more chemotherapeutic agent compounds.

[0082] Particularly preferred, compound (A) is ABTL0812 and compound (B2) is bortezomib - specifically, the cancer is multiple myeloma (see Example 2.1 for an example of this preferred embodiment).

[0083] Bortezomib is a proteasome inhibitor, and different proteasome inhibitors can be said to treat cancer based on similar mechanisms - therefore it is reasonable to believe that the positive experimental data on bortezomib discussed in this article could also be obtained by using other proteasome inhibitors besides bortezomib (such as carfilzomib or isazomib).

[0084] Particularly preferred, compound (A) is ABTL0812 and compound (B2) is olaparib - specifically, the cancer is endometrial cancer (see Example 5.1 for an example of this preferred embodiment).

[0085] Olaparib is a PARP inhibitor, and different PARP inhibitors can be said to treat cancer based on similar mechanisms - therefore it is reasonable to believe that the positive experimental data of olaparib discussed in this article could also be obtained by using other PARP inhibitors (such as enoparib) besides olaparib.

[0086] Particularly preferred, compound (A) is ABTL0812 and compound (B2) is bevacizumab - specifically, the cancer is endometrial cancer (see Example 5.2 for one example of this preferred embodiment).

[0087] Bevacizumab works by inhibiting vascular endothelial growth factor (VEGF) to slow the growth of new blood vessels - and can be regarded as an example of anti-VEGF mAb.

[0088] Therefore, bevacizumab can be regarded as an example of anti-VEGF mAb, and different anti-VEGF mAbs can be said to treat cancer based on similar mechanisms - so it is reasonable to believe that the positive experimental data of bevacizumab discussed in this article make it reasonable that similar positive results can also be obtained by using other anti-VEGF mAbs besides bevacizumab.

[0089] Preferably, the pharmaceutical combination discussed herein is wherein compound (A) is ABTL0812 and wherein:

[0089] - Compound (B2) is bortezomib and the cancer is multiple myeloma;

[0089] - Compound (B2) is olaparib and the cancer is endometrial cancer; or

[0089] - Compound (B2) is bevacizumab and the cancer is endometrial cancer.

[0090] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0091] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0092] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0092] Immunotherapy Agents - Third-State Compounds (B3)

[0093] As known in the art, checkpoint inhibitor therapy is a form of cancer immunotherapy. This therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can weaken the immune response to immune stimuli. Checkpoint inhibitors are molecules that can block immune checkpoint proteins. Therefore, checkpoint inhibitors enhance the immune response that promotes the elimination of cancer cells.

[0094] Currently approved checkpoint inhibitors are usually antibodies that target molecules CTLA4, PD-1 and PD-L1. These checkpoint inhibitors can be called anti-PD1, anti-PDL1 and anti-CTLA4 checkpoint inhibitors.

[0095] The conclusion of work example 6.1 is as follows:

[0095] "These results indicate that, in addition to its anti-cancer effects on tumor cells, ABTL0812 also stimulates the immune system to achieve a pro-inflammatory phenotype, thereby altering the tumor microenvironment and promoting the recruitment of other immune cells such as cytotoxic T lymphocytes. This transforms 'cold' tumors that induce immune system suppression into 'hot' and immunogenic tumors, highlighting the potential combination of ABTL0812, which enhances anti-cancer efficacy by promoting a pro-inflammatory and anti-tumor microenvironment, with specific immune checkpoint inhibitors."

[0096] The conclusion of work example 7 is as follows:

[0096] "...ABTL0812 demonstrates in vivo immunomodulatory effects by inducing the infiltration of T lymphocytes within tumor lesions, which in turn indicates the presence of a pro-inflammatory anti-tumor microenvironment conducive to the infiltration of immune cells to kill cancer cells...highlighting its potential combination with specific immune checkpoint inhibitors to enhance anticancer efficacy."

[0097] Therefore, the experimental data in this paper (see Examples 6-8) provide evidence that ABTL0812 itself has a positive immunomodulatory effect associated with immunotherapy (such as immunotherapy for cancer) - especially a positive immunomodulatory effect associated with the use of immune checkpoint inhibitors, which seems reasonable.

[0098] Tumors can manipulate the PD-1 / PD-L1 immune checkpoint pathway to shut down T cells that target cancer. Therefore, in some embodiments, compound (B3) is a checkpoint inhibitor targeting the PD-1 / PD-L1 pathway, also known as anti-PD-1 or anti-PD-L1, which enables T cells to eliminate cancer cells.

[0099] CTLA-4 is another pathway that can be targeted by checkpoint inhibitors capable of blocking the CTLA-4 receptor. In some embodiments, compound (B3) is a checkpoint inhibitor targeting CTLA-4.

[0100] In some embodiments, checkpoint inhibitors may be antibody-based agents or non-antibody-based agents (e.g., small molecules or peptides).

[0101] In a specific embodiment, the checkpoint inhibitor is a checkpoint inhibitor antibody. Examples of checkpoint inhibitors targeting the PD-1 / PDL-1 pathway include atezolizumab, avelumab, cemiplimab, durvalumab, nivolumab, and pembrolizumab. Ipilimumab is an example of a checkpoint inhibitor targeting the CTLA-4 pathway.

[0102] In another embodiment, the checkpoint inhibitor is a non-antibody-based agent (e.g., a small molecule or peptide). Examples of small molecule checkpoint inhibitors are peptide-based immunomodulators. Specifically, certain macrocyclic peptides have been shown to inhibit PD-1 and PDL-1. Furthermore, hydrolysis-resistant D-peptides have been shown to antagonize PD-L1. Other examples of immunomodulatory small molecules characterized as checkpoint inhibitors are sulfamethoxypyrimidine and sulfamethadiazole derivatives (sulfadiazine), biaryl-derived compounds, and non-peptide molecules converted into peptide-like or amino acid-inspired small molecules.

[0103] In other embodiments, checkpoint inhibitors may target the VISTA and CD47 / SIRPα signaling pathways, which play a role in tumor immune evasion and cancer progression. Examples of non-antibody peptides targeting these pathways are small immunomodulatory molecules with antitumor effects.

[0104] Preferably, compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0104] Checkpoint inhibitor antibody (preferably anti-PD1, anti-PDL1 or anti-CTLA4 checkpoint inhibitor antibody).

[0105] As understood in this context - for any of the preferred enumerated examples of compound (B3), preferably compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0106] Examples of better anti-PD1 checkpoint inhibitor antibodies are nivolumab, pembrolizumab or spartalizumab.

[0107] Positive results were obtained in the working examples herein using an anti-PD1 checkpoint inhibitor antibody that can be considered a counterpart to pembrolizumab. In short, pembrolizumab is intended for human use and is used in a modified form in the working examples herein, optimized for use in the mouse model employed in the working examples.

[0108] Therefore, in a preferred embodiment, the antibody is pembrolizumab, an anti-PD1 checkpoint inhibitor.

[0109] Examples of preferred anti-PDL1 checkpoint inhibitor antibodies are atezolizumab, avelumab, or durvalumab.

[0110] An example of a better anti-CTLA4 checkpoint inhibitor antibody is ipilimumab.

[0111] Example 8 in this document demonstrates positive results related to the use of anti-PD1 checkpoint inhibitor antibodies - therefore, the preferred embodiment relates to the fact that compound (B3) is an anti-PD1 checkpoint inhibitor antibody (preferably nivolumab, pembrolizumab or spartazumab) - specifically, compound (A) is ABTL0812.

[0112] Preferably, the third-state compound (B3) includes two or more different checkpoint inhibitor antibodies (especially when compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812)).

[0113] As will be understood in this context by those skilled in the art, compound (B3) may be used in combination with other cancer treatment-related agents / compounds, such as one or more chemotherapeutic agent compounds.

[0114] Particularly preferred, compound (A) is ABTL0812 and compound (B3) is an anti-PD1 checkpoint inhibitor antibody (preferably pembrolizumab) - specifically, the cancer is lung cancer (see Example 8.1 for an example of this preferred embodiment).

[0115] Particularly preferred, compound (A) is ABTL0812 and compound (B3) is an anti-PD1 checkpoint inhibitor antibody (preferably pembrolizumab), both of which are further combined with at least one compound (B1) (e.g., paclitaxel and carboplatin) and administered to - specifically, the cancer being lung cancer (for an example of this preferred embodiment, see Examples 8.2 and 8.3).

[0116] Preferably, the pharmaceutical combination discussed herein is wherein compound (A) is ABTL0812 and wherein:

[0116] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody (preferably pembrolizumab) and the cancer is lung cancer; or

[0116] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody (preferably pembrolizumab), which is administered in combination with at least one compound (B1), particularly paclitaxel and carboplatin, and the cancer is lung cancer.

[0117] The conclusion of work example 6.7 is as follows (emphasized):

[0117] "ABTL0812 can induce ICD (immunogenic cell death) in tumors, making these tumors more immunogenic and targetable to the immune system, helping to transform 'cold' tumors that induce immune system suppression into 'hot' and immunogenic tumors..."

[0118] The conclusion of work example 6.9 is as follows (emphasized):

[0118] "ABTL0812 promotes the secretion of pro-inflammatory factors and inhibits the release of immunosuppressive factors in cancer cells. Combined with the enhancement of the M1 macrophage phenotype and the inhibition of the M2 phenotype, these data suggest that ABTL0812 can promote a pro-inflammatory anti-tumor environment to exert its effects on immune cells, thereby highlighting its potential combination with other immunotherapies to increase efficacy against tumors, especially in highly immunosuppressive tumors such as pancreatic cancer."

[0119] Therefore, the experimental data presented herein (see Examples 6-8) provide evidence that ABTL0812 itself has a positive immunomodulatory effect to enhance the anticancer efficacy of treatments that interact with immune system responses and their regulation (e.g., intercytokines) other than checkpoint inhibitors. Thus, it will be apparent to those skilled in this art that ABTL0812 can enhance the anticancer effects of immunomodulators.

[0120] Therefore, in some embodiments, compound (B3) is an anticancer immunomodulatory agent compound. The term "anticancer immunomodulatory agent compound" (also referred to herein) is used to refer to an immunomodulatory agent that is capable of targeting and modulating the pathways by which the immune system can attack and eliminate cancer cells. Immunomodulators are a known group of molecules within cancer immunotherapy agents and may include, for example, checkpoint inhibitors, cytokines, agonists, and adjuvants. Regulation of the immune system includes mechanisms that stimulate or suppress the immune system.

[0121] In some embodiments, compound (B3) is an immunotherapeutic compound, an anticancer immunomodulatory compound, and a cytokine. Cytokines are messenger molecules that regulate the maturation, growth, and responsiveness of immune cells. Examples of immunomodulatory cytokines include cytokines targeting the IL-2 / IL-2R pathway and cytokines targeting the IFNAR1 and / or IFNAR2 pathway. Aldesleukin (Proleukin®) is an example of an immunomodulatory cytokine targeting the IL-2 / IL-2R pathway. Examples of immunomodulatory cytokines targeting the IFNAR1 and / or IFNAR2 pathway are interferon α-2a, interferon α-2b (Intron A®), and polyethylene glycol interferon α-2b (Sylatron® / PEG-Intron®). Other examples of cytokines include granulosa macrophage community-stimulating factor (GM-CSF) for the treatment of neuroblastoma.

[0122] In some embodiments, compound (B3) is an immunotherapeutic compound, an anticancer immunomodulatory compound, and a agonist. An agonist is a molecule capable of activating pathways that promote adaptive immune responses. For example, an immunomodulatory agonist can enhance the activation of killer T cells or stimulate the activity of innate immune cells (e.g., dendritic cells).

[0123] In some embodiments, compound (B3) is an immunotherapeutic compound, an anticancer immunomodulatory compound, and an adjuvant. An adjuvant is a molecule capable of activating pathways involved in the innate immune system, stimulating a systemic immune response, and ultimately promoting an adaptive immune response. Examples of immunomodulatory adjuvants are adjuvants targeting TLR-like receptors (e.g., TLR7 or TLR3). Imiquimod and polyICLC (Hiltonol®) are examples of adjuvants targeting TLR-like receptors to treat cancer.

[0124] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0125] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0126] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0126] Radiation Therapy Treatment - Fourth State

[0127] Preferably, radiation therapy is performed with a radiation dose of 2 to 200 Gy, such as 5 to 100 Gy, or more preferably 15 to 85 Gy.

[0128] For lymphoma, preferably, radiation therapy is performed with a radiation dose of 15 to 45 Gy.

[0129] For solid tumors, radiotherapy is preferably performed with a radiation dose of 55 to 85 Gy.

[0130] Preferably, the radiation dose is administered after the administration of compound (A) (preferably ABTL0812) - such as at least one day after the initial administration of compound (A) (preferably ABTL0812).

[0131] As understood in this context - in any embodiment of radiotherapy treatment as described herein, preferably, compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0132] As understood by those skilled in this art in this context, radiation therapy can be combined with the use of related cancer treatment agents / compounds, such as one or more chemotherapeutic agents.

[0133] Regarding the use of fourth-state type radiation therapy - preferably, the cancer is glioblastoma.

[0134] Regarding the use of fourth-state radiotherapy - particularly preferably, compound (A) is ABTL0812 - specifically, the cancer is glioblastoma carcinoma. (For an example of this preferred embodiment, see Example 4 in this document).

[0135] Regarding the use of fourth-state type radiotherapy - preferably, compound (A) is ABTL0812, which is administered in combination with at least one compound (B1) (a chemotherapeutic compound). Specifically, compound (B1) is temozolomide or topotecan. Specifically, the cancer is glioblastoma.

[0136] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0137] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0138] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0138] Optimal Chemotherapy Agent - Fifth State

[0139] Regarding the fifth state as described above - preferably, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0139] Temozolomide;

[0139] Topotecan;

[0139] Fluorouracil;

[0139] Oxaliplatin; and

[0139] Formyltetrahydrofolate.

[0140] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0140] Irinotecan;

[0140] Fluorouracil;

[0140] Oxaliplatin; and

[0140] Tetrahydrofolate.

[0141] In other embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0141] Carboplatin; and

[0141] Paclitaxel.

[0142] As understood in this context - for any of the preferred enumerated examples of compound (B1), preferably compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0143] Preferably, the fifth-state compound (B1) comprises two or more different chemotherapeutic agents (especially when compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812)) - such as preferably, wherein the first-state compound (B1) comprises:

[0143] Irinotecan, folate, oxaliplatin, and fluorouracil; or

[0143] Irinotecan, Topotecan and Cyclophosphamide.

[0144] As will be understood in this context by those skilled in the art, the compound (B1) may be used in combination with other cancer treatment-related agents / compounds, such as one or more targeted therapeutic agents.

[0145] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is temozolomide - specifically, wherein the cancer is neuroblastoma (see Example 1.1 for an example of this preferred embodiment).

[0146] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is topotecan—specifically, the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.2 herein). In other embodiments, compound (A) is ABTL0812 and compound (B1) is topotecan—specifically, the cancer is pancreatic cancer or glioblastoma.

[0147] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is irinotecan—specifically, the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.3 herein). In other embodiments, compound (A) is ABTL0812 and compound (B1) is irinotecan—specifically, the cancer is pancreatic cancer or glioblastoma.

[0148] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is cyclophosphamide - specifically, wherein the cancer is neuroblastoma. (For an example of this preferred embodiment, see Example 1.4 herein).

[0149] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is irinotecan, leucovorin, oxaliplatin and fluorouracil - specifically, wherein the cancer is pancreatic cancer. (For an example of this preferred embodiment, see Example 3.1 herein).

[0150] Particularly preferred, compound (A) is ABTL0812 and compound (B1) is cranberry - specifically, the cancer is endometrial cell carcinoma. (For an example of this preferred embodiment, see Example 3.2 herein).

[0151] Particularly preferred is that compound (A) is ABTL0812 and compound (B1) is temozolomide – specifically, the cancer is glioblastoma. (For an example of this preferred embodiment, see Example 3.3 herein).

[0152] Preferably, the pharmaceutical combination discussed herein is wherein compound (A) is ABTL0812 and wherein:

[0152] - Compound (B1) is temozolomide and the cancer is neuroblastoma;

[0152] - Compound (B1) is topotecan and the cancer is neuroblastoma;

[0152] - Compound (B1) is irinotecan and the cancer is neuroblastoma;

[0152] - Compound (B1) is cyclophosphamide and the cancer is neuroblastoma;

[0152] - Compound (B1) is irinotecan, leucovorin, oxaliplatin and fluorouracil, and the cancer is pancreatic cancer;

[0152] - Compound (B1) is a cranberry and the cancer is endometrial cancer; or

[0152] - The compound (B1) is temozolomide and the cancer is glioblastoma.

[0153] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0154] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0155] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0155] Other specific combinations related to all states in this document

[0156] In the working examples described herein, positive results were obtained by combining compounds (A), (B1) and (B3) (i.e., by a triple combination).

[0157] Therefore, the present invention also relates to a pharmaceutical combination comprising:

[0157] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0157] (i)a can be any integer value between 0 and 7.

[0157] (ii) b can be any integer value between 2 and 7.

[0157] (iii) c can be any integer value between 0 and 7.

[0157] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0157] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0157] (B1): Chemotherapy compound; and

[0157] (B3): Immunotherapy compound,

[0157] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients.

[0158] In a particular embodiment: (i)a can be any integer value between 5 and 7, (ii)b can be any integer value between 2 and 4, and (iii)c can be any integer value between 1 and 5. In another embodiment, R1 is H and R2 is OH.

[0159] In a particular embodiment, compound (A) is at least one compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof:

[0159] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0159] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0159] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0159] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0159] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0159] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0160] Specifically, compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812) or a pharmaceutically acceptable salt thereof. More specifically, compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

[0161] In some embodiments, the cancer is at least one cancer selected from the group consisting of:

[0161] Lung cancer;

[0161] Non-small cell lung cancer;

[0161] Squamous cell carcinoma;

[0161] Adenocarcinoma;

[0161] Endometrial cancer;

[0161] Serous carcinoma of the endometrium;

[0161] Endometrioid carcinoma;

[0161] Pancreatic cancer;

[0161] Glioblastoma;

[0161] Drug-resistant recurrent breast cancer;

[0161] Head and neck cancer;

[0161] Multiple myeloma;

[0161] Neuroblastoma and

[0161] Bile duct cancer.

[0162] In some embodiments, compound (B3) is an immunotherapeutic compound, which is an anticancer immunomodulatory compound. In specific embodiments, compound (B3) is a checkpoint inhibitor. Specific embodiments and examples are described in the section "Immunotherapeutic Agents - Third-State Compound (B3)" of this specification.

[0163] In some embodiments, compound (B3) is at least one immunotherapeutic compound selected from the group consisting of: checkpoint inhibitors, preferably checkpoint inhibitor antibodies. Preferably, the checkpoint inhibitor antibody is an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0164] In some embodiments, compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0164] -An anti-PD1 antibody, preferably, wherein the anti-PD1 antibody is nivolumab, pembrolizumab or spartazumab;

[0164] -An anti-PDL1 antibody, preferably, wherein the anti-PDL1 antibody is atezolizumab, avelumab, or durvalumab;

[0164] -Anti-CTLA4 antibody, preferably ipilimumab.

[0165] Specifically, compound (B3) is an anti-PD1 antibody, preferably, wherein the anti-PD1 antibody is pembrolizumab.

[0166] In some embodiments:

[0166] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, superior to pembrolizumab, and the cancer is lung cancer; or

[0166] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, preferably pembrolizumab, administered in combination with at least one compound (B1), preferably paclitaxel and carboplatin, and for the cancer of lung cancer.

[0167] In some embodiments, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0167] Temozolomide;

[0167] Topotecan;

[0167] Irinotecan;

[0167] Cyclophosphamide;

[0167] Fluorouracil;

[0167] Cisplatin;

[0167] Carboplatin;

[0167] Oxaliplatin;

[0167] Tetrahydrofolate;

[0167] Cranberries;

[0167] Bleomycin;

[0167] Capecitabine;

[0167] Mitomycin B;

[0167] Paclitaxel;

[0167] Albumin-bound paclitaxel;

[0167] Dorcetaxel;

[0167] Gesitabine;

[0167] Methotrexate;

[0167] Pepmetrozine;

[0167] 5-Fluorouracil;

[0167] Cytarabine;

[0167] Mercaptopurine;

[0167] Glucophosphamidon;

[0167] Isapirone;

[0167] Nimustine;

[0167] Carmustine;

[0167] Lomustine;

[0167] Mitoxantrone;

[0167] Etoposide;

[0167] Changchun Xinjian;

[0167] Vincristine; and

[0167] Tamoxifen.

[0168] In a particular embodiment, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0168] Temozolomide;

[0168] Topotecan;

[0168] Irinotecan;

[0168] Cyclophosphamide;

[0168] Fluorouracil;

[0168] Oxaliplatin;

[0168] Tetrahydrofolate; and

[0168] Cranberries.

[0169] Specifically, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0169] Temozolomide;

[0169] Topotecan;

[0169] Fluorouracil;

[0169] Oxaliplatin; and

[0169] Formyltetrahydrofolate.

[0170] In another embodiment, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0170] Irinotecan;

[0170] Fluorouracil;

[0170] Oxaliplatin; and

[0170] Formyltetrahydrofolate.

[0171] In another embodiment, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0171] Carboplatin; and

[0171] Paclitaxel.

[0172] In another embodiment, compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0172] Temozolomide;

[0172] Topotecan;

[0172] Irinotecan;

[0172] Cyclophosphamide;

[0172] Fluorouracil;

[0172] Oxaliplatin;

[0172] Tetrahydrofolate;

[0172] Small cranberry;

[0172] Carboplatin; and

[0172] Paclitaxel.

[0173] Specifically, compound (B1) is paclitaxel and carboplatin.

[0174] In another embodiment, compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil.

[0175] In a specific embodiment, compound (A) is ABTL0812, compound (B1) is paclitaxel and carboplatin, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody.

[0176] In one embodiment, compound (A) is ABTL0812, compound (B1) is irinotecan, leucovorin, oxaliplatin, and fluorouracil, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody. Specifically, compound (B3) is an anti-PD1 checkpoint inhibitor antibody selected from nivolumab, pembrolizumab, and spartazumab, preferably pembrolizumab.

[0177] Particularly preferred, compound (A) is ABTL0812, compound (B1) comprises paclitaxel / carboplatin, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody. In a particular embodiment, the cancer is lung cancer (see Examples 8.2 and 8.3 for an example of one of these preferred embodiments).

[0178] Particularly preferred, compound (A) is ABTL0812, compound (B1) comprises irinotecan, leucovorin, oxaliplatin, and fluorouracil, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody. In a particular embodiment, the cancer is pancreatic cancer (see Example 8.5 for one example of this preferred embodiment).

[0179] Depending on the type of compound (B1), it may be administered, for example, intravenously (e.g., in the case of antibodies) or orally (e.g., in the case of small molecules). Specifically, compound (3) is administered intravenously via an infusion.

[0180] Specifically, compound (A) is administered orally. In a particular embodiment, the dosage of compound (A) is from 200 mg to 7000 mg per day, more preferably from 1500 mg to 5000 mg per day, even more preferably from 3000 mg to 4700 mg per day, and most preferably from 3500 mg to 4300 mg per day.

[0180] Cancer - Related to all states in this article

[0181] Regarding any of the first to fifth states in this article - preferably, cancer is at least one cancer selected from the following groups:

[0181] Lung cancer;

[0181] Non-small cell lung cancer;

[0181] Squamous cell carcinoma;

[0181] Adenocarcinoma;

[0181] Endometrial cancer;

[0181] Serous carcinoma of the endometrium;

[0181] Endometrioid carcinoma;

[0181] Pancreatic cancer;

[0181] Glioblastoma;

[0181] Drug-resistant recurrent breast cancer;

[0181] Head and neck cancer;

[0181] Multiple myeloma;

[0181] Neuroblastoma and

[0181] Bile duct cancer.

[0182] More preferably, the cancer is at least one cancer selected from the following groups:

[0182] Non-small cell lung cancer;

[0182] Squamous cell carcinoma;

[0182] Endometrial cancer;

[0182] Pancreatic cancer;

[0182] Glioblastoma;

[0182] Breast cancer;

[0182] Multiple myeloma;

[0182] Neuroblastoma; and

[0182] Bile duct carcinoma.

[0183] More specifically, cancer is at least one cancer selected from the following groups:

[0183] Lung cancer;

[0183] Endometrial cancer;

[0183] Pancreatic cancer;

[0183] Glioblastoma;

[0183] Breast cancer;

[0183] Neuroblastoma; and

[0183] Bile duct cancer.

[0184] In a specific embodiment, the cancer is a solid tumor. A "solid tumor" or solid cancer is a growth (new cell growth) or lesion (anatomical damage or physiological dysfunction) formed by the abnormal growth of body tissue cells other than blood, bone marrow, or lymphocytes. Solid tumors are composed of abnormally high-quality cells that may originate from different tissue types, such as the liver, colon, breast, or lung, and initially grow in the organ of their cell origin. However, in later stages of the disease, such cancers can spread to other organs through metastatic tumor growth.

[0185] In a particular embodiment, the cancer is a carcinoma, sarcoma, germ cell tumor, or blastoma.

[0186] In a particular embodiment, cancer is a tumor. Tumors are cancers originating from epithelial cells and account for 80% to 90% of all cancer cases because epithelial tissue is the most abundant tissue found in the body. In a particular embodiment, tumors include many of the most common cancers, particularly such as lung cancer, colorectal cancer, pancreatic cancer, laryngeal cancer, tongue cancer, prostate cancer, breast cancer, ovarian cancer, liver cancer, head and neck cancer, esophageal cancer, kidney cancer, endometrial cancer, gallbladder cancer, bladder cancer, or stomach cancer.

[0187] There are two types of cancer: adenocarcinoma and squamous cell carcinoma. Adenocarcinoma develops in epithelial cells or glands, while squamous cell carcinoma originates from squamous epithelium. Adenocarcinoma may affect mucous membranes and is initially perceived as thickened, patchy, white mucous membranes. These adenocarcinomas are rapidly spreading cancers.

[0188] In a particular embodiment, the cancer is adenocarcinoma.

[0189] In a particular embodiment, the cancer is lung cancer, endometrial cancer, or pancreatic cancer.

[0190] In a particular embodiment, the cancer is lung cancer, and more specifically, non-small cell lung cancer.

[0191] In a particular embodiment, the cancer is squamous cell carcinoma.

[0192] In a particular embodiment, the cancer is endometrial cancer, more specifically, endometrioid carcinoma or serous endometrial carcinoma.

[0193] In a particular embodiment, the cancer is pancreatic cancer. More specifically, the pancreatic cancer is exocrine pancreatic cancer (the most common type) or neuroendocrine pancreatic cancer.

[0194] In a particular embodiment, the cancer is cholangiocarcinoma.

[0195] In a particular embodiment, the cancer is breast cancer, and more specifically, drug-resistant recurrent breast cancer.

[0196] In a particular embodiment, the cancer is head and neck cancer.

[0197] In a specific embodiment, the cancer is a sarcoma. A sarcoma is a cancer caused by connective tissue including muscle, bone, cartilage, and fat. In a specific embodiment, the sarcoma is, for example, osteosarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or angioendothelioma (blood vessels), liposarcoma (fat or adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primordial embryonic connective tissue), or mesenchymal tumor or mixed mesodermal tumor (mixed connective tissue type).

[0198] In a specific embodiment, the cancer is a brain cancer. Specifically, the brain cancer is a glioma. More specifically, the glioma is a glioblastoma.

[0199] In a specific embodiment, the cancer is a germ cell tumor. Germ cell tumors refer to germ cell tumors that originate from pluripotent cells and most commonly occur in the testes or ovaries (seminomas and malignant embryonal tumors, respectively).

[0200] In a specific embodiment, the cancer is a blastoma. A blastoma is a cancer originating from immature precursor cells or embryonic tissue. Blastomas are more common in children than in the elderly. In a specific embodiment, the blastoma is, for example, hepatoblastoma, neuroblastoma, ductoblastoma, nephroblastoma, pancreatoblastoma, pleuroblastoma, retinoblastoma, or glioblastoma multiforme. In a specific embodiment, the cancer is a neuroblastoma.

[0201] The cancers that can be treated by the medical combination of the present invention are solid tumors, such as lung cancer, colorectal cancer, pancreatic cancer, laryngeal cancer, tongue cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, head and neck cancer, esophageal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, malignant embryonal carcinoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, epithelial carcinoma, glioma, astrocytoma, neuroblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0202] In a particular embodiment, the cancer is metastatic or advanced cancer.

[0203] In a specific embodiment, the cancer is a hematologic malignancy. The term "hematologic malignancy" refers to a class of cancers affecting the blood, bone marrow, and lymph nodes, including lymphoma, myeloma, and leukemia. Historically, scientists and physicians have classified these diseases based on their location in the body, the appearance of the affected cells under a microscope, and the natural progression of the disease. In leukemia, cancer cells are found circulating in the blood and bone marrow, while in lymphoma, cells tend to aggregate in lymphatic tissue and form masses or tumors. Myeloma is a tumor of the bone marrow and involves a specific subset of white blood cells that produce unique proteins.

[0204] In a particular embodiment, the hematologic malignancy is leukemia, lymphoma or myeloma.

[0205] In a specific embodiment, the hematologic malignancy is leukemia. More specifically, leukemia includes, for example, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and acute monocytic leukemia (AMoL).

[0206] In a particular embodiment, the hematologic malignancy is lymphoma. More specifically, the lymphoma is Hodgkin's lymphoma (HL) or non-Hodgkin's lymphoma (NHL).

[0207] In a specific embodiment, the hematologic malignancy is myeloma, also known as multiple myeloma (i.e., cancer of plasma cells (usually white blood cells) that typically produce antibodies).

[0208] In another specific embodiment, the hematologic malignancy is in the precancerous stage. As used herein, the term "precancerous stage" refers to a hyperproliferative condition or malignant prodromal symptom that can develop into cancer.

[0208] Administration of compound (A) and / or compound (B) - relevant to all states and conditions herein

[0209] As discussed above and with respect to any of the first to fifth states discussed herein, combination therapy is not necessary if the two compounds (A) and (B) are administered simultaneously, for example, as a single composition, or sequentially, for example, as two separate compositions. It is important that, when the second compound / drug is administered, the effective amount of the first compound / drug is already in the patient's body and / or has already exerted its effect in the patient's body.

[0210] When compound (B) is referred to in this document, it should be understood as referring to any one of compound (B1), compound (B2) and / or compound (B3).

[0211] Preferably, the pharmaceutical combination discussed herein may be a single composition comprising both compound (A) and compound (B).

[0212] Compound (A) (especially ABTL0812) is preferred to be administered orally.

[0213] The dosage of compound (A) (especially ABTL0812) is preferably 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0214] Preferably, the daily dose of compound (A) (especially ABTL0812) is given three times a day, with the daily dose being 1200-1400 mg three times a day.

[0215] This article has successfully conducted relevant human clinical studies on ABTL0812, in which the daily dose was 3900mg - administered in the form of 3 doses of 1300mg.

[0216] Therefore, the compound (A) is preferably ABTL0812 and the dosage is 3800 mg to 4000 mg (preferably 3900 mg) - especially when it is administered in three doses of 1300 mg.

[0217] Regarding compound (B), the preferred administration route will generally depend on the compound (B) of interest.

[0218] Those skilled in this technique can routinely determine the best administration route for a particular compound (B) of interest.

[0219] The preferred administration route of the preferred compound (B) is briefly described below:

[0219] Temozolomide; -Preferredly administered via oral capsules or tablets;

[0219] Topotecan; -Preferred to be administered intravenously via infusion;

[0219] Irinotecan; -Preferred to be administered intravenously via infusion;

[0219] Cyclophosphamide; -Preferred to be administered intravenously via infusion;

[0219] Fluorouracil; -Preferred to be administered intravenously via infusion;

[0219] Oxaliplatin; - Preferred to be administered intravenously via infusion;

[0219] Formyltetrahydrofolate; -Preferred to be administered intravenously via infusion;

[0219] Cranberries; -Preferred to be administered intravenously via infusion;

[0219] Bortezomib; -Preferred to be administered intravenously via infusion;

[0219] Olaparib; - Preferably administered orally as a capsule or tablet;

[0219] Bevacizumab; - Preferred intravenous administration via infusion; Anti-PD1 checkpoint inhibitor antibody - Preferred intravenous administration via infusion.

[0219] The present invention in the form of a so-called technical solution / embodiment

[0220] This "technical solution format" is divided into six sub-parts, each specifically addressing the first to fifth states and their embodiments as discussed herein.

[0220] First-state sample and related examples - (B1): Chemotherapy compound - second-line therapy for cancer treatment

[0221] 1. A pharmaceutical combination comprising:

[0221] (A): A polyunsaturated fatty acid compound of formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0221] (i)a can be any integer value between 0 and 7

[0221] (ii) b can be any integer value between 2 and 7.

[0221] (iii)c can be any integer value between 0 and 7.

[0221] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0221] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0221] and

[0221] (B1): Chemotherapy compound,

[0221] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is a second-line therapy for cancer.

[0222] 2. As in technical solution 1, the pharmaceutical combination, wherein

[0222] (i)a can be any integer value between 5 and 7

[0222] (ii)b can be any integer value between 2 and 4, and

[0222] (iii)c can be any integer value between 1 and 5.

[0223] 3. The pharmaceutical combination as described in any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0224] 4. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0224] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0224] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0224] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0224] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0224] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0224] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0225] 5. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0226] 6. The pharmaceutical combination of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0227] 7. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0227] Lung cancer;

[0227] Non-small cell lung cancer;

[0227] Squamous cell carcinoma;

[0227] Adenocarcinoma;

[0227] Endometrial cancer;

[0227] Serous carcinoma of the endometrium;

[0227] Endometrioid carcinoma;

[0227] Pancreatic cancer;

[0227] Glioblastoma;

[0227] Drug-resistant recurrent breast cancer;

[0227] Head and neck cancer;

[0227] Multiple myeloma;

[0227] Neuroblastoma and

[0227] Bile duct cancer.

[0228] 8. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0228] Temozolomide;

[0228] Topotecan;

[0228] Irinotecan;

[0228] Cyclophosphamide;

[0228] Fluorouracil;

[0228] Cisplatin;

[0228] Carboplatin;

[0228] Oxaliplatin;

[0228] Tetrahydrofolate;

[0228] Small cranberry;

[0228] Bleomycin;

[0228] Capecitabine;

[0228] Mitomycin B;

[0228] Paclitaxel;

[0228] Albumin-bound paclitaxel;

[0228] Dorcetaxel;

[0228] Gesitabine;

[0228] Methotrexate;

[0228] Pepmetrozine;

[0228] 5-Fluorouracil;

[0228] Cytarabine;

[0228] Mercaptopurine;

[0228] Glucophosphamidon;

[0228] Isapirone;

[0228] Nimustine;

[0228] Carmustine;

[0228] Lomustine;

[0228] Mitoxantrone;

[0228] Etoposide;

[0228] Changchun New Alkali;

[0228] Vincristine; and

[0228] Tamoxifen.

[0229] 9. The pharmaceutical combination of technical solution 8, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0229] Temozolomide;

[0229] Topotecan;

[0229] Irinotecan;

[0229] Cyclophosphamide;

[0229] Fluorouracil;

[0229] Oxaliplatin;

[0229] Tetrahydrofolate; and

[0229] Cranberries.

[0230] 10. The pharmaceutical combination of technical solution 9, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0230] Temozolomide;

[0230] Topotecan;

[0230] Fluorouracil;

[0230] Oxaliplatin; and

[0230] Formyltetrahydrofolate.

[0231] 11. The pharmaceutical combination of any one of technical solutions 8 to 10, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0232] 12. The pharmaceutical combination as described in technical solution 11, wherein...

[0232] - Compound (B1) is temozolomide and the cancer is neuroblastoma;

[0232] - Compound (B1) is topotecan and the cancer is neuroblastoma;

[0232] - Compound (B1) is irinotecan and the cancer is neuroblastoma.

[0232] - Compound (B1) is cyclophosphamide and the cancer is neuroblastoma.

[0232] - Compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil, and the cancer is pancreatic cancer;

[0232] - Compound (B1) is a cranberry and the cancer is endometrial cancer; or

[0232] - The compound (B1) is temozolomide and the cancer is glioblastoma.

[0233] 13. The pharmaceutical combination of any of the foregoing technical solutions, wherein the pharmaceutical combination is a single composition comprising both compound (A) and compound (B1).

[0234] 14. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is administered orally.

[0235] 15. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the dosage of compound (A) is 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0236] 16. The pharmaceutical combination of any one of technical solutions 14 to 15, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0237] 17. The pharmaceutical combination as described in technical solution 16, wherein...

[0237] Compound (B1) is temozolomide and is administered orally in capsule or tablet form;

[0237] Compound (B1) is topotecan and is administered intravenously via an infusion solution;

[0237] Compound (B1) is irinotecan and is administered intravenously via an infusion solution;

[0237] Compound (B1) is cyclophosphamide and is administered intravenously via an infusion solution;

[0237] Compound (B1) is fluorouracil and is administered intravenously via an infusion solution;

[0237] Compound (B1) is oxaliplatin and is administered intravenously via an infusion solution;

[0237] Compound (B1) is formaldehyde tetrahydrofolate and is administered intravenously via an infusion solution; or

[0237] Compound (B1) is cranberry and is administered intravenously via an infusion.

[0237] Second State and Related Examples - (B2): Targeted Therapeutic Compounds - Targeted Therapy for Cancer

[0238] 1. A pharmaceutical combination comprising:

[0238] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0238] (i)a can be any integer value between 0 and 7

[0238] (ii) b can be any integer value between 2 and 7.

[0238] (iii)c can be any integer value between 0 and 7.

[0238] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0238] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0238] and

[0238] (B2): Targeted therapeutic compound,

[0238] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is a targeted therapy for cancer.

[0239] 2. As in technical solution 1, the pharmaceutical combination, wherein

[0239] (i)a can be any integer value between 5 and 7.

[0239] (ii) b can be any integer value between 2 and 4, and

[0239] (iii)c can be any integer value between 1 and 5.

[0240] 3. The pharmaceutical combination as described in any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0241] 4. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0241] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0241] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0241] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0241] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0241] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0241] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0242] 5. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0243] 6. The pharmaceutical combination of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0244] 7. The pharmaceutical combination of any of the foregoing technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0244] Lung cancer;

[0244] Non-small cell lung cancer;

[0244] Squamous cell carcinoma;

[0244] Adenocarcinoma;

[0244] Endometrial cancer;

[0244] Serous carcinoma of the endometrium;

[0244] Endometrioid carcinoma;

[0244] Pancreatic cancer;

[0244] Glioblastoma;

[0244] Drug-resistant recurrent breast cancer;

[0244] Head and neck cancer;

[0244] Multiple myeloma;

[0244] Neuroblastoma and

[0244] Bile duct cancer.

[0245] 8. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0245] Imatinib;

[0245] Gefitinib;

[0245] Erlotinib;

[0245] Sorafenib;

[0245] Sunitinib;

[0245] Dasatinib;

[0245] Lapatinib;

[0245] Nitinib;

[0245] Proteasome inhibitors (preferably carfilzomib, isazomib, or bortezomib);

[0245] Tamoxifen;

[0245] Janus kinase inhibitors (better than tofacitinib);

[0245] ALK inhibitor (better than crizotinib);

[0245] Bcl-2 inhibitors (preferably omeprazole, navicolax, or gossypol);

[0245] PARP inhibitors (preferably pheniparil or olaparib);

[0245] PI3K inhibitor (better than perifol)

[0245] Apatinib;

[0245] Braf inhibitors (preferably vemurafenib or dabrafenib);

[0245] MEK inhibitor (preferably trametinib);

[0245] CDK inhibitor;

[0245] Hsp90 inhibitor;

[0245] Thalimycin;

[0245] VAL-083 (Dehydrated Euonymus alcohol);

[0245] Vitafrid;

[0245] Serine / threonine kinase inhibitors (preferably sirolimus, everolimus, vemurafenib, trametinib, or dabrafenib); and

[0245] Monoclonal antibody (preferred anti-VEGF mAb; rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab or bevacizumab).

[0246] 9. The pharmaceutical combination of technical solution 8, wherein compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0246] Proteasome inhibitors (preferably carfilzomib, isazomib, or bortezomib);

[0246] PARP inhibitors (preferably enoxaparin or olaparib); and

[0246] Monoclonal antibody (preferably anti-VEGF mAb, rituximab, trastuzumab, alemtuzumab, cetuximab, panitumumab or bevacizumab).

[0247] 10. The pharmaceutical combination of technical solution 9, wherein compound (B2) is at least one targeted therapeutic compound selected from the group consisting of:

[0247] Bortezomib;

[0247] Olapani; and

[0247] Bevacizumab.

[0248] 11. The pharmaceutical combination of any one of technical solutions 8 to 10, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0249] 12. The pharmaceutical combination as described in technical solution 11, wherein...

[0249] - Compound (B2) is bortezomib and the cancer is multiple myeloma;

[0249] - Compound (B2) is olaparib and the cancer is endometrial cancer; or

[0249] - Compound (B2) is bevacizumab and the cancer is endometrial cancer.

[0250] 13. The pharmaceutical combination of any of the foregoing technical solutions, wherein the pharmaceutical combination is a single composition comprising both compound (A) and compound (B2).

[0251] 14. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is administered orally.

[0252] 15. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the dosage of compound (A) is a daily dose of 200 mg to 7000 mg, more preferably a daily dose of 1500 mg to 5000 mg, even more preferably a daily dose of 3000 mg to 4700 mg, and most preferably a daily dose of 3500 mg to 4300 mg.

[0253] 16. The pharmaceutical combination of any one of technical solutions 14 to 15, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0254] 17. The pharmaceutical combination as described in technical solution 16, wherein...

[0254] Compound (B2) is bortezomib and is administered intravenously via an infusion solution;

[0254] Compound (B2) is olaparib and is administered orally as a capsule or tablet; or

[0254] Compound (B2) is bevacizumab and is administered intravenously via an infusion solution.

[0254] Third State and Related Examples - (B3): Immunotherapy Compounds - Immunotherapy for Cancer

[0255] 1. A pharmaceutical combination comprising:

[0255] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0255] (i)a can be any integer value between 0 and 7.

[0255] (ii) b can be any integer value between 2 and 7.

[0255] (iii)c can be any integer value between 0 and 7.

[0255] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0255] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0255] and

[0255] (B3): Immunotherapy compound,

[0255] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients, wherein the treatment is an immunotherapy for cancer.

[0256] 2. As in the pharmaceutical combination of technical solution 1, wherein

[0256] (i)a can be any integer value between 5 and 7

[0256] (ii)b can be any integer value between 2 and 4, and

[0256] (iii)c can be any integer value between 1 and 5.

[0257] 3. The pharmaceutical combination as described in any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0258] 4. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0258] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0258] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0258] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0258] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0258] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0258] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0259] 5. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0260] 6. The pharmaceutical combination of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0261] 7. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0261] Lung cancer;

[0261] Non-small cell lung cancer;

[0261] Squamous cell carcinoma;

[0261] Adenocarcinoma;

[0261] Endometrial cancer;

[0261] Serous carcinoma of the endometrium;

[0261] Endometrioid carcinoma;

[0261] Pancreatic cancer;

[0261] Glioblastoma;

[0261] Drug-resistant recurrent breast cancer;

[0261] Head and neck cancer;

[0261] Multiple myeloma;

[0261] Neuroblastoma and

[0261] Bile duct cancer.

[0262] 8. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0262] A checkpoint inhibitor antibody, preferably wherein the checkpoint inhibitor antibody is an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0263] 9. The pharmaceutical combination of technical solution 8, wherein compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0263] -An anti-PD1 antibody, preferably, wherein the anti-PD1 antibody is nivolumab, pembrolizumab or spartazumab;

[0263] -An anti-PDL1 antibody, preferably, wherein the anti-PDL1 antibody is atezolizumab, avelumab or durvalumab;

[0263] -Anti-CTLA4 antibody, preferably ipilimumab.

[0264] 10. The pharmaceutical combination of technical solution 9, wherein compound (B3) is an anti-PD1 antibody and the anti-PD1 antibody is pembrolizumab.

[0265] 11. The pharmaceutical combination of any one of technical solutions 8 to 10, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0266] 12. The pharmaceutical combination as described in technical solution 11, wherein...

[0266] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, superior to pembrolizumab, and the cancer is lung cancer; or

[0266] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, preferably pembrolizumab, administered in combination with at least one compound (B1), preferably paclitaxel and carboplatin, and for the cancer of lung cancer.

[0267] 13. The pharmaceutical combination of any of the foregoing technical solutions, wherein the pharmaceutical combination is a single composition comprising both compound (A) and compound (B3).

[0268] 14. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is administered orally.

[0269] 15. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the dosage of compound (A) is a daily dose of 200 mg to 7000 mg, more preferably a daily dose of 1500 mg to 5000 mg, even more preferably a daily dose of 3000 mg to 4700 mg, and most preferably a daily dose of 3500 mg to 4300 mg.

[0270] 16. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) (especially ABTL0812) is administered before the administration of the immunotherapy compound (B3).

[0271] 17. A pharmaceutical combination of any one of technical solutions 14 to 16, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0272] 18. The pharmaceutical combination as described in technical solution 17, wherein...

[0272] Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, preferably pembrolizumab, and is administered intravenously via an infusion solution.

[0272] Fourth-state and related embodiments - Radiotherapy treatment of cancer

[0273] 1. A pharmaceutical composition comprising:

[0273] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0273] (i)a can be any integer value between 0 and 7

[0273] (ii) b can be any integer value between 2 and 7.

[0273] (iii)c can be any integer value between 0 and 7.

[0273] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0273] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0273] This pharmaceutical composition is used for the treatment of cancer in human patients, wherein the treatment is radiotherapy for cancer.

[0274] 2. The pharmaceutical composition as described in technical solution 1, wherein...

[0274] (i)a can be any integer value between 5 and 7.

[0274] (ii)b can be any integer value between 2 and 4, and

[0274] (iii)c can be any integer value between 1 and 5.

[0275] 3. The pharmaceutical composition of any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0276] 4. The pharmaceutical composition of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0276] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0276] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0276] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0276] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0276] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0276] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0277] 5. The pharmaceutical composition of any of the foregoing technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0278] 6. The pharmaceutical composition of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0279] 7. The pharmaceutical composition of any of the foregoing technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0279] Lung cancer;

[0279] Non-small cell lung cancer;

[0279] Squamous cell carcinoma;

[0279] Adenocarcinoma;

[0279] Endometrial cancer;

[0279] Serous carcinoma of the endometrium;

[0279] Endometrioid carcinoma;

[0279] Pancreatic cancer;

[0279] Glioblastoma;

[0279] Drug-resistant recurrent breast cancer;

[0279] Head and neck cancer;

[0279] Multiple myeloma;

[0279] Neuroblastoma and

[0279] Bile duct cancer.

[0280] 8. The pharmaceutical composition of any of the foregoing technical solutions, wherein the radiotherapy treatment is performed with a radiation dose of 2 to 200 Gy, preferably 5 to 100 Gy or even more preferably 15 to 85 Gy.

[0281] 9. The pharmaceutical composition of technical solution 8, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0282] 10. The pharmaceutical composition of any of the foregoing technical solutions, wherein the radiation dose is administered after the administration of compound (A), preferably at least one day after the initial administration of compound (A).

[0283] 11. The pharmaceutical composition of technical solution 10, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812) or a pharmaceutically acceptable salt thereof.

[0284] 12. The pharmaceutical composition of any of the foregoing technical solutions, wherein compound (A) is administered orally.

[0285] 13. The pharmaceutical composition of any of the foregoing technical solutions, wherein the dosage of compound (A) is 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0286] 14. The pharmaceutical composition of any one of technical solutions 12 to 13, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0287] 15. The pharmaceutical composition of any of the foregoing technical solutions, wherein the cancer is glioblastoma.

[0288] 16. The pharmaceutical composition of claim 15, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0288] Fifth State Sample and Related Examples - Preferred (B1) Chemotherapy Compound - Common Cancer Treatment

[0289] 1. A pharmaceutical combination comprising:

[0289] (A): A polyunsaturated fatty acid compound of the formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0289] (i)a can be any integer value between 0 and 7

[0289] (ii) b can be any integer value between 2 and 7.

[0289] (iii)c can be any integer value between 0 and 7.

[0289] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0289] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0289] and

[0289] (B1): Chemotherapy compound,

[0289] This pharmaceutical combination is intended for the simultaneous, separate, or sequential treatment of cancer in human patients, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0289] Temozolomide;

[0289] Topotecan;

[0289] Irinotecan;

[0289] Cyclophosphamide;

[0289] Fluorouracil;

[0289] Oxaliplatin;

[0289] Tetrahydrofolate; and

[0289] Cranberries.

[0290] 2. As in the pharmaceutical combination of technical solution 1, wherein

[0290] (i)a can be any integer value between 5 and 7

[0290] (ii)b can be any integer value between 2 and 4, and

[0290] (iii)c can be any integer value between 1 and 5.

[0291] 3. The pharmaceutical combination as described in any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0292] 4. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0292] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0292] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0292] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0292] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0292] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0292] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0293] 5. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0294] 6. The pharmaceutical combination of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0295] 7. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0295] Lung cancer;

[0295] Non-small cell lung cancer;

[0295] Squamous cell carcinoma;

[0295] Adenocarcinoma;

[0295] Endometrial cancer;

[0295] Serous carcinoma of the endometrium;

[0295] Endometrioid carcinoma;

[0295] Pancreatic cancer;

[0295] Glioblastoma;

[0295] Drug-resistant recurrent breast cancer;

[0295] Head and neck cancer;

[0295] Multiple myeloma;

[0295] Neuroblastoma and

[0295] Bile duct cancer.

[0296] 8. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0296] Temozolomide;

[0296] Topotecan;

[0296] Fluorouracil;

[0296] Oxaliplatin; and

[0296] Formyltetrahydrofolate.

[0297] 9. The pharmaceutical combination of technical solution 8, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0298] 10. As in technical solution 9, the pharmaceutical combination, wherein...

[0298] - Compound (B1) is temozolomide and the cancer is neuroblastoma;

[0298] - Compound (B1) is topotecan and the cancer is neuroblastoma;

[0298] - Compound (B1) is irinotecan and the cancer is neuroblastoma;

[0298] - Compound (B1) is cyclophosphamide and the cancer is neuroblastoma;

[0298] - Compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil, and the cancer is pancreatic cancer;

[0298] - Compound (B1) is a cranberry and the cancer is endometrial cancer; or

[0298] - The compound (B1) is temozolomide and the cancer is glioblastoma.

[0299] 11. The pharmaceutical combination of any of the foregoing technical solutions, wherein the pharmaceutical combination is a single composition comprising both compound (A) and compound (B1).

[0300] 12. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is administered orally.

[0301] 13. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the dosage of compound (A) is a daily dose of 200 mg to 7000 mg, more preferably a daily dose of 1500 mg to 5000 mg, even more preferably a daily dose of 3000 mg to 4700 mg, and most preferably a daily dose of 3500 mg to 4300 mg.

[0302] 14. The pharmaceutical combination of any one of technical solutions 12 to 13, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0303] 15. The pharmaceutical combination as described in technical solution 14, wherein...

[0303] Compound (B1) is temozolomide and is administered orally in capsule or tablet form;

[0303] Compound (B1) is topotecan and is administered intravenously via an infusion solution;

[0303] Compound (B1) is irinotecan and is administered intravenously via an infusion solution;

[0303] Compound (B1) is cyclophosphamide and it is administered intravenously via an infusion solution;

[0303] Compound (B1) is fluorouracil and is administered intravenously via an infusion solution;

[0303] Compound (B1) is oxaliplatin and is administered intravenously via an infusion solution;

[0303] Compound (B1) is formaldehyde tetrahydrofolate and is administered intravenously via an infusion solution; or

[0303] Compound (B1) is cranberry and is administered intravenously via an infusion.

[0303] Other specific combinations (A) and combinations of more than one compound (B) that are associated with all states in this document.

[0304] 1. A pharmaceutical combination comprising:

[0304] (A): A polyunsaturated fatty acid compound of formula COOR1-CHR2-(CH2)a-(CH=CHCH2)b-(CH2)c-CH3, its pharmaceutically acceptable salt, or a combination thereof, wherein

[0304] (i)a can be any integer value between 0 and 7.

[0304] (ii) b can be any integer value between 2 and 7.

[0304] (iii) c can be any integer value between 0 and 7.

[0304] (iv)R1 is H, Na, K, CH3, CH3-CH2 or PO(O-CH2-CH3)2, and

[0304] (v)R2 is OH, OCH3, O-CH2COOH, CH3, Cl, CH2OH, OPO(O-CH2-CH3)2, N(OH)2, F, HCOO or N(OCH2CH3)2;

[0304] (B1): Chemotherapy compound; and

[0304] (B3): Immunotherapy compound,

[0304] This combination of medicines is used simultaneously, separately or sequentially for the treatment of cancer in human patients.

[0305] 2. As in technical solution 1, the pharmaceutical combination, wherein

[0305] (i)a can be any integer value between 5 and 7.

[0305] (ii)b can be any integer value between 2 and 4, and

[0305] (iii)c can be any integer value between 1 and 5.

[0306] 3. The pharmaceutical combination as described in any of the aforementioned technical solutions, wherein R1 is H and R2 is OH.

[0307] 4. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (A) is at least one compound selected from the group consisting of:

[0307] COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812),

[0307] COOH-CHOH-(CH2)6-(CH=CH-CH2)3-CH3(183A1),

[0307] COOH-CHOH-(CH2)3-(CH=CH-CH2)3-(CH2)3-CH3(183A2),

[0307] COOH-CHOH-(CH2)2-(CH=CH-CH2)4-(CH2)3-CH3(204A1),

[0307] COOH-CHOH-(CH2)2-(CH=CH-CH2)5-CH3(205A1) and

[0307] COOH-CHOH-CH2-(CH=CH-CH2)6-CH3(226A1).

[0308] 5. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0309] 6. The pharmaceutical combination of technical solution 5, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812).

[0310] 7. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the cancer is at least one cancer selected from the group consisting of:

[0310] Lung cancer;

[0310] Non-small cell lung cancer;

[0310] Squamous cell carcinoma;

[0310] Adenocarcinoma;

[0310] Endometrial cancer;

[0310] Serous carcinoma of the endometrium;

[0310] Endometrioid carcinoma;

[0310] Pancreatic cancer;

[0310] Glioblastoma;

[0310] Drug-resistant recurrent breast cancer;

[0310] Head and neck cancer;

[0310] Multiple myeloma;

[0310] Neuroblastoma and

[0310] Bile duct cancer.

[0311] 8. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0311] Checkpoint inhibitor antibody, preferably wherein the checkpoint inhibitor antibody is an anti-PD1 antibody, an anti-PDL1 antibody, or an anti-CTLA4 antibody.

[0312] 9. The pharmaceutical combination of technical solution 8, wherein compound (B3) is at least one immunotherapeutic compound selected from the group consisting of:

[0312] -Anti-PD1 antibody, preferably, wherein the anti-PD1 antibody is nivolumab, pembrolizumab or spartazumab;

[0312] -An anti-PDL1 antibody, preferably, wherein the anti-PDL1 antibody is atezolizumab, avelumab or durvalumab;

[0312] - Anti-CTLA4 antibody, preferably ipilimumab.

[0313] 10. The pharmaceutical combination of technical solution 9, wherein compound (B3) is an anti-PD1 antibody, preferably wherein the anti-PD1 antibody is pembrolizumab.

[0314] 11. The pharmaceutical combination of any one of technical solutions 8 to 10, wherein compound (A) is COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3(ABTL0812) or a pharmaceutically acceptable salt thereof.

[0315] 12. As in the pharmaceutical combination of technical solution 11, wherein...

[0315] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, superior to pembrolizumab, and the cancer is lung cancer; or

[0315] - Compound (B3) is an anti-PD1 checkpoint inhibitor antibody, preferably pembrolizumab, administered in combination with at least one compound (B1), preferably paclitaxel and carboplatin, and for the cancer of lung cancer.

[0316] 13. The pharmaceutical combination of any of the foregoing technical solutions, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0316] Temozolomide;

[0316] Topotecan;

[0316] Irinotecan;

[0316] Cyclophosphamide;

[0316] Fluorouracil;

[0316] Cisplatin;

[0316] Carboplatin;

[0316] Oxaliplatin;

[0316] Tetrahydrofolate;

[0316] Small cranberries;

[0316] Bleomycin;

[0316] Capecitabine;

[0316] Mitomycin B;

[0316] Paclitaxel;

[0316] Albumin-bound paclitaxel;

[0316] Dorcetaxel;

[0316] Gesitabine;

[0316] Methotrexate;

[0316] Pepmetrozine;

[0316] 5-Fluorouracil;

[0316] Cytarabine;

[0316] Mercaptopurine;

[0316] Glucophosphamidon;

[0316] Isapirone;

[0316] Nimustine;

[0316] Carmustine;

[0316] Lomustine;

[0316] Mitoxantrone;

[0316] Etoposide;

[0316] Changchun New Alkali;

[0316] Vincristine; and

[0316] Tamoxifen.

[0317] 14. The pharmaceutical combination of technical solution 13, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0317] Temozolomide;

[0317] Topotecan;

[0317] Irinotecan;

[0317] Cyclophosphamide;

[0317] Fluorouracil;

[0317] Oxaliplatin;

[0317] Tetrahydrofolate; and

[0317] Cranberries.

[0318] 15. The pharmaceutical combination of technical solution 13, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0318] Temozolomide;

[0318] Topotecan;

[0318] Fluorouracil;

[0318] Oxaliplatin; and

[0318] Formyltetrahydrofolate.

[0319] 16. The pharmaceutical combination of technical solution 13, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0319] Irinotecan;

[0319] Fluorouracil;

[0319] Oxaliplatin; and

[0319] Formyltetrahydrofolate.

[0320] 17. The pharmaceutical combination of technical solution 13, wherein compound (B1) is at least one chemotherapeutic compound selected from the group consisting of:

[0320] Carboplatin; and

[0320] Paclitaxel.

[0321] 18. The pharmaceutical combination of technical solution 17, wherein compound (B1) is paclitaxel and carboplatin.

[0322] 19. The pharmaceutical combination of technical solution 16, wherein compound (B1) is irinotecan, methyltetrahydrofolate, oxaliplatin and fluorouracil.

[0323] 20. A pharmaceutical combination of any one of technical solutions 1 to 19, wherein compound (A) is ABTL0812, compound (B1) is paclitaxel and carboplatin, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody.

[0324] 21. A pharmaceutical combination of any one of technical solutions 1 to 19, wherein compound (A) is ABTL0812, compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil, and compound (B3) is an anti-PD1 checkpoint inhibitor antibody.

[0325] 22. The pharmaceutical combination of any one of technical solutions 20 to 21, wherein compound (B3) is an anti-PD1 checkpoint inhibitor antibody selected from nivolumab, pembrolizumab and spartazumab, preferably pembrolizumab.

[0326] 23. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (B1) is administered intravenously via an infusion solution.

[0327] 24. The pharmaceutical combination of any of the aforementioned technical solutions, wherein compound (A) is administered orally.

[0328] 25. The pharmaceutical combination of any of the aforementioned technical solutions, wherein the dosage of compound (A) is 200 mg to 7000 mg per day, more preferably 1500 mg to 5000 mg per day, even more preferably 3000 mg to 4700 mg per day, and most preferably 3500 mg to 4300 mg per day.

[0329] 26. A pharmaceutical combination of any one of technical solutions 1 to 7, 13 to 19 and 23 to 25, wherein compound (B3) is an anticancer immunomodulatory compound.

[0330] 27. The pharmaceutical combination of technical solution 26, wherein compound (B3) is an anticancer immunomodulatory compound selected from the group consisting of: interleukins, checkpoint inhibitors, agonists and adjuvants.

[0330] Example

[0330] Example 1: Combination of ABTL0812 with different / additional chemotherapeutic agents: In vitro analysis

[0330] 1.1: Cell viability analysis of ABTL0812 cells alone or in combination with temozolomide in neuroblastoma.

[0331] Objective: To investigate the potential synergistic effect of ABTL0812 added to temozolomide in neuroblastoma cell lines SK-N-BE(2) and LA1-5S. Temozolomide is a common chemotherapeutic agent used as a backbone of second-line treatment for, for example, high-risk neuroblastoma. Therefore, it is of interest to know whether there is any synergistic effect between ABTL0812 and temozolomide.

[0332] Methods: LA1-5S and SK-N-BE(2) cells were cultured with increasing concentrations of temozolomide (500 μM, 1000 μM, and 1500 μM) and ABTL0812 at a fixed concentration below IC50 (30 μM for SK-N-BE(2) and 40 μM for LA1-5S). Cells were treated in IMDM containing 0.5% FBS for 48 hours. Cell viability was assessed by crystal violet analysis. Six replicates were performed for different doses, and the results shown are the average of two independent experiments. Statistical analysis was performed using GraphPad Prism® 5.0 software according to the T-test principle.

[0333] Results: The addition of 30 μM or 40 μM ABTL0812 increased the cytotoxicity of temozolomide. This increase was statistically significant at all concentrations (** p < 0.01; *** p < 0.001) - see Figure 1 in this paper.

[0334] Conclusion: ABTL0812 enhanced the cytotoxicity of temozolomide in in vitro neuroblastoma cell lines SK-N-BE(2) and LA1-5S. These results support in vivo studies of the combination of the two drugs.

[0334] 1.2: Cell viability analysis of ABTL0812 cells alone or in combination with topotecan in neuroblastoma

[0335] Objective: To investigate the potential synergistic effect of ABTL0812 added to topotecan in the neuroblastoma cell line LA1-5S. Topotecan is a common chemotherapeutic agent used as a backbone of second-line treatment for, for example, high-risk neuroblastoma. Therefore, it is of interest to know whether there is any synergistic effect between ABTL0812 and topotecan.

[0336] Methods: LA1-5S cells were cultured with increasing concentrations of topotecan (0.5 μM, 1 μM, and 2 μM) and a fixed concentration of ABTL0812 (30 μM). Cells were treated in IMDM containing 0.5% FBS for 72 hours. Cell viability was assessed by crystal violet analysis. Data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using GraphPad Prism® 5.0 software based on the t-test principle.

[0337] Results: The addition of 30 μM ABTL0812 significantly increased the cytotoxicity of topotecan. (Compared with the mediator, *p<0.05, **p<0.01; compared with matched concentrations of each drug as a single agent, $p<0.05). See Figure 2 in this paper.

[0338] Conclusion: In the in vitro neuroblastoma cell line LA1-5S, ABTL0812 enhanced the cytotoxicity of topotecan. These results support in vivo studies of the combination of the two drugs.

[0338] 1.3: Cell viability analysis of ABTL0812 cells alone or in combination with irinotecan in neuroblastoma.

[0339] Objective: To investigate the potential synergistic effect of ABTL0812 added to irinotecan in the neuroblastoma cell line LA1-5S. Irinotecan is a common chemotherapeutic agent used as a backbone of second-line treatment for, for example, high-risk neuroblastoma. Therefore, it is of interest to know whether there is any synergistic effect between ABTL0812 and irinotecan.

[0340] Methods: LA1-5S cells were cultured with increasing concentrations of irinotecan (4 μM, 8 μM, and 16 μM) and a fixed concentration of ABTL0812 (30 μM). Cells were treated in IMDM containing 0.5% FBS for 72 hours. Cell viability was assessed by crystal violet analysis. Data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using GraphPad Prism® 5.0 software according to the t-test principle (* p < 0.05; ** p < 0.01; *** p < 0.001).

[0341] Results: The addition of 30 μM ABTL0812 significantly increased irinotecan cytotoxicity. (Compared with the mediator, * p < 0.05, ** p < 0.01; compared with ABTL0812 as a single agent, # p < 0.05; compared with matched concentrations of each drug as a single agent, $ p < 0.05). See Figure 3 in this paper.

[0342] Conclusion: In the in vitro neuroblastoma cell line LA1-5S, ABTL0812 enhanced the cytotoxicity of irinotecan. These results support in vivo studies of the combination of the two drugs.

[0342] 1.4: Cell viability analysis of ABTL0812 cells alone or in combination with cyclophosphamide in neuroblastoma.

[0343] Objective: To investigate the potential synergistic effect of ABTL0812 added to cyclophosphamide in the neuroblastoma cell line LA1-5S. Cyclophosphamide is a common chemotherapeutic agent used as a backbone of second-line treatment for, for example, high-risk neuroblastoma. Therefore, it is of interest to know whether there is any synergistic effect between ABTL0812 and cyclophosphamide.

[0344] Methods: LA1-5S cells were cultured with increasing concentrations of cyclophosphamide (1 μM, 1.5 μM, and 2 μM) and ABTL0812 (30 μM) at a fixed IC50 concentration. Cells were treated in IMDM containing 0.5% FBS for 72 hours. Cell viability was assessed by crystal violet analysis. Data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using GraphPad Prism® 5.0 software according to the t-test principle (* p < 0.05; ** p < 0.01; *** p < 0.001).

[0345] Results: The addition of 30 μM ABTL0812 significantly increased the cytotoxicity of 1 μM cyclophosphamide. (p < 0.05 compared with matched concentrations of each drug as a single agent). See Figure 4 in this paper.

[0346] Conclusion: In the in vitro neuroblastoma cell line LA1-5S, ABTL0812 enhanced the cytotoxicity of cyclophosphamide. These results support in vivo studies of the combination of the two drugs.

[0346] Example 2: Combination of ABTL0812 with targeted therapy: In vitro analysis

[0346] 2.1: Cell viability analysis of ABTL0812 cells alone or in combination with bortezomib in multiple myeloma

[0347] Objective: To investigate the potential synergistic effect of ABTL0812 added to bortezomib in multiple myeloma cell lines JJN-3 and OPM2. Bortezomib is a class 1 proteasome inhibitor approved for the treatment of patients with multiple myeloma. Therefore, it is of interest to know whether there is any additive effect between the two drugs.

[0348] Methods: JJN-3 and OPM2 cells were seeded in 24-well plates and treated with ABTL0812 (5 μM), bortezomib (0.5 and 1 nM, respectively), or a combination of the two drugs, and incubated in an incubator for 48 hours (0.5% FBS). Cell viability was studied by MTT assay and the combination index was determined to assess possible synergistic effects.

[0349] Results: JJN-3 cells were co-cultured with ABTL0812 at 5 μM (below IC50 concentration) and bortezomib at 0.5 nM (below IC50 concentration), respectively, which induced approximately 10% and 15% cell death. When the two drugs were combined, cell death was enhanced, inducing approximately 40% cell death. Cell viability was assessed by MTT assay in all cases, and the combination index (CI) was calculated according to the methods of Chou and Talalay (Chou 2006; Chou 2010) to assess synergistic effects, as follows: CI = (D)1 / (Dx)1 + (D)2 / (Dx)2, where CI < 1, = 1, and > 1 represent synergistic, additive, and antagonistic effects, respectively. In the denominator, (Dx)1 is the "individual" D1×% of the inhibitory system, and (Dx)2 is the "individual" D2×% of the inhibitory system. In the numerator, the "combination" (D)1 and (D)2 also inhibited ×. The CI for this combination was 0.049, indicating a very high synergistic effect. In OPM2 cells, this was co-cultured with ABTL0812 at 5 μM (below IC50 concentration) and bortezomib at 1 nM (below IC50 concentration), inducing approximately 20% and 10% cell death, respectively. When the two drugs were combined, cell death was enhanced, inducing approximately 55% cell death. The combination index was 0.50, indicating a synergistic effect – see Figure 5 in this paper.

[0350] Conclusion: ABTL0812 and bortezomib exhibit a strong synergistic effect in in vivo multiple myeloma cell lines JJN-3 and OPM2. The anticancer activity was enhanced when the two drugs were combined at concentrations below the IC50, thus demonstrating a synergistic effect between the two drugs. These results open up opportunities for in vivo combinations of the two drugs.

[0350] Example 3: Combination of ABTL0812 with different chemotherapeutic agents: In vivo analysis

[0350] 3.1: Anticancer activity of ABTL0812 alone or in combination with FOLFIRINOX (5-FU, folate, irinotecan, and oxaliplatin) in a human pancreatic cancer xenograft model implanted in nude mice MiaPAca2 cells.

[0351] Test system: Nu / nu female mice.

[0352] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with FOLFIRINOX, a standard care therapy for pancreatic cancer.

[0353] Methods: Tumor formation was induced by injecting 5 × 10⁶ MiaPaca2 cells into one side of the abdomen of mice. When the tumor reached a volume of approximately 100 mm³, the animals were uniformly randomized and started different treatments. ABTL0812 was administered orally at a dose of 120 mg / kg / day. FOLFIRINOX chemotherapy was administered intraperitoneally once a week for a total of four times. 5-FU (30 mg / kg), folate (50 mg / kg), irinotecan (50 mg / kg), and oxaliplatin (2.5 mg / kg) were administered intraperitoneally on different days. 5-FU and folate were administered on Tuesdays, and irinotecan and oxaliplatin were administered on Thursdays. Tumor volume and body weight were monitored three times a week.

[0354] Results: Compared with the Folfirinox, ABTL0812, and mediator groups, the combination of ABTL0812 and Folfirinox significantly increased the therapeutic potential of chemotherapy and demonstrated a reduction in maximum tumor volume. Statistical analysis showed that the combination therapy significantly improved tumor growth reduction compared with Folfirinox alone, which is the standard of care for the treatment of advanced pancreatic cancer (p<0.001, by t-test). In addition, no reduction in body weight or blood count (not shown) was observed in any treatment group (including ABTL0812 and Folfirinox administered together with each other), indicating that this combination was not toxic. See Figure 6 in this paper.

[0355] Conclusion: In a human pancreatic cancer xenograft model using MiaPaca2 cells implanted in nude mice, ABTL0812 significantly enhanced the anticancer effect of Folirinox without increasing toxicity. Folirinox is the standard of care for patients with advanced pancreatic cancer; therefore, these results indicate that the combination therapy of ABTL0812 and Folirinox may have clinical significance in the treatment of pancreatic cancer.

[0355] 3.2: Anticancer activity of ABTL0812 alone or in combination with cranberry in a human endometrial cancer xenograft model implanted with Ishikawa cells from nude mice.

[0356] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with cranberry, a reference second-line treatment for endometrial cancer.

[0357] Methods: Tumor formation was induced by injecting 4 × 10⁶ Ishikawa cells into one side of the abdomen of mice. When the tumor reached a volume of approximately 50 mm³, the animals were uniformly randomized and started different treatments. ABTL0812 was administered orally at a dose of 120 mg / kg / day. Cranberries were administered intraperitoneally once a week. Tumor volume and body weight were monitored three times a week.

[0358] Results: Compared with control animals, ABTL0812 and cranberry significantly reduced tumor volume (ANOVA, followed by a t-test). The efficacy of ABTL0812 was essentially similar to that observed with cranberry treatment. Interestingly, ABTL0812 enhanced the antitumor effect of docetaxel. Statistical analysis showed that this combination therapy significantly improved the reduction in tumor growth compared with cranberry alone (*p<0.05, by t-test). Furthermore, no reduction in body weight or blood count (not shown) was observed in any treatment group (including ABTL0812 and cranberry administered together with other treatment groups), indicating that this combination was not toxic. See Figure 7 in this paper.

[0359] Conclusion: In an endometrial cancer xenograft model derived from Ishikawa cells, ABTL0812 reduced tumor growth. In this model, ABTL0812 exhibited effects similar to those of SOC cranberry. ABTL0812 enhanced the antitumor activity of cranberry without toxicity. These results suggest that the combination therapy of ABTL0812 and cranberry may have clinical significance in the treatment of endometrial cancer.

[0359] 3.3: Anticancer activity of ABTL0812 alone or in combination with temozolomide in a human glioblastoma orthotopic xenograft model using U87MG cells implanted in nude mouse brains.

[0360] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with temozolomide, a reference drug for the treatment of glioblastoma.

[0361] Methods: Mouse brains were injected with luciferase-transfected U87MG cells. Five days after cell seeding, animals were randomized and treatment began. 240 mg / kg ABTL0812 was administered orally for 5 days / week; and 32 mg / kg temozolomide was administered orally for the first 5 days. Tumors were measured by quantifying the bioluminescence intensity (BLI) of the region of interest.

[0362] Results: ABTL0812 and temozolomide, as single agents, significantly increased disease-free survival in animals with glioblastoma tumors in the brain. Interestingly, the combination of ABTL0812 and temozolomide was significantly more effective than single treatment. See Figure 8 in this paper.

[0363] Conclusion: ABTL0812, as a single agent, increased disease-free survival in mice with tumors and enhanced the antitumor activity of temozolomide. These results indicate that the combination therapy of ABTL0812 and temozolomide may have clinical significance in the treatment of glioblastoma.

[0363] Example 4: Combination of ABTL0812 and radiotherapy: In vivo analysis

[0363] 4.1: Anticancer activity of ABTL0812 alone or in combination with radiotherapy in a human glioblastoma subcutaneous xenograft model using U87MG and T98G cells implanted in nude mice.

[0364] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with radiotherapy, the main therapeutic strategy for glioblastoma.

[0365] Methods: 1 × 10⁶ U87MG or T98G cells were subcutaneously injected into each side of the ventral region of mice to induce tumor formation. When the tumor reached a volume of 0.8–1.3 cm³, the animals were uniformly randomized and started different treatments. 240 mg / kg ABTL0812 was administered orally for 5 days / week, and a single dose of 4 Gy of radiation therapy was administered on day 3.

[0366] Results: ABTL0812 as a single agent significantly reduced the growth of subcutaneous glioblastoma tumors. Furthermore, the combination of ABTL0812 and radiotherapy was significantly more effective than either ABTL0812 or radiotherapy as a single treatment. See Figure 9 in this article.

[0367] Conclusion: ABTL0812, as a single agent, reduces the growth of glioblastoma tumors and enhances the antitumor activity of radiotherapy. These results indicate that the combination therapy of ABTL0812 and radiotherapy may have clinical significance in the treatment of glioblastoma.

[0367] 4.2: Anticancer activity of ABTL0812 alone or in combination with radiotherapy in a human glioblastoma orthotopic xenograft model using U87MG cells implanted in the brains of nude mice.

[0368] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with radiotherapy, the main therapeutic strategy for glioblastoma.

[0369] Methods: Mouse brains were injected with luciferase-transfected U87MG cells. Five days after cell seeding, animals were randomized and treatment began. 240 mg / kg ABTL0812 was administered orally for 5 days per week, followed by a single 4 Gy radiation therapy on day 10. Tumors were measured by quantifying the bioluminescence intensity (BLI) of the region of interest.

[0370] Results: ABTL0812 and radiotherapy as single agents significantly increased disease-free survival in animals with glioblastoma tumors in the brain. Interestingly, the combination of ABTL0812 and radiotherapy was significantly more effective than single treatment. See Figure 10 in this paper.

[0371] Conclusion: ABTL0812, as a single agent, increased disease-free survival in mice with tumors and enhanced the antitumor activity of radiotherapy. These results suggest that the combination therapy of ABTL0812 and radiotherapy may have clinical significance in the treatment of glioblastoma.

[0371] Example 5: Combination of ABTL0812 with targeted therapy: In vivo analysis

[0371] 5.1: Anticancer activity of ABTL0812 alone or in combination with olaparib in a human endometrial cancer model implanted in nude mice using Ishikawa cells.

[0372] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with olaparib, a reference drug for the treatment of endometrial cancer.

[0373] Methods: Tumor formation was induced by injecting 4 × 10⁶ Ishikawa cells into one side of the abdomen of mice. When the tumor reached a volume of approximately 50 mm³, the animals were uniformly randomized and started different treatments. ABTL0812 was administered orally at a dose of 120 mg / kg / day. Olaparib was administered orally at a dose of 50 mg / kg / day. Tumor volume and body weight were monitored three times a week.

[0374] Results: Compared with control animals, ABTL0812 and olaparib significantly reduced tumor volume (ANOVA, followed by a t-test). The efficacy of ABTL0812 was practically similar to that observed with olaparib treatment. Interestingly, ABTL0812 enhanced the antitumor effect of docetaxel. Statistical analysis showed that this combination therapy significantly improved the reduction in tumor growth compared with olaparib alone (**p<0.01, by t-test). Furthermore, no reduction in body weight or blood count (not shown) was observed in any treatment group (including ABTL0812 and olaparib administered together with each other), indicating that this combination was not toxic. See Figure 11 in this paper.

[0375] Conclusion: In an endometrial cancer xenograft model derived from Ishikawa cells, ABTL0812 reduced tumor growth. In this model, ABTL0812 exhibited efficacy similar to olaparib treatment. ABTL0812 enhanced the antitumor activity of olaparib without toxicity. These results suggest that combination therapy with ABTL0812 and olaparib may have clinical significance in the treatment of endometrial cancer.

[0375] 5.2: Anticancer activity of ABTL0812 alone or in combination with bevacizumab in a human endometrial cancer model implanted in nude mice using Ishikawa cells.

[0376] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with bevacizumab, which has shown potential efficacy in endometrial cancer.

[0377] Methods: Tumor formation was induced by injecting 4 × 10⁶ Ishikawa cells into one side of the abdomen of mice. When the tumor reached a volume of approximately 50 mm³, the animals were uniformly randomized and started different treatments. ABTL0812 was administered orally at a dose of 120 mg / kg / day. Bevacizumab was administered intraperitoneally at a dose of 100 μg every four days for a total of four administrations. Tumor volume and body weight were monitored three times a week.

[0378] Results: Compared with control animals, ABTL0812 and bevacizumab significantly reduced tumor volume (ANOVA, followed by a t-test). The efficacy of ABTL0812 was practically similar to that observed with bevacizumab treatment. Interestingly, ABTL0812 enhanced the antitumor effect of docetaxel. Statistical analysis showed that this combination therapy significantly improved the reduction in tumor growth compared with bevacizumab alone (*p<0.05, by t-test). Furthermore, no reduction in body weight or blood count (not shown) was observed in any treatment group (including ABTL0812 and bevacizumab administered together with each other), indicating that this combination was not toxic. See Figure 12 in this paper.

[0379] Conclusion: In an endometrial cancer xenograft model derived from Ishikawa cells, ABTL0812 reduced tumor growth. In this model, ABTL0812 exhibited efficacy similar to bevacizumab treatment. ABTL0812 enhanced the antitumor activity of bevacizumab without toxicity. These results suggest that combination therapy with ABTL0812 and bevacizumab may have clinical significance in the treatment of endometrial cancer.

[0379] Example 6: Immunomodulatory effects of ABTL0812: In vitro analysis

[0379] 6.1: Immunomodulatory effects of ABTL0812 on human THP-1 macrophages by enhancing the pro-inflammatory phenotype of M1 and inhibiting the anti-inflammatory phenotype of M2.

[0380] Objective: To investigate the immunomodulatory effect of ABTL0812 on macrophage polarization into the M1 phenotype (pro-inflammatory and anti-tumor) and the M2 phenotype (anti-inflammatory and pro-tumor), and this immunomodulatory effect will affect the tumor microenvironment after ABTL0812 treatment.

[0381] Methods: THP-1 mononuclear spheroids grown in suspension were cultured with PMA for 24 hours to induce attachment to discs and differentiation into macrophages. Once THP-1 macrophages differentiated, they were polarized into M1 macrophages by incubation with LPS in the presence of ABTL0812 (50 or 100 μM) for 6 or 24 hours. Simultaneously, the differentiated macrophages were polarized into M2 macrophages by incubation with IL-4 and IL-13 in the presence of ABTL0812 (50 μM) for 24 hours. Subsequently, the polarized macrophages were lysed, total RNA was extracted, reverse transcribed into cDNA, and the mRNA levels of IL1β, TNFα (M1 marker), and IL-10 (M2 marker) were assessed by RT-qPCR using specific probes.

[0382] Results: The immunomodulatory effect of ABTL0812 significantly enhanced the levels of IL-1β and TNFα mRNA in macrophages polarized to M1, and significantly inhibited the level of IL-10 mRNA in macrophages polarized to M2 (t-test: **p<0.01 and ***p<0.001). When differentiated macrophages were cultured together with ABTL0812 alone without polarization, ABTL0812 significantly induced the expression of IL-1β, thus highlighting its immunomodulatory effect on human THP-1 cells. See Figure 13 in this article.

[0383] Conclusion: ABTL0812 enhances the polarization of human THP-1 monocytes to M1 and significantly increases the gene expression of IL-1β and TNF-α, thereby promoting a pro-inflammatory environment that will exert anti-tumor effects. Furthermore, ABTL0812 inhibits the polarization of human THP-1 monocytes to M2 and significantly reduces the gene expression of IL-10, avoiding M2 macrophage-mediated immunosuppression, a common mechanism used by tumor cells to evade the immune system. These results indicate that, in addition to its anti-cancer effects on tumor cells, ABTL0812 also stimulates the immune system to achieve a pro-inflammatory phenotype, recruiting other immune cells such as cytotoxic T lymphocytes, thereby transforming "cold" tumors that induce immune system suppression into "hot" and immunogenic tumors. This highlights the potential combination of ABTL0812, which enhances anti-cancer efficacy by promoting a pro-inflammatory and anti-tumor microenvironment, with specific immune checkpoint inhibitors.

[0383] 6.2: Induction of PDL1 expression in cancer cells treated with ABTL0812

[0384] Objective: To investigate the immunomodulatory effect of ABTL0812, alone or in combination with IFNγ, a well-described master regulator of PDL1 expression, on PDL1 expression in cancer cells.

[0385] Methods: Human cancer cell lines were cultured together with 100 μM ABTL0812 for 48 hours and collected for staining with a fluorescently labeled anti-PDL1 antibody. After staining with the anti-PDL1 antibody, the cells were flow-through cytometer (Facs Canto) to analyze the PDL1 content in the cancer cells. Additionally, Panc-1 cells were cultured together with ABTL0812 (50 μM), the master regulator of PDL1 expression IFNγ (2.5 ng / ml), or a combination of both. Subsequently, the cells were collected, stained with the anti-PDL1 antibody, and PDL1 expression was analyzed by flow-through cytometer.

[0386] Results: ABTL0812 induced PDL1 expression in all tested cancer cell lines (with similar efficacy), increasing from baseline in 34% (MiaPaca2 cells) to 86% (Capan-2 cells) (T-test *p<0.05, **p<0.01). When analyzing PDL1 levels in Panc-1 cells co-cultured with IFNγ, with or without ABTL0812, IFNγ induced significantly higher PDL1 levels compared to ABTL0812 (T-test **p<0.01). Interestingly, cells co-cultured with both ABTL0812 and IFNγ induced PDL1 levels similar to the sum of the effects mediated by ABTL0812 and IFNγ. Compared with ABTL0812 treatment, the combination of ABTL0812 and IFNγ induced significantly higher PDL1 levels (T-test ***p<0.001), but this increase was not significant compared with IFNγ treatment, indicating that the two drugs have an additive effect on PDL1 performance when administered together. See Figure 14 in this article.

[0387] Conclusion: ABTL0812 induces PDL1 expression in human pancreatic cancer and endometrial cancer cells. Compared with ABTL0812, IFNγ, the main regulator of PDL1 expression, induces higher PDL1 levels. However, when both drugs are administered together, there is an additive effect on PDL1 expression, inducing higher levels. ABTL0812 is cytotoxic to cancer cells and stimulates macrophages to polarize towards a pro-inflammatory phenotype, particularly producing high levels of IFNγ. These results highlight the potential combination of ABTL0812 and immune checkpoint inhibitors, as the mediated induction of PDL1 levels would allow immune checkpoint inhibitors to target cancer cells.

[0387] 6.3: Induction of a pro-inflammatory environment in macrophages

[0388] Objective: After evaluating the immunomodulatory effects of ABTL0812 on human macrophages, investigate the effects of ABTL0812 on cancer cells and how the conditioned medium of cancer cells treated with ABTL0812 affects the viability and polarization of human macrophages.

[0389] Methods: MiaPaca2 cells were co-cultured with 40 μM ABTL0812 for 72 hours, and the conditioned medium (RPMI) of these ABTL0812-treated cells was collected. Simultaneously, human THP-1 cells were induced to attach to discs and differentiate into macrophages by co-culturing with PMA (phorbol myristate acetate) for 24 hours. Subsequently, the conditioned medium of ABTL0812-treated MiaPaca2 cells was transferred to PMA-activated THP-1 macrophages and cultured for 24 hours for RT-qPCR analysis of the M1 phenotypic markers IL-1β and TNF-α, or cultured for 48 hours for cell viability studies using MTT assay. For M1 marker analysis, cells were collected, RNA was extracted, reverse transcribed into cDNA, and the mRNA levels of IL-1β and TNF-α were assessed by RT-qPCR.

[0390] Results: Previous results showed that ABTL0812 with an IC50 of 50 μM was cytotoxic to MiaPaca2 cells and induced the release of various factors into the culture medium. When this culture medium containing ABTL0812-treated MiaPaca2 cells was transferred to activated THP-1 macrophages, it induced metabolic activation, thereby significantly increasing the survival rate of THP-1 cells. Furthermore, ABTL0812-modulated culture medium induced the polarization of THP-1 macrophages toward a pro-inflammatory anti-tumor phenotype, significantly increasing the gene expression of IL-1β and TNF-α, thus confirming the immunomodulatory effect of ABTL0812 mediated by its action on cancer cells. See Figure 15 in this article.

[0391] Conclusion: ABTL0812 exhibits immunomodulatory effects not only through its direct action on macrophage polarization but also through its action on cancer cells. This is because ABTL0812-regulated culture medium for MiaPaca2 cells not only increases the viability and metabolic activity of human THP-1 macrophages but also induces M1 polarization by increasing the expression of IL-1β and TNF-α in these macrophages. Combined with ICD induction (Example 6.7) and inhibition of immunosuppressive factor secretion (Example 6.6), these data strongly suggest that ABTL0812 can immunomodulate the tumor microenvironment through its anticancer effect on cancer cells (which subsequently transforms "cold" tumors into "hot" tumors that can be targeted by the immune system), in addition to its direct action on human macrophages. Therefore, its potential combination with immune checkpoint inhibitors to enhance anticancer efficacy is highlighted.

[0391] 6.4: Induction of PDL1 expression in cancer cells treated with ABTL0812

[0392] Objective: To investigate the immunomodulatory effect of ABTL0812 on the expression of PDL1 in cancer cells, which may expose cancer cells to immune checkpoint inhibitor immunotherapy.

[0393] Methods: Human pancreatic cancer cell lines (MiaPaca2, Panc-1, Capan-2, and SU.86.86) and human endometrial cancer cell lines (Ishikawa, ANC3, Hec-1A, Ark1, and Ark2) were cultured with ABTL0812 at doses ranging from 0 to 80 μM for 24 hours. Cells were then collected and stained with anti-PDL1 antibody for flow cytometry analysis of the percentage of PDL1-positive cells.

[0394] Results: ABTL0812 induced PDL1 expression in all tested cancer cell lines. In pancreatic cancer cells, the expression increased in 34% to 86% of cases, and in endometrial cancer cells, the expression increased in 10% to 35% of cases (T-test *p<0.05, **p<0.01). See Figure 19 in this paper.

[0395] Conclusion: ABTL0812 induces PDL1 expression in human pancreatic cancer and endometrial cancer cells, potentially enabling immune checkpoint inhibitors to target cancer cells. These results support the potential synergistic effect of ABTL0812 with immunotherapy for cancer treatment.

[0395] 6.5: Inhibition of PD1 expression in primary human CD4 and CD8 T cells from healthy donor peripheral blood treated with ABTL0812 in vitro.

[0396] Objective: To investigate the immunomodulatory effect of ABTL0812 on PD1 expression in primary human T cells activated by CD3 and CD28 or unactivated by CD3 and CD28.

[0397] Methods: Human PBMCs were purified from peripheral blood of healthy donors using Ficoll and cultured in vitro. Human T cells were activated by co-culturing with antibodies against IL-2, CD3, and CD28 for 10 days, followed by treatment with ABTL0812 for 6 hours. Finally, the PD1 content expressed in the T cell membrane was analyzed by flow cytometry using specific antibodies. Results are expressed as % of positive cells and are presented as the mean of three independent experiments (t-test, **p<0.01 and ***p<0.001).

[0398] Results: ABTL0812 induced inhibition of PD1 expression in both unactivated and activated human primary CD4 and CD8 T lymphocytes. See Figure 20 in this paper.

[0399] Conclusion: ABTL0812 induces inhibition of PD1 expression in both activated and inactivated primary human CD4 and CD8 T cells purified from healthy donors. This may potentially help enhance the immune system against cancer cells by blocking PD1-mediated immunosuppression and T cell inactivation.

[0399] 6.6: Immunomodulatory effects of ABTL0812 on the secretory proteome of human pancreatic cancer cells

[0400] Objective: To investigate the immunomodulatory effect of ABTL0812 on the secretory proteome of cancer cells by analyzing secretory factors in culture medium using a protein microarray that detects up to 38 different chemokines.

[0401] Methods: Human pancreatic cancer cells were treated with 100 μM ABTL0812 for 24 hours, and the culture medium was collected for incubation using the RayBio C series human chemokine antibody array C1 (RayBiotec). The culture medium was then incubated with a membrane containing antibodies against 38 different chemokines. Subsequently, the membrane was incubated with a secondary antibody and further developed using HRP substrate. The signal intensity was assessed using densitometric assays, and the results of repeated assessments in three different organisms are graphically presented.

[0402] Results: The immunomodulatory effects of ABTL0812 induced reductions in the following: immunosuppressive chemokine CXCL6 (associated with immunosuppression, invasion, and poor prognosis); CXCL16 (promotes tumor invasion and is upregulated in pancreatic cancer); angiopoietin (promotes immunosuppression and angiogenesis); and CCL5 (promotes Treg tumor invasion and immunosuppression). See Figure 21 in this article.

[0403] Conclusion: ABTL0812 promotes the inhibition of the release of immunosuppressive factors in human pancreatic cancer cells. These data indicate that the effect of ABTL0812 on the secretory proteome of cancer cells modulates the tumor microenvironment towards a more pro-inflammatory and anti-tumor phenotype, thereby inhibiting the secretion of immunosuppressive factors.

[0403] 6.7: Immunomodulatory effects of ABTL0812, which induces immunogenic cell death, on human pancreatic cancer cells.

[0404] Objective: To investigate the immunomodulatory effect of ABTL0812 on cancer cells by evaluating the induction of immunogenic cell death (ICD).

[0405] Methods: Human pancreatic cancer cells were treated with ABTL0812 at progressively increasing concentrations (0 to 150 μM) for 24 hours. ICD markers of extracellular Hmgb1 and ATP, surface calreticulin, and activation of apoptotic proteases 3 and 8 were assessed by ELISA (Hmgb1 and ATP), flow cytometry (calreticulin), and immunodiffusion (apoptotic proteases 3 and 8). For extracellular Hmgb1 and ATP, culture media from ABTL0812-treated cells were collected and co-cultured with specific antibodies for further colorimetric detection. For surface calreticulin, cancer cells were collected and co-cultured with specific antibodies for further flow cytometry. For activation of apoptotic proteases 3 and 8, cancer cells were collected, protein lysates were obtained, and co-cultured with specific antibodies for further immunodiffusion and quantification were finally performed using densitometric assays.

[0406] Results: ABTL0812 immunomodulatory effects induced dose-dependent increases in all ICD markers: extracellular Hmgb1 and ATP, surface calreticulin, and activation of apoptotic proteases 3 and 8, as detected by ELISA, luciferase assay, flow cytometry, and fluorescent substrate analysis (t-tests: **p<0.01 and ***p<0.001, respectively). See Figure 22 in this paper. Similar results were obtained using different human pancreatic cancer cell lines; in Figure 23, results in MiaPaca2 cells are shown as a representative experiment.

[0407] Conclusion: ABTL0812 induces ICD in human pancreatic cancer cells, as indicated by dose-dependent increases in the following ICD markers: extracellular Hmgb1 and ATP, surface calreticulin, and activation of apoptotic proteases 3 and 8. These results suggest that ABTL0812 can induce ICD in tumors, making these tumors more immunogenic and targetable to the immune system, helping to transform "cold" tumors that induce immunosuppression into "hot" and immunogenic tumors. This data supports the potential combination of ABTL0812, which enhances anticancer efficacy, with immunotherapy.

[0407] 6.8: Immunomodulatory effects of ABTL0812 on human immortalized THP-1 and human primary macrophages by enhancing the pro-inflammatory phenotype of M1 and inhibiting the anti-inflammatory phenotype of M2.

[0408] Objective: To investigate the immunomodulatory effects of ABTL0812 on immortalized and primary macrophages polarized into the M1 phenotype (pro-inflammatory and anti-tumor) and the M2 phenotype (anti-inflammatory and pro-tumor), and to study the effects of this immunomodulatory effect on the tumor microenvironment after ABTL0812 treatment.

[0409] Methods: THP-1 mononuclear globules grown in suspension were induced to attach to discs and differentiate into macrophages by co-culturing with PMA for 24 hours. Mononuclear globules were then co-cultured with 20 ng / mL M-CSF1 for 7 days to differentiate into macrophages. Simultaneously, whole blood was collected from healthy donors, and circulating mononuclear globules were purified using immunomagnetic separation. Mononuclear globules were selected using an anti-CD14 antibody bound to magnetic beads, retained in the magnetic column, and further eluted for in vitro culture. After obtaining activated macrophages (immortified and primary), these activated macrophages were polarized into M1 cells by co-culturing with LPS+IFNγ in the presence of 100 μM ABTL0812 for 6 hours. Simultaneously, differentiated macrophages were polarized into M2 cells by co-culturing with IL-4 and IL-13 in the presence of ABTL0812 (100 μM) for 24 hours. Subsequently, polarized macrophages were lysed, total RNA was extracted, reverse transcribed into cDNA, and the mRNA levels of IL1β, TNFα (M1 marker), and IL-10 (M2 marker) were assessed by RT-qPCR using specific probes.

[0410] Results: The immunomodulatory effect of ABTL0812 significantly enhanced the levels of IL-1β and TNFα mRNA in macrophages polarized to M1, and significantly inhibited the level of IL-10 mRNA in macrophages polarized to M2 (t-test: **p<0.01 and ***p<0.001). When differentiated macrophages were cultured together with ABTL0812 alone without polarization, ABTL0812 significantly induced the expression of IL-1β, thus highlighting its immunomodulatory effect on human THP-1 cells. See Figure 24 in this article.

[0411] Conclusion: ABTL0812 enhances the polarization of both human THP-1 cells and primary human macrophages towards M1, significantly increases the gene expression of IL-1β and TNFα, thereby promoting a pro-inflammatory environment that exerts anti-tumor effects. Furthermore, ABTL0812 inhibits the polarization of human THP-1 monocytes towards M2, significantly reduces the gene expression of IL-10, and avoids M2 macrophage-mediated immunosuppression, a common mechanism used by tumor cells to evade the immune system. These results indicate that, in addition to its anti-cancer effects on tumor cells, ABTL0812 also stimulates the immune system to achieve a pro-inflammatory phenotype, recruiting other immune cells such as cytotoxic T lymphocytes, thus transforming "cold" tumors that induce immunosuppression into "hot" and immunogenic tumors. These results support the potential combination of ABTL0812, which enhances anti-cancer efficacy by promoting a pro-inflammatory and anti-tumor microenvironment, with immunotherapy.

[0411] 6.9: Immunomodulatory effects of ABTL0812 on the secretory proteome of human immortalized THP1 macrophages and human primary macrophages by protein microarray analysis

[0412] Objective: To investigate the immunomodulatory effect of ABTL0812 on the secretory proteome of immortalized and primary macrophages by analyzing secretory factors in the culture medium using a protein microarray that detects up to 42 different cytokines after treatment with ABTL0812.

[0413] Methods: Whole blood was collected from healthy donors, and circulating mononuclear globules were purified using immunomagnetic separation. Mononuclear globules were selected using an anti-CD14 antibody bound to magnetic beads, retained in the magnetic column, and further eluted for in vitro culture. Mononuclear globules were cultured with 20 ng / mL M-CSF1 for 7 days to differentiate into macrophages. Once the primary mononuclear globules differentiated into macrophages, these macrophages were polarized to M1 by incubation with LPS in the presence of ABTL0812 (50 or 100 μM) for 6 or 24 hours. Simultaneously, differentiated macrophages were polarized to M2 by incubation with IL-4 and IL-13 in the presence of ABTL0812 (50 μM) for 24 hours. THP-1 mononuclear globules grown in suspension were induced to attach to discs and differentiate into macrophages by incubation with PMA for 24 hours. Once THP-1 cells differentiated into macrophages, these macrophages were polarized into M1 cells by incubating with LPS in the presence of ABTL0812 (50 μM) for 6 or 24 hours. Simultaneously, differentiated macrophages were polarized into M2 cells by incubating with IL-4 and IL-13 in the presence of ABTL0812 (50 μM) for 24 hours. M1, M2, and differentiated macrophages (M0) treated with ABTL0812 were cultured using the RayBio C series human intercytokine antibody array C3 (RayBiotec). The culture medium was co-cultured with a PVDF (polyvinylidene fluoride) membrane containing antibodies against 42 different intercytokines. After culture, the membrane was co-cultured with secondary antibodies and further developed using HRP (horseradish peroxidase) substrate. Signal intensity was assessed using densitometric assays, and the results of repeated assessments in three different organisms are graphically presented.

[0414] Results: ABTL0812 immunomodulatory effects induced a decrease in immunosuppressive chemokines and an upregulation of various pro-inflammatory factors such as IL-1β and TNF-α. Among all immunosuppressive cytokines suppressed after ABTL0812 treatment, five were commonly found in immortalized THP1 cells and primary macrophages: IL-10 (associated with immunosuppression, invasion, and poor prognosis in various cancers); CCL22 (associated with immunosuppression, invasion, and poor prognosis in various cancers); CCL17 (associated with immunosuppression, invasion, and poor prognosis in various cancers); CCL8 (associated with immunosuppression, proliferation, and invasion); and CCL7 (associated with immunosuppression and invasion). See Figure 25 in this paper.

[0415] Conclusion: ABTL0812 promotes the secretion of pro-inflammatory factors and inhibits the release of immunosuppressive factors in cancer cells. Combined with the enhancement of the M1 macrophage phenotype and the inhibition of the M2 phenotype (Example 6.8), these data suggest that ABTL0812 can promote a pro-inflammatory anti-tumor environment to exert its effects on immune cells. These results support the potential combination of ABTL0812 with immunotherapy to enhance the therapeutic effect against tumors, especially in highly immunosuppressive tumors such as pancreatic cancer.

[0415] 6.10: ABTL0812 increases the cytotoxicity of T cells in cancer cells through immunomodulatory effects.

[0416] Objective: To investigate the immunomodulatory effect of ABTL0812 on co-cultures of cancer cells and activated T cells.

[0417] Methods: Human PBMCs were purified from peripheral blood of healthy donors using Fico and cultured in vitro. Human T cells were activated by co-culturing with IL-2, CD3, and CD28 antibodies for 10 days. Simultaneously, Ishikawa endometrial cancer cells were treated with 50 μM ABTL0812 for 6 hours and then co-cultured with activated T lymphocytes for 24 hours. Cell viability was then assessed by MTT assay. Untreated Ishikawa cells served as a control. Results are presented as the mean of three different experiments (t-test * p < 0.05).

[0418] Results: Compared with cancer cells not treated with ABTL0812, ABTL0812 enhanced the cytotoxic effect of activated T cells on cancer cells, which is related to the promoting effect of the pro-inflammatory environment mediated by ABTL0812 on cancer cells. See Figure 26 in this article.

[0419] Conclusion: ABTL0812 promotes the activation of the immune system in cancer cells, thereby enhancing the cytotoxic effect of activated primary cells on cancer cells.

[0419] Example 7: Immunomodulatory effects of ABTL0812: In vivo analysis

[0419] 7.1: Induction of PDL1 expression in cancer cells treated with ABTL0812 in a human lung cancer xenograft model implanted with H157 cells and a human pancreatic cancer model implanted with MiaPaca2 cells.

[0420] Objective: To verify the induction of PDL1 expression by ABTL0812 observed in vitro in two in vivo models: the human squamous NSLC cell line H157 implanted in nude mice and the human pancreatic cancer cell line MiaPAca2.

[0421] Methods: Tumor formation was induced by injecting 4 × 10⁶ H157 cells or 5 × 10⁶ MiaPAca2 cells into one side of the abdomen of mice. When the tumor reached a volume of approximately 50 mm³, the animals were uniformly randomized and different treatments were initiated. ABTL0812 was administered orally at a dose of 120 mg / kg / day for three weeks. After treatment, the animals were sacrificed, the tumors were extracted, and the PDL1 protein content in the cancer cells was analyzed by Western ink dot method.

[0422] Results: ABTL0812 increased the protein content of PDL1 in tumors in vivo, further validating previous in vivo results (Figure 14). These results highlight the potential combination of ABTL0812 with immune checkpoint inhibitors, as the mediated induction of PDL1 levels would allow immune checkpoint inhibitors to target cancer cells.

[0422] 7.2: Induction of T cell infiltration in uterine tumor lesions of female mice with endometrial cancer treated with ABTL0812

[0423] Objective: To verify the anticancer efficacy and immunomodulatory effects of ABTL0812 in a syngeneic model of endometrial cancer induced by PTEN deficiency in epithelial cells, which ultimately leads to the proliferation and development of endometrial intraepithelial neoplasia.

[0424] Methods: Tamoxifen was injected into mice to induce PTEN loss. Three weeks after tamoxifen administration, when the animals developed hyperplasia, they were administered ABTL0812 or a mediator at 120 mg / kg daily for the remaining three weeks. At that time, animals treated with the mediator (6 weeks after tamoxifen injection) developed tumor-like lesions, which were quantified by immunohistochemistry of extracted uterus. After ABTL0812 treatment, the animals were sacrificed, and the uterus was extracted, embedded in paraffin, and used for further immunohistochemical analysis. This immunohistochemical analysis was performed by hematoxylin-eosin staining for carcinogenesis assessment, or by anti-CD3 staining to assess T lymphocyte infiltration within the tumor lesion, which indicates the immunomodulation of the tumor microenvironment.

[0425] Results: Mice with a history of endometrial cancer treated with ABTL0812 showed a significant reduction in the development of endometrial intraepithelial neoplasia (EIN) and inhibited the progression of proliferative cancer. Specifically, hematoxylin-eosin staining analysis showed that 80% of ABTL0812-treated mice exhibited EIN compared to 80% of ABTL0812-treated animals with proliferative disease. When analyzing the expression of CD3 T lymphocytes in the treated uterus, ABTL0812 induced CD3 T lymphocyte infiltration within the tumor lesion, while ABTL0812-treated animals showed CD3 T lymphocytes in the surrounding stroma of the tumor lesion without infiltrating into the tumor. See Figure 23 in this paper.

[0426] Conclusion: The in vivo immunomodulatory effects of ABTL0812 demonstrate how it induces T lymphocyte infiltration within tumor lesions, indicating the presence of a pro-inflammatory anti-tumor microenvironment conducive to immune cell infiltration to kill cancer cells. These data correlate with favorable efficacy results, including the ability of ABTL0812 to inhibit the progression of proliferative endometrial cancer, and the manifestation of intraepithelial neoplasia-like lesions in mediated animals, highlighting its potential for enhanced anti-cancer efficacy in combination with immune checkpoint inhibitors.

[0426] Example 8: Combination of ABTL0812 with immunotherapy agents: In vivo analysis

[0426] 8.1: Anticancer activity of ABTL0812, alone or in combination with anti-PD1, in a mouse lung cancer model using LLC1 cells implanted in C57BL6 mice.

[0427] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with a reference anti-PD1 antibody for lung cancer, based on survival rate and endpoints against tumors larger than 800 mm3 or on the differentiation of clinical signs of poisoning or disease. Pembrolizumab is an anti-PD1 checkpoint inhibitor for human use—a modified form used in this example, optimized for the mouse model used in this example.

[0428] Methods: Lewis lung carcinoma cells (LLC1) were a highly tumorigenic mouse cell line originally derived from the lungs of C57BL mice implanted with primary Lewis lung cancer. These cells could be subcutaneously grown in syngeneic C57BL6 mice, where they developed into highly aggressive tumors and could be used to evaluate immunotherapy treatment. Tumor formation was induced by injecting 0.25 × 10⁶ LLC1 cells into one side of the abdomen of C57BL6 mice. When the tumor reached approximately 50 mm³, the animals were randomly assigned to treatment groups (n=7) and different treatments were initiated. ABTL0812 was administered orally at a dose of 120 mg / kg / day. Anti-PD1 antibody (100 μg / dose) was administered intraperitoneally every three days for a total of four administrations. Tumor volume and body weight were monitored three times per week. The endpoint indicator is based on tumors exceeding 800 mm3, or clinical signs of poisoning, suffering, or illness that indicate the need for euthanasia in animals.

[0429] Results: Compared with the mediator and anti-PD1 treatment groups, administration of ABTL0812 alone slightly increased mouse survival, showing a 15% survival rate after 14 days of treatment, compared to 0% survival in the mediator and anti-PD1 groups. Interestingly, the dual combination of ABTL0812 + anti-PD1 antibody showed the best survival rate, with a 38% survival rate after 14 days of treatment. After 9 days of treatment, the mediator group showed a 29% survival rate, the anti-PD1 group showed a 15% survival rate, the ABTL0812 group showed a 43% survival rate, and the ABTL0812 + anti-PD1 treatment showed a 62% survival rate. See Figure 16 in this paper.

[0430] Conclusion: Compared with treatments with the mediator, anti-PD1, and ABTL0812, the combination treatment with ABTL0812 and anti-PD1 (an immune checkpoint inhibitor) significantly increased mouse survival. After 14 days of treatment, administration of ABTL0812 alone slightly increased mouse survival, but its effect was higher over a shorter period. These data indicate a synergistic effect between ABTL0812 and anti-PD1 treatment, leading to increased mouse survival and highlighting its potential combination for human patients.

[0430] 8.2: Anticancer activity of ABTL0812, alone or in combination with anti-PD1 / paclitaxel / carboplatin, in a mouse lung cancer model using LLC1 cells implanted in C57BL6 mice.

[0431] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with reference therapies for the treatment of human lung cancer, namely anti-PD1 antibody and carboplatin / paclitaxel, thereby evaluating the potential synergistic effect between ABTL0812 and anti-PD1 + carboplatin / paclitaxel treatment in terms of tumor volume reduction in subcutaneously grown LLC1 xenografts.

[0432] Methods: Lewis lung cancer cells (LLC1) were a highly tumorigenic mouse cell line originally derived from the lungs of C57BL mice implanted with primary Lewis lung cancer. These cells could be subcutaneously grown in syngeneic C57BL6 mice, where they developed into highly aggressive tumors and could be used to evaluate immunotherapy treatment. Tumor formation was induced by injecting 0.25 × 10⁶ LLC1 cells into one side of the abdomen of C57BL6 mice. The day after tumor implantation, animals were assigned to the treatment group and treatment began (n=5). ABTL0812 was administered orally at a dose of 120 mg / kg / day; anti-PD1 antibody was administered intraperitoneally (ip) every three days for a total of five times; carboplatin and paclitaxel were administered intraperitoneally once a week at doses of 15 mg / kg and 5 mg / kg, respectively, for a total of three to four times. Tumor volume and body weight were monitored three times a week.

[0433] Results: Compared with control animals, ABTL0812 and anti-PD1 + paclitaxel / carboplatin treatment significantly reduced tumor volume (ANOVA, followed by t-test *p<0.05). The efficacy of ABTL0812 was practically similar to that observed with docetaxel treatment. Interestingly, ABTL0812 enhanced the antitumor effect of docetaxel. Statistical analysis showed that this combination therapy significantly improved the reduction in tumor growth compared with docetaxel alone (p<0.001, by t-test). In addition, no reduction in body weight or blood count (not shown) was observed in any treatment group (including ABTL0812 and docetaxel administered together with each other), indicating that this combination was not toxic. See Figure 17 in this paper.

[0434] Conclusion: Regarding the literature description of LLC1 xenografts, anti-PD1 treatment is ineffective in these tumors, but the combination with paclitaxel / carboplatin showed a significant reduction in tumor volume compared to the mediator control group. This is because chemotherapy treatment makes tumor cells immunogenic and recognizable by the immune system, and anti-PD1 antibodies enhance this effect. Administration of ABTL0812 alone showed similar efficacy to anti-PD1 + paclitaxel / carboplatin, but when administered in the triple combination of ABTL0812 + anti-PD1 + paclitaxel / carboplatin, this triple combination induced a significant reduction in tumor volume compared to anti-PD1 + paclitaxel / carboplatin, further confirming the potential synergistic effect of ABTL0812. ABTL0812 also acts as an immunomodulator that induces a pro-inflammatory anti-tumor microenvironment that synergistically with immune checkpoint inhibitors to induce a higher tumor volume reduction. These results indicate that the combination therapy of ABTL0812 plus anti-PD1 + paclitaxel / carboplatin (the standard treatment for lung cancer patients) may have clinical significance in the treatment of lung cancer.

[0434] 8.3: Anticancer activity of ABTL0812, alone or in combination with anti-PD1 / paclitaxel / carboplatin, in a mouse lung cancer model using LLC1 cells from C57BL6 mice injected intraperitoneally.

[0435] Objective: To investigate the antitumor activity of ABTL0812, alone and in combination with reference therapies for the treatment of human lung cancer, namely anti-PD1 antibody and carboplatin / paclitaxel, and to evaluate the potential synergistic effect between ABTL0812 and anti-PD1 + carboplatin / paclitaxel treatment in terms of tumor volume reduction in subcutaneously growing LLC1 tumors.

[0436] Methods: Lewis lung cancer cells (LLC1) were a highly tumorigenic mouse cell line originally derived from the lungs of C57BL mice implanted with primary Lewis lung cancer. These cells could grow intraperitoneally in syngeneic C57BL6 mice, where they developed into highly aggressive tumors attaching to the intestine and could be used to evaluate immunotherapy treatment. One × 10⁶ LLC1 cells were injected intraperitoneally into the C57BL6 mice to induce tumor formation. The day after tumor implantation, animals were assigned to the treatment group and treatment began (n=2). ABTL0812 was administered orally at a dose of 120 mg / kg / day; anti-PD1 antibody was administered intraperitoneally every three days for a total of five times; paclitaxel and carboplatin were administered intraperitoneally once a week at doses of 15 and 5 mg / kg, respectively, for a total of three to four times. After 14 days of treatment, the animals were euthanized, and the tumors growing in the intestine were collected.

[0437] Results: In a xenograft model of LLC1 cells grown intraperitoneally from C57BL6 mice, the combination of ABTL0812 and anti-PD1+paclitaxel / carboplatin significantly reduced tumor volume, showing approximately half the size, compared with control, ABTL0812, and anti-PD1+paclitaxel / carboplatin treatment. See Figure 18 in this article.

[0438] Conclusion: LLC1 cells can grow intraperitoneally in C57BL6 mice, developing into highly invasive tumors that attach to the intestine. Compared with mediator, ABTL0812, and anti-PD1 + paclitaxel / carboplatin treatment, the triple combination of ABTL0812 + anti-PD1 + paclitaxel / carboplatin reduced tumor growth. These results suggest that the combination therapy of ABTL0812 plus anti-PD1 + paclitaxel / carboplatin (the standard treatment for lung cancer patients) may have clinical significance in the treatment of lung cancer.

[0438] 8.4: Anticancer activity and tumor microenvironment immune regulation mediated by ABTL0812 in a mouse pancreatic cancer model using MT5 cells implanted in C57BL6 mice.

[0439] Objective: To investigate the antitumor activity and in vivo tumor immunomodulatory effects of ABTL0812 alone and compare it with reference anti-PD1 antibodies for treating different types of human cancer. Anticancer efficacy will be assessed by tumor volume reduction, and tumor microenvironment immunomodulation will be assessed by tumor immune cell infiltration analysis. Pembrolizumab is an anti-PD1 checkpoint inhibitor for human use—a modified form is used in this example, optimized for the mouse model used in this example.

[0440] Methods: MT5 cells are a highly tumorigenic mouse cell line with mutations in KRAS and p53, originally derived from the pancreas of KRAS-p53-Cre (KPC) mice carrying a triple gene transfection of pancreatic duct adenocarcinoma. These cells can be subcutaneously grown in syngeneic C57BL6 mice, where they develop into highly aggressive tumors and can be used to evaluate immunotherapy treatment. Two × 10⁶ MT5 cells were injected into one side of the abdomen of C57BL6 mice to induce tumor formation. When the tumor reached approximately 50 mm³, the animals were randomly assigned to treatment groups (n=9) and different treatments were initiated. The treatment groups were the mediator, ABTL0812, and anti-PD1. ABTL0812 was administered orally at a dose of 480 mg / kg / day. 200 μg / dose of anti-PD1 antibody was administered intraperitoneally every three days. Tumor volume and body weight were monitored three times per week. At the end of the treatment, mice were euthanized, tumors were collected, and single-cell suspensions were obtained by digestion with a culture medium containing collagenase and lipase, followed by treatment with trypsin and DNA hydrolase. Spleens were collected from the treated mice, crushed using a filter, and treated with trypsin and DNA hydrolase to obtain single cells. After obtaining the cell suspensions, cancer cells and immune cells infiltrating the tumors were stained with specific antibodies targeting different immune cell subsets, and further analyzed using flow cytometry. The combinations used were: Th1 cells = CD45+ CD4+ CCR4- CXCR3+, Th2 cells = CD45+ CD4+ CCR4+ CXCR3-, bone marrow cells = CD45+ CD11b+ Ly6C+, and NK cells = CD45+ NK1.1+.

[0441] Results: ABTL0812 alone demonstrated anticancer efficacy against MT5 tumors: it significantly reduced tumor volume compared to the MT5-treated group and showed similar efficacy to antiPD1 treatment alone. None of the treatments showed any change in mouse body weight or any clinical signs of poisoning, pain, or disease. Furthermore, ABTL0812 induced an increase in intratumoral bone marrow cells, which was associated with its ability to enhance the M1 phenotype in vitro; this increase in bone marrow cells was accompanied by an increase in the percentage of intratumoral NK cells (cells with anticancer activity). Additionally, the spleens of mice treated with ABTL0812 showed an increased Th1 / Th2 ratio, an increase indicating a pro-inflammatory immune system response (p<0.001). See Figure 27 in this paper.

[0442] Conclusion: By modulating the tumor microenvironment towards a more pro-inflammatory and anti-tumor environment, ABTL0812 demonstrated anti-cancer efficacy in a mouse pancreatic cancer model using MT5 cells. ABTL0812 increased the Th1 / Th2 ratio in the spleen, indicating a pro-inflammatory environment in the spleen, commonly used as an indicator of mouse immune system activation. Therefore, ABTL0812 induced an increase in intratumoral bone marrow cells and NK cells, indicating pro-inflammatory anti-tumor immune infiltration. Importantly, this immunomodulatory effect mediated by ABTL0812 was significantly higher compared to anti-PD1 treatment. Pancreatic cancer is considered a highly immunosuppressive and low-immunogenic tumor, and immunotherapy alone has not shown promising results in this type of cancer. These data suggest that ABTL0812 can more effectively promote the transformation of cold pancreatic tumors into hot and more immunogenic tumors than anti-PD1, thus highlighting its potential for enhanced anti-cancer efficacy in combination with immunotherapy and chemotherapy.

[0442] 8.5: In a mouse pancreatic cancer model using MT5 cells implanted in C57BL6 mice, the anticancer activity and tumor microenvironment immune regulation mediated by the combination of ABTL0812 with anti-PD1 and FOLFIRINOX.

[0443] Objective: To investigate the antitumor activity and in vivo tumor immunomodulatory effects of ABTL0812 in combination with anti-PD1 and Folfirinox. Previous studies have demonstrated the ability of ABTL0812 to enhance the efficacy of Folfirinox, the standard-of-care treatment for human advanced pancreatic cancer, in a xenograft model of human pancreatic cancer using MiaPaca2 cells implanted in nude mice (Example 3.1). Based on the results of ABTL0812-mediated tumor microenvironment modulation in vivo (Example 8.4), it was decided to test the triple combination to evaluate its efficiency in tumor volume reduction. Pembrolizumab is an anti-PD1 checkpoint inhibitor for human use—a modified form was used in this example, optimized for the mouse model used in this example.

[0444] Methods: As in Example 8.4, 2 × 10⁶ MT5 cells were injected into one side of the abdomen of C57BL6 mice to induce tumor formation. When the tumor reached a volume of approximately 50 mm³, the animals were randomly assigned to treatment groups (n=9) and different treatments were initiated. The treatment groups were methyltransferase, anti-PD1, ABTL0812 + Folfirinox, and the triple combination ABTL0812 + anti-PD1 + Folfirinox. ABTL0812 was administered orally at a dose of 480 mg / kg / day. Anti-PD1 antibody was administered intraperitoneally every three days at a dose of 200 μg / dose. The Folfirinox chemotherapy combination was administered intraperitoneally once a week for a total of four times. Mice were administered 30 mg / kg of 5-FU, 50 mg / kg of leucovorin, 50 mg / kg of irinotecan, and 2.5 mg / kg of oxaliplatin intraperitoneally over two separate days. 5-FU and leucovorin were administered on Tuesday, and irinotecan and oxaliplatin on Thursday. Tumor volume and body weight were monitored three times a week. At the end of treatment, mice were euthanized, tumors were collected, and single-cell suspensions were obtained by digestion with a culture medium containing collagenase and lipase, followed by treatment with trypsin and DNA hydrolase. After obtaining the cell suspensions, cancer cells and immune cells infiltrating the tumors were stained with specific antibodies targeting different immune cell subsets, and further analyzed using flow cytometry. The combination used was: bone marrow cells = CD45+ CD11b+ Ly6C+ and CD8 cells = CD45+ CD3- CD8+.

[0445] Results: Compared with the other treatments, the triple combination treatment of ABTL0812, anti-PD1, and FOLFIRINOX showed the highest antitumor activity, with the most significant reduction in tumor volume. None of the treatments showed any change in mouse body weight or any clinical signs of poisoning, pain, or disease. When analyzing tumor immune infiltration, the anticancer efficacy against MT5 tumors was associated with a significant increase in intratumoral bone marrow cells and CD8 cells, which demonstrated anticancer activity and promoted a pro-inflammatory phenotype. None of the other treatments showed a significant increase in CD8 anticancer cells (p<0.001). See Figure 28 in this paper.

[0446] Conclusion: By modulating the tumor microenvironment towards a more pro-inflammatory and anti-tumor environment, the combination of ABTL0812 with anti-PD1 and FOLFIRINOX demonstrated enhanced anti-cancer efficacy in a mouse pancreatic cancer model using MT5 cells. ABTL0812 increased intratumoral bone marrow cells, which was associated with an increase in CD8 anti-cancer immune cells, which could be explained by a more pro-inflammatory and anti-cancer environment. Pancreatic cancer is considered a highly immunosuppressive and low-immunogenic tumor, and immunotherapy alone has not shown very promising results in treating this type of cancer. These data suggest that the triple combination of ABTL0812 + anti-PD1 and FOLFIRINOX can provide a more effective alternative for treating this type of cancer.

[0446] References

[0447] 1: EP2409963B1 (Lipopharma - applied in 2010)

[0448] 2: Erazo et al.; Clinical Cancer Research; 22(10) May 15, 2016

[0449] 3:WO2018 / 210830A1 (Ability Pharmaceuticals)

Claims

1. A pharmaceutical combination comprising: (A): COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812) or a pharmaceutically acceptable salt thereof; and (B3): an immunotherapeutic compound that is a checkpoint inhibitor, selected from the group consisting of anti-PD1 antibodies and anti-PDL1 antibodies; the pharmaceutical combination is intended for use simultaneously, separately or sequentially in the treatment of cancer in human patients, wherein the treatment is an immunotherapy for cancer.

2. The pharmaceutical combination as described in claim 1, wherein: The anti-PD1 antibody is nivolumab, pembrolizumab, or spartalizumab; the anti-PDL1 antibody is atezolizumab, avelumab, or durvalumab.

3. The pharmaceutical combination as described in claim 1, wherein compound (B3) is an anti-PD1 antibody.

4. The pharmaceutical combination as claimed in claim 1, wherein compound (A) is the sodium salt of COOH-CHOH-(CH2)6-(CH=CH-CH2)2-(CH2)3-CH3 (ABTL0812).

5. The pharmaceutical combination as claimed in claim 1, wherein the cancer is at least one cancer selected from the group consisting of: lung cancer; non-small cell lung cancer; squamous cell carcinoma; adenocarcinoma; endometrial cancer; pancreatic cancer; glioblastoma; drug-resistant recurrent breast cancer; head and neck cancer; multiple myeloma; neuroblastoma; bile duct cancer; colorectal cancer; laryngeal cancer; tongue cancer; prostate cancer; breast cancer; ovarian cancer; liver cancer; esophageal cancer; gallbladder cancer; bladder cancer; and stomach cancer.

6. The pharmaceutical combination as described in claim 5, wherein the endometrial cancer is serous endometrial carcinoma or endometrioid carcinoma.

7. A pharmaceutical combination as described in any one of claims 1 to 6, wherein the pharmaceutical combination is a single composition comprising both compound (A) and compound (B3).

8. A pharmaceutical combination as described in any of claims 1 to 6, wherein compound (A) is administered prior to the administration of compound (B3).

9. A pharmaceutical combination as described in any one of claims 1 to 6, wherein the pharmaceutical combination further comprises at least one compound (B1), wherein compound (B1) is a chemotherapeutic compound.

10. The pharmaceutical combination as described in claim 9, wherein compound (B1) is selected from the group consisting of: temozolomide; topotecan; irinotecan; cyclophosphamide; fluorouracil; cisplatin; carboplatin; oxaliplatin; leucovorin; cranberry; bleomycin; capecitabine; mitomycin B; paclitaxel; albumin-bound paclitaxel; docetaxel; gemcitabine; methotrexate; pemetrexed; cytarabine; mercaptopurine; glucosamine; ixabepilone; nimustine; carmustine. Lomustine; Mitoxantrone; Etoposide; Vincristine; Vincristine; and Tamoxifen.

11. The pharmaceutical combination as described in claim 9, wherein compound (B1) is selected from the group consisting of: temozolomide; topotecan; irinotecan; cyclophosphamide; fluorouracil; oxaliplatin; methyltetrahydrofolate; cranberry; carboplatin; and paclitaxel.

12. The pharmaceutical combination as described in claim 11, wherein compound (B1) is paclitaxel and carboplatin.

13. The pharmaceutical combination as described in claim 11, wherein compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil.

14. The pharmaceutical combination as claimed in claim 11, wherein compound (B1) is paclitaxel and carboplatin, compound (B3) is an anti-PD1 checkpoint inhibitor antibody, and the cancer is lung cancer or endometrial cancer.

15. The pharmaceutical combination as claimed in claim 11, wherein compound (B1) is irinotecan, folate, oxaliplatin and fluorouracil, compound (B3) is an anti-PD1 checkpoint inhibitor antibody, and the cancer is pancreatic cancer.

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