A niclosamide containing compound for the treatment of proliferative diseases and modulation of the immune system

Abstract

The invention relates to the field of 26S proteasome inhibition and modulation by niclosamide and to a method for treating diseases related to increased proliferation, immune system, inflammation and neurodegenerative disorders by inhibition and modulation of the 26S proteasome in living cells, and to a niclosamide-containing compound for use in this treatment.

Description

[0001] A NICLOSAMIDE CONTAINING COMPOUND FOR THE TREATMENT OF PROLIFERATIVE DISEASES AND MODULATION OF THE IMMUNE SYSTEM

[0002] Technical Field

[0003] The invention relates to the field of 26S proteasome inhibition and modulation using niclosamide. It specifically relates to methods of treating diseases associated with increased cell proliferation, immune system dysregulation, inflammation, and neurodegenerative disorders through the inhibition and modulation of the 26S proteasome in living cells. Additionally, the invention encompasses a niclosamide- containing compound for use in such treatments.

[0004] Prior Art

[0005] Despite recent advances in treatment approaches, cancer is still one of the leading causes of death worldwide. Compounds belonging to the benzimidazole class have been found to exert cytostatic and cytotoxic effects on colorectal cancer (CRC) and hepatocellular carcinoma (HCC)-derived cell lines and in vivo models (1).

[0006] Similarly, the halogenated salicylanilide compounds niclosamide, raffoxanilide and closantel have been reported to affect cancer cells. These compounds were found to reduce tumor burden and liver metastasis in mouse models mimicking sporadic CRC. Furthermore, niclosamide showed significant anticancer activity in pancreas and esophagus (2).

[0007] It is important to underline that the potential antitumor effects of various anthelmintic drugs are not limited to the aforementioned cytostatic and proapoptotic properties, as several studies have highlighted their ability to modulate host immunity (3). Immunological effects such as the "production of proinflammatory cytokines and chemokines in response to LPS" and the "regulation of T-cells" have been described in relation to niclosamide involvement.

[0008] It has also been reported that niclosamide affects the activation of other immune cell subsets such as dendritic cells (DCs) and induces the secretion of proinflammatory cytokines (i.e. TNF-_ and IL-6) and chemokines (e.g. MIP1, MCP1) (4).

[0009] However, it is also known that some findings suggest that niclosamide instead inhibits antigen-specific activation of T cells by DCs (the primary cytotoxic T-cell response against cancer) due to down-regulation of costimulatory and major histocompatibility complex (MHC) molecules on the cell membrane.

[0010] Patent document EP2699319B1 mentions a formulation containing niclosamide for the treatment of cancer.

[0011] Patent document EP3062808B1 mentions a formulation containing niclosamide for the treatment of prostate cancer.

[0012] Despite the above applications and many other findings, the reason for both the tumor suppressor effect and the immune modulatory effect of niclosamide has not been fully elucidated.

[0013] Detailed Description of the Invention

[0014] In our ongoing work on the present invention, we have determined that niclosamide causes a dramatic decrease in the chemotrypsin activity of the 20S / 26S Proteasome in HepG2 cells, approximately 3-fold, at 2 hours of incubation time in the cell (Figure 1). In our ongoing study, we found that Niclosamide also statistically significantly decreased the proteasome activities of trypsin and caspase-like effects (Fig. 2 and 3). In our studies with T98G glioblastoma cells, we determined that niclosamide significantly decreased chemotrypsin, trypsin and caspase-like activities in a similar manner (Data not shown).

[0015] Cell cycle-related proteins, such as p53, p27, p21, belong to the group of intrinsically disordered proteins (IDPs). The lack of a stable 3D structure of these proteins makes them more susceptible to modulation by the proteasome system than other ordered proteins. Proteasome system is known to play a more important role in the degradation of these protein types. The proteasome system includes the ubiquitin-proteasome system (UPS) and the ubiquitin-independent proteasome system (UIPS). The UPS requires ubiquitination of proteins before transporting them to the 26S proteasome, consisting of the 19S regulatory particle (19S RP) and the 20S core particle (20S CP). In contrast, the UIPS bypasses the 19S structures of the 26S and primarily includes the 20S core and regulators such as PA28s. While the 26S proteasome degrades ubiquitinated proteins, 20S-mediated proteolysis does not require ubiquitination. It directly degrades misfolded, oxidatively damaged, intrinsically disordered proteins (IDPs). P-amyloid (Alzheimer’s disease), Tau (Alzheimer’s disease), a-synuclein (Parkinson's disease), and cell cycle-related proteins such as p53, p27, and p21 are IDPs and targets of the 20S proteasome (5). In the next study, we investigated how p53, p27, p21, proteins were affected by the proteasome inhibition induced by niclosamide. The results showed that niclosamide increased all p53, p27, p21, proteins (Figure 4).

[0016] The expected effect of the increase in p53, p27, and p21 proteins, which are related to cell proliferation and apoptosis, is the cessation of cell proliferation and / or a decrease in the number of cells, with or without apoptosis. Therefore, when we investigated the proliferation of HepG2 cells treated with niclosamide 48 hours later using the MTT assay, it was determined that the proliferation of HepG2 cells decreased significantly (Figure 5).

[0017] In our ongoing studies in our laboratory, we have observed that increased proteasome activity induces changes in the amount or activity ofNFkB and STAT3. In our next experiment, we investigated how NFkB-Luc signaling changes in the presence and absence of niclozamide in HepG2-pRetro- NFkB-Luc cells expressing continuous NFkB-Luc, which we had previously established in our laboratory. We used the chemical celastrol, which is known to inhibit NFkB, as a negative control to check the functionality of the NFkB-Luc construct. The results showed that niclozamide significantly increased NFkB-Luc signaling (Figure 6).

[0018] In one embodiment, the invention describes compounds comprising a niclosamide, a niclosamide-like and a pharmaceutically acceptable carrier that reduces 20S and / or 26S proteasome activity below basal levels.

[0019] The invention also provides compounds that can be used for the treatment of many proliferative diseases, including cancer, by inhibition or modulation of the 20S and / or 26S proteasome.

[0020] The invention provides compounds for the inhibition of the proteolytic activity of the immunoproteasome in addition to the 20S proteasome and the 26S proteasome system by the use of niclosamide and / or salts thereof.

[0021] The invention also provides compounds that can be used for the treatment of proliferative diseases, including cancer, and for the arrest of cell proliferation due to the dramatic increase of tumor suppressor proteins such as p21, p27 and p53 by inhibition or modulation of the 20S and / or 26Sproteasome.

[0022] This method of reducing proteasome activity is through direct or indirect inhibition of the 20S and / or 26S proteasome.

[0023] This method of reducing proteasome activity modulates proteasome activity through the ubiquitin-dependent proteasome system (UPS) and / or ubiquitin- independent proteasome system (UIPS) pathways. The methods of the present invention may be used to treat a patient in need. In some embodiments, such a patient includes those with cancer, a neurodegenerative disease, an autoimmune disease, an infectious disease, or an inflammatory disease.

[0024] In some embodiments, an autoimmune disease includes one from the following group of diseases: alopecia areata, ankylosing spondylitis, arthritis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune inner ear disease (also known as Meniere's disease), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, Behget's disease, Crohn's disease, type 1 diabetes mellitus, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, inflammatory bowel disease, lupus nephritis, multiple sclerosis, myasthenia gravis, pemphigus, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, rheumatic fever, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus (SLE), ulcerative colitis, vitiligo, and Wegener's granulomatosis.

[0025] In some embodiments, cancer includes one of the types of cancer specified in this paragraph; leukemia; carcinoma of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, prostate, head, neck and skin; hematopoietic tumors of the lymphoid lineage, acute lymphocytic leukemia; B-cell lymphoma; Burkett's lymphoma; hematopoietic tumors of the myeloid lineage, acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, fibrosarcoma, rhabdomyasarcoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; neuroblastoma and glioma.

[0026] In some embodiments, the inflammation includes one of the disease types mentioned in this paragraph; rheumatoid arthritis, spondyloatopathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, juvenile arthritis, bronchitis, bursitis, gastritis, inflammatory bowel disease, ulcerative colitis, acne vulgaris, asthma, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis and interstitial cystitis. These Figures are as follows;

[0027] Figure 1. Effects of niclosamide on the chemotrypsin activity in HepG2 cell

[0028] Figure 2. Effects of niclosamide on the trypsin activity in HepG2 cell.

[0029] Figure 3. Effects of niclosamide on the caspase-like activity in HepG2 cell.

[0030] Figure 4. Effect of niclosamide on p21, p53 and p27 protein amounts in HepG2 cells

[0031] Figure 5. Effect of niclosamide on HepG2 cell proliferation after 48 hours (MTT assay result).

[0032] Figure 6. Effect of niclosamide on NFkB-Luc fusion protein in HepG2 cells containing Retro-X- NFkB-Luc construct

[0033] REFERENCES

[0034] 1. Blaszczak-Swiatkiewicz, K. Antiproliferative Aspect of Benzimidazole Derivatives’ Activity and Their Impact on NF-kappaB, Expression. Molecules 2019, 24, 3902

[0035] 2. Ye, T.; Xiong, Y.; Yan, Y.; Xia, Y.; Song, X.; Liu, L.; Li, D.;Wang, N.; Zhang, L.; Zhu, Y.; et al. The anthelmintic drug niclosamide induces apoptosis, impairs metastasis and reduces immunosuppressive cells in breast cancer model. PLoS ONE 2014, 9,

[0036] 3. Gargantilla, M.; Persoons, L.; Kauerova, T.; Del Rio, N.; Daelemans, D.; Priego, E.M.; Kollar, P.; Perez-Perez, M.J. Hybridization Approach to Identify Salicylanilides as Inhibitors of Tubulin Polymerization and Signal Transducers and Activators of Transcription 3 (STAT3). Pharmaceuticals 2022, 15, 835.

[0037] 4. Wu, C.S.; Li, Y.R.; Chen, J.J.; Chen, Y.C.; Chu, C.L.; Pan, I.H.; Wu, Y.S.; Lin, C.C. Antihelminthic niclosamide modulates dendritic cells activation and function. Cell. Immunol. 2014, 288, 15-23.

[0038] 5. Peter E. Wright & H. Jane Dyson, Intrinsically disordered proteins in cellular signalling and regulation. Nature Reviews Molecular Cell Biology volume 16, pages 18-29 (2015)

Claims

CLAIMS1. The invention describes a composition for use in reducing proteasome activity and / or regulating the protein homeostasis network using a niclosamide molecule and a pharmaceutically acceptable carrier.

2. A compound of claim 1 wherein the use of the compound and / or salts thereof inhibits the proteolytic activity of the 20S proteasome, 26S proteasome and immunoproteasome.

3. A compound according to claim 1, wherein the niclosamide causes the activity of the proteasome (20S and / or 26S) to decrease below basal levels.

4. A compound according to claim 1 or 2, wherein the niclosamide is an antagonist that directly or indirectly inhibits the 20S and / or 26S proteasome.

5. A compound according to claim 1, wherein in particular the chemotrypsinlike activity of the 20S proteasome and the 26S proteasome is inhibited.

6. A compound according to claim 1, wherein in particular the trypsin-like activity of the 20S proteasome and the 26S proteasome is inhibited.

7. A compound according to claim 1, wherein in particular the caspase-like activity of the 20S proteasome and the 26S proteasome is inhibited.

8. A compound according to claim 1, wherein the chymotrypsin-like, trypsinlike and caspase-like activities of the 20S proteasome and the 26S proteasome are simultaneously inhibited.

9. A compound according to any one of claims 1 to 3, wherein the niclosamide reduces proteasome activities in a UPS (Ubiquitin-ATP -Dependent Proteolysis) dependent and / or UPS independent manner.

10. A compound according to any one of claims 1 to 8, characterized in that the compound is present in an amount to effectively inhibit proteasome function in a cell or a mammal.

11. A compound according to any one of claims 1 to 8, wherein the niclosamide provides methods of increasing the amount cell proliferation-related proteins p21 p27 and p53 within the cell by decreasing proteasome activity.

12. A compound according to any one of claims 1 to 8, wherein the niclosamide provides compositions and methods of preventing and / or treating cell proliferative diseases, including cancer.

13. The compound according to claim 12, wherein said cell proliferative diseases include, but are not limited to, proteionopathic cell proliferative diseases, disorders and / or various conditions characterized by abnormal cell growth, preferably abnormally increased cell proliferation, cancer, cancer metastasis, immune-mediated reactions and diseases (e.g, transplant rejection, graft vs host disease, immune reaction to gene therapy, autoimmune diseases, pathogen-induced immune dysregulation, etc.), certain circulatory diseases and certain neurodegenerative diseases.

14. A compound according to claim 12 or 13, wherein said cancer is a cancer as set forth in this paragraph, including, but not limited to; carcinoma, leukemias and lymphomas such as cutaneous T-cell lymphomas, peripheral T-cell lymphomas, human T-cell lymphotropic virus-associated lymphomas such as adult T-cell leukemia / lymphoma, B-cell lymphoma, acute lymphocytic leukemia, acute non-lymphocytic leukemias, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndrome, mesothelioma, head and neck cancers (e.g. genitourinary cancers (e.g. prostate, bladder, kidney, uterus, ovary, testis, rectal and colon), lung cancer, breast cancer, liver cancer, colon cancer, small bowel cancer, pancreatic cancer, melanoma and other skin cancers, gastric cancer, central nervous system neoplasms, neuroectodermal cancer, spinal axis tumors, glioma, meningioma and pituitary adenoma, liver cancer and thyroid cancer and / or brain tumors, childhood solid tumors such asneuroblastoma, retinoblastoma, Wilms tumor, bone tumors and soft tissue sarcomas and their metastases15. A compound according to any one of claims 1 to 8, wherein the niclosamide provides compositions and methods of preventing and / or treating immune system related diseases by providing immune system modulation.

16. A compound according to claim 15, wherein the immune system-related diseases set forth in this paragraph include, but are not limited to; ankylosing spondylitis, arthritis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune inner ear disease (also known as Meniers disease), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune hepatitis, Bechet's disease, Crohn's disease, diabetes mellitus type 1, glomerulonephritis, Graves' disease, Guillain- Barre syndrome, inflammatory bowel disease, lupus nephritis, multiple sclerosis, myasthenis gravis, pemphigus, pemic anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, Raynaud's Phenomenon includes rheumatic fever, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemiclupus erythematosus (SLE), ulcerative colitis, vitiligo and Wegener's granulomatosis.

17. A compound according to claim 15, wherein said immune-mediated responses are inflammation-related diseases, including, but not limited to, those set forth in this paragraph; rheumatoid arthritis, spondylopathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, juvenile arthritis, bronchitis, bursitis, gastritis, inflammatory bowel disease, ulcerative colitis, acne vulgaris, asthma, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel disease, inflammatory pelvic disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis.