Methods of treating inflammation

Abstract

The present disclosure relates to anti-inflammatory agents, particularly to compounds, compositions, and methods for treating inflammation and inflammation-related conditions, such as arthritis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to U.S. Provisional Application No. 63 / 333,281, filed on April 21, 2022, the entire disclosure of which is incorporated herein by reference.

[0003] Reference to sequence listings submitted electronically

[0004] The computer-readable nucleotide / amino acid sequence listing submitted contemporaneously and identified as follows is incorporated by reference in its entirety: 16 kilobyte xml document, named "83864-388941_SL.xml", created on April 17, 2023. Technical Field

[0005] The present disclosure relates to anti-inflammatory agents, and more particularly to compounds, compositions and methods for treating inflammation and inflammation-related disorders, such as arthritis. Background Art

[0006] Since its discovery as a bioactive molecule in the late 1980s, nitric oxide (NO) has been found to play an important role as a signaling molecule in many parts of the organism and as a cytotoxic or regulatory effector molecule of the innate immune response. NO, synthesized by nitric oxide synthase (NOS), is the smallest known bioactive molecule and can be produced by a variety of cells. NO plays an important role in neurotransmission, vascular function, host defense, and immune regulation.

[0007] Three NOS isoforms have been identified: neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). After the enzyme activation of the constitutively expressed endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS), the signal molecule NO is synthesized on demand in a relatively short period of time (seconds to minutes). In contrast, inducible NO synthase (iNOS) is only expressed after cell activation and then produces NO in a relatively long period of time (hours to days). iNOS can be produced by a variety of cells after being induced by cytokines or other stimulants. Therefore, the regulated short pulse synthesis and constant NO production distinguish the physiological and pathophysiological effects of NO.

[0008] NO is an important pro-inflammatory mediator that has an effect on the immune system. In fact, NO plays a dual role in the immune-inflammatory process. On the one hand, NO can kill microorganisms and has a protective effect on the body, helping to fight various viruses, such as herpes simplex virus (HSV). On the other hand, NO can damage normal tissue cells to produce pathogenic effects, and is widely involved in various diseases, such as the manifestation of Borna disease. According to existing studies, macrophages and other effector cells, including neutrophils, monocytes and endothelial cells are the main effector cells involved in the antimicrobial effect of NO.

[0009] The iNOS gene is under the transcriptional control of cytokines, lipopolysaccharide (LPS) and other inflammatory mediators. Abnormal expression of iNOS plays a key role in many inflammatory diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, multiple sclerosis, systemic sclerosis, Sjögren's syndrome ( syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, celiac disease, glomerulonephritis, dilated cardiomyopathy, cutaneous lupus erythematosus, systemic lupus erythematosus, dermatitis, apical periodontitis, and psoriasis. For example, NO produced by iNOS appears to be critically involved in the pathogenesis of OA and contributes to OA pathogenesis by regulating ECM homeostasis and cytokine expression, leading to oxidative damage and chondrocyte apoptosis. Using immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization, iNOS expression has been described in rheumatoid arthritis (RA), multiple sclerosis (MS), and Sjögren's syndrome.

[0010] Many studies have suggested similar roles for iNOS activity in human disease. However, it remains to be shown whether inhibition of iNOS-derived NO in human patients will protect against tissue-destructive processes such as OA, RA, MS type 1 diabetes, etc. Thus, there is a significant unmet need for improved treatments for a variety of inflammatory diseases, including rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc. Summary of the invention

[0011] In one aspect, the present disclosure provides a method of treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc., comprising administering a therapeutically effective amount of a compound of the formula

[0012]

[0013] or a pharmaceutically acceptable salt thereof.

[0014] In another aspect, the present disclosure provides a pharmaceutical composition comprising a compound of the formula

[0015]

[0016] or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient. In some embodiments, the compound in the pharmaceutical composition is taken in a therapeutically effective amount for treating inflammatory diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0017] In another aspect, the present disclosure provides compounds of the formula

[0018]

[0019] Or the use of a pharmaceutically acceptable salt thereof for preparing a medicament for treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0020] Other embodiments, features and advantages of the present disclosure will be apparent from the following embodiments and through practicing the present disclosure. The compounds, methods and compositions of the present disclosure may be described as any of the embodiments listed below. It should be understood that any embodiment described herein may be used in combination with any other embodiment described herein to the extent that the embodiments do not contradict each other.

[0021] 1. A method for treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc., comprising administering a therapeutically effective amount of a compound of the formula

[0022]

[0023] or a pharmaceutically acceptable salt thereof.

[0024] 2. The method according to item 1, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0025] 3. The method according to item 1, wherein the inflammatory disease is osteoarthritis (OA).

[0026] 4. The method according to any one of items 1 to 3, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

[0027] 5. The method according to any one of items 1 to 4, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

[0028] 6. The method according to any one of items 1 to 5, wherein the compound is administered orally.

[0029] 7. The method according to any one of items 1 to 6, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0030] 8. The method according to any one of items 1 to 7, wherein the therapeutically effective amount of the compound is administered once a week, twice a week, once a day (QD), twice a day (BID) or three times a day (TID).

[0031] 9. The method according to any one of items 1 to 8, wherein the therapeutically effective amount of the compound inhibits NO production in the patient.

[0032] 10. The method according to any one of items 1 to 9, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

[0033] 11. The method according to any one of items 1 to 10, further comprising administering one or more additional therapeutic agents to the patient.

[0034] 12. The method according to item 10, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

[0035] 13. The method according to item 10, wherein the one or more additional therapeutic agents are selected from the group consisting of aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, icam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxoprozin, mefenamic, celecoxib, cortisone, prednisone, and methylprednisone.

[0036] 14. A pharmaceutical composition comprising a compound of the formula

[0037]

[0038] or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient, wherein the compound is taken in a therapeutically effective amount for treating inflammatory diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0039] 15. The pharmaceutical composition according to item 14, wherein the amount of the compound in the composition is from about 1 mg to about 1000 mg.

[0040] 16. The pharmaceutical composition according to item 14 or 15, wherein the therapeutically effective amount of the compound inhibits NO production in a patient.

[0041] 17. The pharmaceutical composition according to any one of items 14 to 16, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

[0042] 18. The pharmaceutical composition according to any one of items 14 to 17, wherein the composition is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

[0043] 19. The pharmaceutical composition according to any one of items 14 to 18, wherein the composition is administered orally.

[0044] 20. A compound of the formula

[0045]

[0046] Or the use of a pharmaceutically acceptable salt thereof for preparing a medicament for treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0047] 21. The use according to item 20, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0048] 22. The use according to item 20, wherein the inflammatory disease is osteoarthritis (OA).

[0049] 23. The use according to any one of items 20 to 22, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

[0050] 24. The use according to any one of items 20 to 23, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

[0051] 25. The use according to any one of items 20 to 24, wherein the compound is administered orally.

[0052] 26. The use according to any one of items 20 to 25, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0053] 27. The use according to any one of items 20 to 26, wherein the therapeutically effective amount of the compound is administered once a week, twice a week, once a day (QD), twice a day (BID) or three times a day (TID).

[0054] 28. The use according to any one of items 20 to 27, wherein the therapeutically effective amount of the compound inhibits NO production in the patient.

[0055] 29. The use according to any one of items 20 to 28, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

[0056] 30. The use according to any one of items 20 to 29, wherein the treatment further comprises administering one or more additional therapeutic agents to the patient.

[0057] 31. The use according to item 30, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

[0058] 32. The use according to item 30, wherein the one or more additional therapeutic agents are selected from the group consisting of: aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone and methylprednisolone.

[0059] 33. A compound of the formula

[0060]

[0061] Or a pharmaceutically acceptable salt thereof, for use in a method for treating an inflammatory disease in a subject, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0062] 34. The compound according to item 33, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0063] 35. The compound according to item 33, wherein the inflammatory disease is osteoarthritis (OA).

[0064] 36. The compound according to any one of clauses 33 to 35, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

[0065] 37. The compound according to any one of items 33 to 36, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

[0066] 38. The compound according to any one of items 33 to 37, wherein the compound is administered orally.

[0067] 39. The compound according to any one of items 33 to 38, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0068] 40. The compound according to any one of items 33 to 39, wherein the method comprises administering a therapeutically effective amount of the compound once a week, twice a week, once a day (QD), twice a day (BID) or three times a day (TID).

[0069] 41. The compound according to any one of items 33 to 40, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit NO production in a patient.

[0070] 42. The compound according to any one of items 33 to 41, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit iNOS gene expression in the patient.

[0071] 43. The compound according to any one of items 33 to 42, further comprising administering one or more additional therapeutic agents to the patient.

[0072] 44. A compound according to item 43, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

[0073] 45. The compound according to item 43, wherein the one or more additional therapeutic agents are selected from the group consisting of: aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone and methylprednisolone. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is a graph showing the different effects of Compound (I) and Compound (II) on cell growth of primary human chondrocytes. Concentrations up to 5 mM were tested for each drug. IL-1β (IL-1B): 5 ng / mL.

[0075] Figure 2is a graph showing the comparison of NDX2 and NAG in IL-6 production in primary human chondrocytes. Concentrations of 1.25 mM and 2.5 mM were tested for each drug. IL-1β (IL-1B): 5 ng / mL.

[0076] FIG. 3A to FIG. 3B Shown is a comparison of NDX2 and NAG on Cox-2 expression in primary human chondrocytes as determined by western blot. Figure 3A Is a gel showing the effect of NDX2 and NAG on Cox-2 expression in primary human chondrocytes (lane 1: NT; lane 2: IL-1β; lane 3: 2 mM IL-1β+NDX2; lane 4: 2 mM IL-1β+NAG). Figure 3B is a graph obtained from semi-quantitative analysis of gel images. IL-1β: 5 ng / mL.

[0077] Figure 4 The graph shows the effect of NDX2 on the mRNA level of IL-6 in primary human chondrocytes. IL-1β (IL-1B): 5 ng / mL.

[0078] Figure 5 The graph shows the effect of NDX2 on the mRNA level of Cox-2 in primary human chondrocytes. IL-1β (IL-1B): 5 ng / mL.

[0079] Figure 6 is a graph showing the effect of nitrite production in primary human chondrocytes. IL-1β(IL-1B): 5ng / mL.

[0080] Figure 7 is a graph showing the effects of NDX1 and NDX2 on cell growth of mouse macrophage cell line RAW264.7 cells. Concentrations up to 5 mM were tested for each drug.

[0081] Figure 8 is a graph showing the comparison of NDX1, NDX2 and NAG in IL-6 production in RAW264.7 cells. Concentrations up to 5 mM were tested for each drug. LPS: 100 ng / mL.

[0082] Fig. 9It is a gel image showing the comparison of iNOS and expression of NDX1, NDX2 and NAG in mouse RAW264.7 macrophages by western blotting. LPS: 100 ng / mL. Lane 1: NT; Lane 2: LPS; Lane 3: 0.1 mM LPS+NDX1; Lane 4: 1 mM LPS+NDX1; Lane 5: 0.5 mM LPS+NDX2; Lane 6: 5 mM LPS+NDX2; Lane 7: 0.5 mM LPS+NAG; Lane 8: 5 mM LPS+NAG.

[0083] Fig.10 Is a gel image showing the comparison of NDX1, NDX2 and NAG in Cox-2 expression in mouse RAW264.7 macrophages by Western blotting. LPS: 100 ng / mL. Lane 1: NT; Lane 2: LPS; Lane 3: 0.1 mM LPS + NDX1; Lane 4: 1 mM LPS + NDX1; Lane 5: 0.5 mM LPS + NDX2; Lane 6: 5 mM LPS + NDX2; Lane 7: 0.5 mM LPS + NAG; Lane 8: 5 mM LPS + NAG.

[0084] Fig.11 is a graph showing comparison of NDX1, NDX2 and NAG in iNOS expression in mouse RAW264.7 macrophages as determined by RT-PCR analysis. LPS: 100 ng / mL.

[0085] Fig.12 is a graph showing the comparison of NDX1, NDX2 and NAG in IL1β (IL1B) expression in mouse RAW264.7 macrophages determined by RT-PCR analysis. LPS: 100 ng / mL.

[0086] Fig.13 is a graph showing comparison of NDX1, NDX2 and NAG in nitrite production in LPS-activated mouse RAW264.7 macrophages. LPS: 100 ng / mL.

[0087] Fig.14 is a graph showing comparison of MPO activity from lung tissues of mice treated with various methods. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV. N=8.

[0088] Fig.15is a graph showing comparison of leukocyte migration to the peritoneal cavity from mice treated with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV. N=8.

[0089] Figures 16A to 16D show a comparison of the migration of leukocytes from mice treated with various treatments to the peritoneal cavity. Figure 16A shows images of the migration of leukocytes from mice treated with the following treatments to the peritoneal cavity: LPS: 10 mg / kg, IP; NAG: 200 mg / kg, IV. N=8. White arrows: neutrophils; red arrows: monocytes. Figure 16B shows images of the migration of leukocytes from mice treated with the following treatments to the peritoneal cavity: LPS: 10 mg / kg, IP; NDX1: 200 mg / kg, IV. N=8. White arrows: neutrophils; red arrows: monocytes. Figure 16C shows images of the migration of leukocytes from mice treated with the following treatments to the peritoneal cavity: LPS: 10 mg / kg, IP; NDX2: 200 mg / kg, IV. N=8. White arrows: neutrophils; red arrows: monocytes. Figure 16D shows a graph showing a comparison of the migration of leukocytes from mice treated with various treatments to the peritoneal cavity. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV. N=8.

[0090] Fig.17 is a graph showing comparison of serum IL6 levels in mice treated with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV. N=8.

[0091] Fig.18 is a graph showing comparison of serum TNFa levels in mice treated with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV. N=8.

[0092] Fig.19 is a graph showing comparison of serum IL6 levels in mice treated with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV. N=8.

[0093] Fig. 20 is a graph showing comparison of serum TNFa levels in mice treated with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV. N=8.

[0094] Fig.21 is a graph showing a comparison of the effects of NDX1, NDX2 and NAG on cell growth of primary mouse peritoneal macrophages. Concentrations up to 1 mM were tested for each drug. No drug was added to the untreated group (NT). There were eight replicates in each group (n=8). *P<0.05 compared to the LPS group. The experiment was repeated twice.

[0095] Fig. 22 is a graph showing the comparison of IL-6 production by NDX1, NDX2 and NAG in primary mouse peritoneal macrophages. Various concentrations (0.01 mM, 0.1 mM and 1 mM) were tested for NDX1 and NDX2, while 0.1 mM and 1 mM NAG were used as controls. LPS: 100 ng / mL. There were eight replicates in each group (n=8). *P<0.05 compared to the LPS group. The experiment was repeated twice.

[0096] Fig.23 is a graph showing the comparison of TNFα produced by NDX1, NDX2 and NAG in primary mouse peritoneal macrophages. Various concentrations (0.01 mM, 0.1 mM and 1 mM) were tested for NDX1 and NDX2, while 0.1 mM and 1 mM NAG were used as controls. LPS: 100 ng / mL. There were eight replicates in each group (n=8). *P<0.05 compared to the LPS group. The experiment was repeated twice. DETAILED DESCRIPTION

[0097] Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the particular embodiments described, and therefore may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing particular embodiments and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims.

[0098] For the sake of brevity, the disclosed disclosures (including patents) listed in this specification are incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the disclosure belongs.

[0099] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims are intended to exclude any optional elements. Therefore, this statement is intended to be combined with the recitation of claimed elements as a prerequisite basis for the use of such exclusive terminology as "solely," "only," and the like, or the use of a "negative" limitation.

[0100] As used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0101] To provide a more concise description, some quantitative expressions given herein are not limited by the term "about". It should be understood that, regardless of whether the term "about" is explicitly used, each quantity given herein is intended to refer to the actual given value, and it is also intended to refer to the approximate value of the given value based on the reasonable inference of a person of ordinary skill in the art, including equivalent values ​​and approximate values ​​obtained due to the experimental and / or measurement conditions of the given value.

[0102] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used to implement or test the present disclosure, preferred methods and materials are now described.

[0103] It should be understood that certain features of the present disclosure described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination.

[0104] Representative Examples

[0105] In some embodiments, the present disclosure provides a method of treating a subject suffering from an inflammatory disease, comprising administering a therapeutically effective amount of a compound of the formula

[0106]

[0107] In some embodiments, the present disclosure provides compounds of the formula

[0108]

[0109] or a pharmaceutically acceptable salt thereof for use in the preparation of a medicament for treating a subject suffering from an inflammatory disease. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of the formula

[0110]

[0111] or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient.

[0112] As used herein, the term "subject" or "patient" refers to a human being, or in the case of veterinary applications, it can be a laboratory animal, farm animal, livestock or wild animal. The methods described herein can be applied to subjects, including but not limited to humans, laboratory animals, such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; livestock, such as dogs, cats and rabbits; agricultural animals, such as cattle, horses, pigs, sheep, goats.

[0113] As used herein, the term "therapeutically effective amount" refers to the amount of a drug or agent that induces a biological or medical response in a subject (i.e., a tissue system, animal or human) sought by a researcher, veterinarian, doctor or other clinician, including but not limited to relieving the symptoms of the inflammatory disease being treated. In some embodiments, the therapeutically effective amount is the amount of an active agent that can treat or relieve an inflammatory disease or the symptoms of an inflammatory disease at a reasonable benefit / risk ratio applicable to any medical treatment. In some embodiments, the therapeutically effective amount is the amount of an active agent that can serve as a disease-modifying drug for the inflammatory disease being treated. In some embodiments, the therapeutically effective amount is the amount of an inactive prodrug that, when converted via normal metabolic processes, produces an amount of an active drug that can induce a biological or medical response in the subject being sought. In some embodiments, the therapeutically effective amount is the amount of an active agent that can cause a change in the activity of a biological target in a subject to which the active agent is administered. In some embodiments, the therapeutically effective amount is the amount of an active agent that can inhibit NO production in a patient's body. In some embodiments, the therapeutically effective amount is the amount of an active agent that can inhibit iNOS gene expression in a subject's body.

[0114] It will be appreciated that inflammation and inflammatory processes can occur in conjunction with a wide range of diseases and disease states. It will be appreciated that there are two types of inflammation: acute and chronic. While acute inflammation can be beneficial because it can indicate that a subject is fighting an infection and / or can help speed up the healing process from an injury or disease, chronic inflammation caused by the immune system sending chemical messengers that drive the inflammatory process for a longer amount of time can cause debilitating pain and disease. It will be appreciated that chronic inflammation is associated with heart disease, diabetes, cancer, arthritis, and intestinal diseases (e.g., Crohn's disease and ulcerative colitis). In some embodiments, the disease to be treated in connection with the present disclosure can be any disease in which the pro-inflammatory mediator nitric oxide (NO) interacts with the tissues of a subject to produce a pathogenic effect and / or drive the disease. In some embodiments, diseases in which the pro-inflammatory mediator nitric oxide (NO) may play a role include neurodegeneration in many diseases of the nervous system, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and ischemic brain injury (stroke). In some embodiments, the disease to be treated in connection with the present disclosure may be any disease in which abnormal expression of the iNOS gene plays a role in the development of the disease. Exemplary diseases include inflammatory diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, multiple sclerosis, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, celiac disease, glomerulonephritis, dilated cardiomyopathy, cutaneous lupus erythematosus, systemic lupus erythematosus, dermatitis, apical periodontitis, psoriasis, etc. In some embodiments, the inflammatory disease can be rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

[0115] It should be understood that the methods, uses, compositions or compounds described herein can be administered in any mode of administration known in the art. As used herein, "administering" includes all ways of introducing the compounds and compositions described herein into a subject, including but not limited to oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, etc. The methods, uses, compositions or compounds described herein can be administered in unit dosage forms and / or formulations containing pharmaceutically acceptable conventional non-toxic carriers, adjuvants and / or vehicles.

[0116] In some embodiments, the methods, uses, compositions or compounds described herein can be administered orally. Formulations suitable for oral administration include solid formulations, such as tablets, capsules containing particles, liquids or powders, lozenges (including liquid-filled), chewable tablets, multi-particles and nanoparticles, gels, solid solutions, liposomes, films, oval suppositories, sprays and liquid formulations.

[0117] Liquid formulations include suspensions, solutions, syrups and elixirs. These formulations can be used as fillers in soft or hard capsules and typically contain a carrier such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifiers and / or suspending agents. Liquid formulations can also be prepared by reconstituting a solid, such as a solid from a sachet.

[0118] Binders are generally used to impart cohesive qualities to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugar, polyethylene glycol, natural and synthetic gums, polyvinyl pyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous, etc.), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dihydrated calcium phosphate.

[0119] Tablets may also optionally contain surfactants, such as sodium lauryl sulfate and polysorbate 80; and glidants, such as silicon dioxide and talc. When present, surfactants may comprise 0.2% to 5% by weight of the tablet, while glidants may comprise 0.2% to 1% by weight of the tablet.

[0120] Tablets generally also contain lubricants, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and a mixture of magnesium stearate and sodium lauryl sulfate. Lubricants generally account for 0.25% to 10% by weight of the tablet, preferably 0.5% to 3% by weight.

[0121] Other possible ingredients include antioxidants, colorants, flavoring agents, preservatives and taste masking agents. Exemplary tablets contain up to about 80% drug, about 10% to 25% to about 90% binder, about 0% to about 85% diluent, about 2% to about 10% disintegrant, and about 0.25% to about 10% lubricant.

[0122] Tablet blends may be compressed directly or by roller compression to form tablets. Tablet blends or portions of blends may alternatively be wet, dry or melt granulated, melt agglomerated or extruded prior to tableting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in H. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (Marcel Dekker, New York, 1980).

[0123] Solid formulations for oral administration can be formulated as immediate and / or modified release formulations. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release formulations.

[0124] In some embodiments, the methods, uses, compositions or compounds described herein can be directly administered to the bloodstream, muscle or internal organs. Suitable modes for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous administration.

[0125] In some embodiments, the methods, uses, compositions or compounds described herein may be co-administered or co-formulated with one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone and methylprednisone.

[0126] Any effective regimen can be used to administer the compounds and compositions described herein. For example, the compounds and compositions described herein can be administered as a single dose, or the dose can be divided and administered as a multiple dose daily regimen. In addition, a staggered regimen, such as one to five days a week, can be used as an alternative to daily treatment. In some embodiments, in the methods, uses, compounds or compositions described herein, multiple doses are administered to the subject. In some embodiments, multiple doses (preferably about 2 to about 80 doses) of a compound or composition as described herein are administered to the subject, for example, at intervals of 8 to 72 hours or at intervals of 8 to 12 hours.

[0127] Any suitable treatment course of the compound of formula (I) or (II) described herein may be used. In one embodiment, individual doses and dosing regimens are selected to provide a total dose of about 1 mg to about 1000 mg mg or about 200 mg to about 1000 mg administered during a given day. In some embodiments, the compound of formula (I) or (II) is administered in a single daily dose (QD) or twice daily dose (BID) or three times daily dose (TID) in the methods or uses described herein. In some embodiments, the compound of formula (I) or (II) is administered in a dose of about 300 mg to about 900 mg per dose in a twice daily dose (BID) in the methods or uses described herein. In some embodiments, the compound of formula (I) or (II) is administered in a dose of about 600 mg to about 800 mg in a twice daily dose (BID) in the methods or uses described herein. In some embodiments, the compound of formula (I) or (II) is administered in a dose of about 700 mg in a twice daily dose (BID) in the methods or uses described herein. In some embodiments, the compound of formula (I) or (II) is administered in the methods or uses described herein for a period of several days, a week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, etc. In some embodiments, the compound of formula (I) or (II) is administered daily in the methods or uses described herein for 10 to 45 days, or until the patient's state indicates that treatment is stopped, as observed by the treating physician. In some embodiments, the compound of formula (I) or (II) is administered daily in the methods or uses described herein for 10 to 20 days, or until the patient's state indicates that treatment is stopped, as observed by the treating physician. In some embodiments, the compound of formula (I) or (II) is administered daily in the methods or uses described herein for 25 to 35 days, or until the patient's state indicates that treatment is stopped, as observed by the treating physician. In some embodiments, the compound of formula (I) or (II) is administered daily in the methods or uses described herein for about 30 days, or until the patient's state indicates that treatment is stopped, as observed by the treating physician.

[0128] It should be understood that the unit daily dosage of the compound of Formula (I) or Formula (II) may vary significantly depending on the patient's condition, the inflammatory disease being treated, the route of administration of the compound of Formula (I) or Formula (II), and the possibility of co-administration of additional therapeutic agents, as described herein. The effective amount to be administered to a patient is based on body surface area, mass, and the physician's assessment of the patient's condition.

[0129] The examples and formulations provided below further illustrate and illustrate specific aspects of the embodiments of the present disclosure. It should be understood that the scope of the present disclosure is not limited in any way by the scope of the following examples. Compounds of formula (I) and (II) (also referred to as NDX1 and NDX2, respectively) can be prepared according to the methods described in WO2022 / 051575, the entire contents of which are incorporated herein by reference. N-acetylglucosamine (also referred to as NAG) can be purchased from commercial sources, such as Sigma-Aldrich (St. Louis, MO, USA).

[0130] Example 1: Determination of the non-toxic dose range of NDX in human chondrocyte cultures

[0131] Primary Culture of Human Chondrocytes:

[0132] Normal cartilage tissue. Articular cartilage was collected from the femoral condyle and tibial plateau, which was obtained from the autopsy service of UVA Hospital and approved by the Institutional Review Board. All tissue samples were graded according to the modified Mankin scale, and only cartilage without signs of osteoarthritis was used as the source of chondrocytes. The time interval between death and the time of collecting cartilage from these knee joints in the laboratory was at least 24h and the range was up to 96h. Cartilage scraps were collected in the operating room and placed in tissue culture medium (DMEM, 10% FBS, penicillin, streptomycin) and transported to the laboratory at 4°C. Tissue was processed in the laboratory within 24 hours after collection.

[0133] Chondrocytes isolated from cartilage were digested by collagenase and maintained in continuous monolayer cultures in DMEM containing 10% FBS. Cell viability after separation of chondrocytes from normal cartilage by collagenase digestion was >95%, as determined by trypan blue stain (Sigma-Aldrich). Experiments were performed with primary or first generation cells. Trypsin was used for cell passage if necessary.

[0134] Cell proliferation analysis

[0135] Human chondrocytes were seeded in 96-well plates (5×104 cells / well) and incubated overnight with culture medium. Cells were then treated as needed and cell proliferation assays were performed using Roche Cell Proliferation Reagent WST-1 (Fisher Scientific Company, Nazareth, PA) following the encapsulation instructions provided by the manufacturer. No IL-1β and other drugs were added to the untreated group (NT). There were six replicates in each group (n=6). The experiment was repeated twice. The results are shown in Figure 1 middle.

[0136] WST-1 analysis showed that NDX1 was dose-dependently toxic to human chondrocytes in the presence of IL-1β (IL-1B). NDX1 exhibited significant cytotoxicity. NDX2 and NAG could prevent the inhibition of cell proliferation induced by IL-1β (n=6).

[0137] Data Analysis:

[0138] The values ​​obtained are presented as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni / Dunnet's test. The statistical significance level between two groups was set at P < 0.05.

[0139] Example 2: Evaluation of the preventive effect of NDX on IL-1β activation in human chondrocyte cultures

[0140] Cell treatment

[0141] Primary cultures of human chondrocytes were seeded in 96-well plates (5×104 cells / well) and treated with different drugs for 24 h. Both culture medium and cells were collected for the following analysis.

[0142] Detection of IL-6 content

[0143] The amount of IL-6 in the culture medium was measured by a commercial ELISA kit (Sigma-Aldrich) following the instructions provided by the manufacturer. Briefly, the culture medium was gradually mixed with different analytical reagents. The resulting solution was read at 450 nm on a microplate reader. The ELISA test showed that NDX2 significantly inhibited the production of IL-6 by human chondrocytes stimulated with IL-1B, where its activity was comparable to that of NAG (n=6). No IL-1β and other drugs were added to the untreated group (NT). The experiment was repeated twice. The results are shown in Figure 2 middle.

[0144] Western blot analysis

[0145] Protein was prepared from cells grown in 6-well plates (approximately 500,000 cells / well) and protein concentration was determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Samples containing 100 μg of protein were run on a 10% SDS-polyacrylamide gel at a constant current of 80 V and electrotransferred to a nitrocellulose membrane (Thermo Scientific) at a constant voltage of 10 V overnight. The membrane was blocked for 1 hour at room temperature with 5% fatty acid free bovine serum albumin (BSA) fraction V (Roche Diagnostics, Indianapolis, IN, USA) in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20), washed, and incubated overnight at 4°C in 5% BSA in TBST containing each specific primary antibody against COX-2 (Santa Cruz Biotechnology). β-actin was used as a loading control. The membrane was then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling) at room temperature for 1 hour, followed by mixing the chemiluminescent substrate for the HRP antibody and enhancer solution (Thermo Scientific) in a 1:1 ratio. The membrane was incubated in the dark with autoradiographic film (Genesee Scientific, San Diego, CA, USA), and the film was developed to observe the bands. The grayscale image on the film was then scanned using a flatbed scanner (300-400 dpi). Densitometric determination was performed in Photoshop. Using the magic wand tool in the tool tray, the area of ​​each band was selected, and the average histogram was recorded, and then a graph was drawn. IL-1β and other drugs were not added to the untreated group (NT). The experiment was repeated twice.

[0146] Western blotting showed that NDX2 significantly inhibited Cox-2 expression in human chondrocytes stimulated with IL-1β, where its activity was comparable to that of NAG. Figure 3A and Figure 3B middle.

[0147] Gene expression analysis

[0148] Total RNA was extracted and purified from cells using the RNeasy kit (QIAGEN Sciences, Valencia, CA, USA) according to the manufacturer's protocol. During RNA purification, DNA was digested using RNase-free DNase. TM cDNA Synthesis Kit and iQ TM Synthesis of cDNA from total RNA and quantitative PCR were performed using the SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA). Target genes included COX-2 and IL-6. The 18s ribosomal RNA gene was used as an internal control. Primer sequences are listed as follows: for 18s rRNA, 5'-GTGACCAGTTCACTCTTG GT-3' (forward) (SEQ ID NO: 1), 5'-CATTGGAAGTGAAGCGTTTCG-3' (reverse) (SEQ ID NO: 2); for Cox-2, 5'-TGCATTCTTTGCCCAGCACT-3' (forward) (SEQ ID NO: 3), 5'-AAAGG CGCAGTTTACGCTGT-3' (reverse) (SEQ ID NO: 4); and for IL-6, 5'-GGTACATCCTCGA CGGCATCT-3' (forward) (SEQ ID NO: 5), 5'-GTGCCTCTTTGCTGCTTTCAC-3' (reverse) (SEQ ID NO: 6). Threshold cycle (CT) values ​​were calculated using amplification curves. Data were analyzed using the 2-ΔΔCT method, with 18s rRNA serving as a reference. Gene expression was normalized to the control group in each experiment and expressed as a fold change. No IL-1β and other drugs were added to the untreated group (NT). There were four replicates (n=4) in each group. The experiment was repeated twice.

[0149] RT-PCR analysis showed that NDX2 could inhibit the increase in mRNA levels of pro-inflammatory genes IL-6 and COX-2 induced by IL-1β (n=4). Figure 4 and Figure 5 middle.

[0150] Detection of nitrite production

[0151] The Griess reagent system is based on a chemical reaction using p-aminobenzenesulfonamide and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. This system detects nitrite production. In the experiments disclosed herein, nitric oxide detection was performed by a commercial kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's manual. Briefly, all experimental samples (culture medium in each group) and standards containing a dilution series of a nitrite standard reference curve were mixed with 50 μL of p-aminobenzenesulfonamide solution and incubated in a 96-well plate at room temperature in the absence of light for 10 minutes. Then 50 μl of NED solution was added to each well and incubated at room temperature in the absence of light for 10 minutes. The optical density (OD) was measured at 530 nm on a microplate reader within 30 minutes. IL-1β and other drugs were not added to the untreated group (NT). There were six replicates (n=6) in each group. Repeat the experiment twice.

[0152] Using Griess reagent, it was found that NDX2 dose-dependently inhibited the production of nitric oxide in human chondrocytes stimulated with IL-1β (n=6). The results are shown in Figure 6 middle.

[0153] Data analysis

[0154] The values ​​obtained are presented as mean ± SD and analyzed using one-way ANOVA followed by Bonferroni / Dunnett's test. The statistical significance level between the two groups was set at P < 0.05.

[0155] Example 3: Mouse macrophage analysis to determine the non-toxic dose range of NDX to mouse macrophage RAW264.7 cells.

[0156] Cell culture and treatment

[0157] Mouse RAW264.7 macrophage cell line (ATCC; Manassas, VA, USA) was maintained in DMEM medium containing 10% FBS and 1% antibiotic mixture. For cell treatment, different doses of test drugs were added to the culture 0.5 h before adding 100 ng / mL LPS (Sigma-Aldrich Co., St Louis, MO, USA), and then the cells were co-treated with LPS for 24 h.

[0158] Cytotoxicity assay

[0159] Macrophages in the control and treatment groups were inoculated on 96-well plates for 24 hours, and the number of cells was counted using the WST-1 kit (Fisher Scientific). After removing the supernatant, 150 μL of fresh culture medium was added to each well. Then 15 μL of cell proliferation reagent WST-1 was added and incubated with the cells in the dark at 37°C in a humidified atmosphere of 5% carbon dioxide for 3 hours. Each sample was measured at 450nm on a microplate reader. No drug was added to the untreated group (NT). There were eight replicates (n=8) in each group. The experiment was repeated twice.

[0160] WST-1 analysis showed that NDX1 had a dose-dependent toxicity to the mouse macrophage cell line RAW264.7 cells; NDX2 and NAG showed less toxicity than NDX1 (n=8). The results are shown in Figure 7 middle.

[0161] Statistical analysis

[0162] Data are expressed as mean ± SD. Statistical evaluation was performed by analysis of variance (ANOVA) using SPSS15.0 software. Student t-test (two-tailed) was performed to compare the differences between the two groups, and p < 0.05 was considered significant.

[0163] Example 4: Evaluation of the effect of NDX on mouse macrophage RAW264.7 cells activated by LPS

[0164] IL-6 content detection

[0165] Following the instructions provided by the manufacturer, the amount of IL-6 in the culture medium from the different treatments was measured by a commercial ELISA kit (Sigma-Aldrich). Briefly, cells were seeded in 96-well plates and subjected to various treatments. The culture medium was collected and gradually mixed with different analytical reagents. The resulting solution was read at 450nm on a microplate reader. There were eight replicates in each group (n=8). *P<0.05 compared to the LPS group. The experiment was repeated twice.

[0166] ELISA test showed that all three drugs tested (NDX1, NDX2 and NAG) could significantly reduce the production of IL-6 by LPS-activated mouse RAW264.7 macrophages, and NDX1 treatment was the most effective among them (n=8). The results are shown in Figure 8 middle.

[0167] Western blot analysis

[0168] Cellular protein was prepared from cells grown in 6-well plates (approximately 500,000 cells / well) using a commercial cell lysis buffer (Thermo Scientific). Protein concentration was then determined using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Samples containing 100 μg of protein were run on a 10% SDS-polyacrylamide gel at a constant current of 80 V and electrotransferred to a nitrocellulose membrane (Thermo Scientific) at a constant voltage of 10 V overnight. The membrane was blocked with 5% fatty acid free bovine serum albumin (BSA) fraction V (Roche Diagnostics, Indianapolis, Indiana, USA) in TBST solution (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 hour at room temperature, rinsed, and incubated overnight at 4°C in 5% BSA in TBST solution containing each specific primary antibody against mouse iNOS and Cox-2 (both from Novus Biologicals, LLC). β-actin was used as an internal reference. The membrane was then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling) at room temperature for 1 hour, followed by mixing the chemiluminescent substrate and enhancer solution of the HRP antibody (Thermo Scientific) in a 1:1 ratio. The membrane was incubated with autoradiographic film (Genesee Scientific, San Diego, CA, USA) in the dark, and the film was developed to observe the bands. The grayscale images on the film were then scanned using a flatbed scanner (300-400 dpi). Densitometry was performed in Photoshop. Using the magic wand tool in the tool tray, the area of ​​each band was selected and the average histogram was recorded and then plotted. All Western blotting was performed in duplicate. No LPS and other drugs were added to the untreated group (NT).

[0169] Western blot analysis revealed that NAG>NDX2>NDX1 was used to inhibit Cox-2 expression, and NDX1>NDX2>NAG was used to inhibit iNOS expression. The results are shown in Figures 9 and 10 middle.

[0170] Gene expression analysis

[0171] Total RNA was purified using the RNeasy kit (Qiagen, Valencia, CA, USA) according to the protocol provided by the manufacturer and stored at -80°C. TM cDNA Synthesis Kit and iQ TMSYBR Green Supermix kit (Bio-Rad Laboratories, Helix, California, USA) was used to synthesize cDNA from total RNA and quantitative PCR. Target genes included iNOS, IL-6 and IL-1β. The 18s ribosomal RNA gene was used as an internal control. The critical cycle (CT) value was calculated using the amplification curve. The data were analyzed using the 2-ΔΔCT method, with 18s rRNA serving as a reference. Gene expression was normalized to the control group in each experiment and expressed as a change factor. The primer sequences are listed as follows: for 18s rRNA, 5'-GTGACCAGTTCACTCTTGGT-3' (forward) (SEQ ID NO: 1), 5'-CATTGGAAGTGAA GCGTTTCG-3' (reverse) (SEQ ID NO: 2); for iNOS, 5'-GAGGGATGCCTTCCGCAGCTG-3' (forward) (SEQ ID NO: 7), 5'-GAATCGAACCCTGATTCCCCGTC-3' (reverse) (SEQ ID NO: 8); for IL-1β, 5'-CAACCAACAAGTGATATTCTCCATG-3' (forward) (SEQ ID NO: 9), 5'-GATCCACACTCTCCAGCTGCA-3' (reverse) (SEQ ID NO: 10); and for IL-6, 5'-GAGT CCTTCAGAGAGATACAG-3' (forward) (SEQ ID NO: 11), 5'-TGGTCTTGGTCCTTAGCC-3' (reverse) (SEQ ID NO:12).

[0172] RT-PCR showed that NDX1 (0.1 mM and 0.5 mM) could inhibit the mRNA expression of both iNOS and IL1β, while NAG (0.5 mM) could inhibit the mRNA expression of IL1β but not iNOS. NDX2 (0.5 mM) did not inhibit the mRNA expression of iNOS or IL1β (n=4). The results are shown in Figure 11 to Figure 12 middle.

[0173] Detection of nitrite production

[0174] The Griess reagent system is based on a chemical reaction using p-aminobenzenesulfonamide and N-1-naphthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. This system detects nitrite production. In the experiments disclosed herein, nitric oxide detection was performed by a commercial kit (Promega, Madison, Wisconsin, USA) according to the manufacturer's manual. Briefly, all experimental samples and standards containing a dilution series of a nitrite standard reference curve were mixed with 50 μL of p-aminobenzenesulfonamide solution and incubated in a 96-well plate at room temperature in the absence of light for 10 minutes. Then 50 μL of NED solution was added to each well and incubated for 10 minutes at room temperature in the absence of light. The optical density (OD) was determined at 530 nm on a microplate reader within 30 minutes. LPS and other drugs were not added to the untreated group (NT). The experiment was repeated twice. There were six replicates (n=6) in each group. The experiment was repeated twice.

[0175] The nitrite test showed that NDX1 (0.2mM, 1mM and 5mM) and NDX2 (0.5mM and 5mM) could significantly inhibit nitric oxide production (n=6), while NAG (0.5-5mM) could not. The results are shown in Fig.13 middle.

[0176] Example 5: Determination of the anti-inflammatory activity of NDX1 and NDX2 against LPS-induced systemic inflammation in mice.

[0177] animal

[0178] Male C57BL6 / J mice (8 to 10 weeks old, 20-25 g) purchased from Charles River were used in this study. Mice were raised in the animal facility of the University of Virginia (UVa) School of Medicine. All experimental procedures were approved by the UVa School of Medicine Animal Research Ethics Committee and in accordance with the National Committee for Control of Animal Experiments (CONCEA) guidelines.

[0179] Animal models

[0180] Moderate endotoxemia caused by LPS was induced by a single dose (10 mg / Kg, ip) of LPS (E. coli 0111:B4, Sigma Chemical Co., St. Louis, MO, United States) 30 min after intravenous injection of NAG or NDX1 or NDX2 or vehicle (saline). Two doses (200 mg / kg body weight and 300 mg / kg body weight) of NAG, NDX1 and NDX2 were tested. Animals without any treatment were used to obtain baseline data. Six hours after LPS treatment, inflammation analysis was performed as described below. A total of 40 animals were randomly divided into 5 groups (8 mice / group): untreated (NT); 10 mg / Kg LPS plus saline (LPS); 10 mg / Kg LPS plus NAG (LPS+NAG); 10 mg / Kg LPS plus NDX1 (LPS+NDX1); and 10 mg / Kg LPS plus NDX2 (LPS+NDX2). The experiment was repeated twice.

[0181] Migration of leukocytes into the peritoneal cavity

[0182] Peritoneal cells were collected by lavage with ice-cold Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD, USA). Two methods were used: 1) Peritoneal cells were cultured in DMEM supplemented with 10% FBS and antibiotics in a CO2 incubator for 24 h. WST-1 analysis was then performed to count the number of cells; 2) Peritoneal cells were fixed in 70% ethanol at 4°C overnight for use. The cell suspension (100 μL) was then centrifuged at 1,500 rpm for 10 minutes at 4°C on a glass cover of a 96-well plate, stained with a fluorescent DNA dye DAPI that displays cell nuclei, assessed by a Cytation 5 image reader and analyzed by Gen5 software.

[0183] Myeloperoxidase activity assay

[0184] Leukocyte migration to the lung was assessed using a myeloperoxidase kinetic colorimetric assay. Tissue samples were collected in 50 mM K2HPO4 buffer (pH 6.0) containing 13.72 mM hexadecyltrimethylammonium bromide (HTAB) and stored at -80°C until analysis. Samples were homogenized using a Tissue-Tearor, and the homogenate was centrifuged (13.000 rpm, 2 min, 4°C). Supernatants were analyzed spectrophotometrically for myeloperoxidase activity at 450 nm.

[0185] Migration of leukocytes to the lungs and peritoneum

[0186] Sequestration of circulating leukocytes is an event that can impair an appropriate response to infection. To compare the effects of NDX1 and NDX2 with NAG on leukocyte infiltration in the lung, myeloperoxidase (MPO) activity was measured. Fig.14 The results in confirmed that, among the three compounds tested, NDX1 was the most effective in reducing leukocyte sequestration in the lungs of LPS mice. In addition to reducing leukocyte sequestration in the lungs, WST-1 test or DAPI staining indicated that NDX1 together with LPS also significantly reduced the migration of leukocytes to the peritoneal cavity compared to LPS alone, while NAG and NDX2 could not. The results indicated that the sequestered leukocytes were mainly neutrophils (Figure 16A, Figure 16B, Figure 16C, white arrows) and a small number of monocytes larger than neutrophils (Figure 16A, Figure 16B, Figure 16C, red arrows). The results are shown in Fig.15 , Figure 16A, Figure 16B, Figure 16C and Figure 16D.

[0187] Determination of cytokine levels

[0188] According to the manufacturer's instructions, ELISA was used to determine the concentration of TNFa and IL-6 in serum. Briefly, serum from each animal was collected and gradually mixed with different analytical reagents. The resulting solution was read at 450nm on a microplate reader.

[0189] In vivo experiments showed that NDX1 was most effective in reducing the inflammatory response to LPS in mice.

[0190] Serum levels of inflammatory cytokines

[0191] The production of inflammatory cytokines coordinates the response to infectious agents through the activation and recruitment of immune cells. To determine the production of inflammatory mediators, serum concentrations of IL-6 and TNFa were quantified. LPS increased serum levels of IL-6 and TNFa, and all three tested compounds, NDX1, NDX2 and NAG, at a dose of 300 mg / kg were able to significantly reduce serum levels of IL-6 and TNFa in LPS- mice. The results are shown in Fig.17 , Fig.18 , Fig.19 and Fig. 20 There was no significant difference in the effects among the three compounds at this dose. However, NDX1 showed significantly higher inhibitory activity on IL6 and TNFa production compared to NAG or NDX2 at a reduced dose of 200 mg / kg. The results are shown in Fig.21 , Fig. 22 , Fig.23 And Figure 24.

[0192] Example 6: Evaluation of the anti-inflammatory activity of NDX1 and NDX2 in LPS-activated primary mouse peritoneal macrophages

[0193] Experimental Group

[0194] 1. Untreated (NT)

[0195] 2.LPS 100 ng / mL

[0196] 3. LPS 100 ng / mL + NDX 1 dose 1*

[0197] 4. LPS 100 ng / mL + NDX 1 dose 2*

[0198] 5. LPS 100 ng / mL + NDX 1 dose 3*

[0199] 6. LPS 100 ng / mL + NDX2 dose 1

[0200] 7. LPS 100 ng / mL + NDX2 dose 2

[0201] 8. LPS 100 ng / mL + NDX2 dose 3

[0202] 9.LPS 100 ng / mL + NAG dose 2

[0203] 10.LPS 100 ng / mL + NAG dose 3

[0204] (*Dose 1, 2, and 3 without cytotoxicity in the cytotoxicity analysis were selected as the low dose, intermediate dose, and high dose)

[0205] Cell culture and WST-1 assay

[0206] Peritoneal macrophages were isolated from the peritoneal cavity and cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, VT, USA) and 100 IU / mL penicillin G and 100 μg / mL streptomycin at 37°C in a humidified atmosphere of 5% carbon dioxide. The cells were grown in 96-well plates and co-treated with 100 ng / ml LPS (Sigma-Aldrich) 0.5 hours after the addition of BNAG and NAG. Twenty-four hours later, the number of cells was counted by using the WST-1 kit (Fisher Scientific). After removing the supernatant, 150 μl of new culture medium was added to each cell. Then 15 μl of cell proliferation reagent WST-1 was added and incubated with the cells in the dark at 37°C in a humidified atmosphere of 5% carbon dioxide for 3 hours. Each sample was measured at 450 nm on a microplate reader.

[0207] WST-1 analysis showed that 100 ng / mL of LPS significantly increased cell growth of mouse primary peritoneal macrophages. The combination of each of the three test compounds NDX1, NDX2 or NAG at different doses up to 1 mM with 100 ng / mL LPS had no significant effect on cell number compared to LPS alone. The results are shown in Fig.21 ELISA test showed that 100ng / mL LPS significantly increased the production of IL6 and TNFa in mouse primary peritoneal macrophages. All three drugs significantly reduced the production of IL-6 and TNFa in LPS-activated macrophages, and NDX1 was the most effective. The results are shown in Fig. 22 and Fig.23 middle.

[0208] Detection of IL-6 and TNFα levels

[0209] Following the instructions provided by the manufacturer, the amount of IL-6 and TNFα was measured by commercial ELISA kits (Sigma-Aldrich). Briefly, cells were seeded in 96-well plates and subjected to various treatments. Culture medium was collected and gradually mixed with different analytical reagents. The resulting solution was read at 450 nm on a microplate reader.

[0210] Statistical analysis

[0211] All data are reported as mean ± SD. One-way ANOVA test with multiple comparisons was performed. In each figure, the p value indicates whether the difference between the selected control group and the rest of the sample is significant [P value <0.05 (*)]. The results were obtained using the GraphPadPrims program.

[0212] In vitro experiments demonstrated that NDX1 was effective in reducing the inflammatory response to LPS in primary mouse peritoneal macrophages.

[0213] Example 7

[0214] In Examples 5 and 6, two compounds, NDX1 and NDX2, were examined as modified molecules obtained from NAG. The anti-inflammatory properties of their parental molecules (using an in vivo model of systemic inflammation in mice and an in vitro model of inflammation in primary mouse peritoneum) were all induced by LPS. Although all three compounds tested at a dose of 300 mg / kg showed significant inhibitory effects on the elevated levels of serum cytokines IL-6 and TNFα production induced by LPS, NDX1 exhibited the highest potency among the three compounds at a dose of 200 mg / kg. NDX1 also exhibited the strongest inhibitory effect on LPS-induced migration of leukocytes to the lungs and peritoneal cavity. In vitro experiments revealed that NDX1, NDX2, and NAG could significantly reduce IL-6 and TNFα production by LPS-activated mouse peritoneal macrophages, and NDX1 was the most effective among them. Without being bound by any theory, additional studies using human cells / tissues and specific animal disease models may provide necessary information regarding whether NDX1 is a viable strategy to target inflammation in a variety of pathologies including, but not limited to, osteoarthritis, rheumatoid arthritis, inflammatory bowel disease, and cancer.

Claims

1. A method for treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc., comprising administering a therapeutically effective amount of a compound of the formula or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the inflammatory disease is rheumatoid arthritis (RA).

3. The method of claim 1, wherein the inflammatory disease is osteoarthritis (OA).

4. The method of any one of claims 1 to 3, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

5. The method of any one of claims 1 to 4, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

6. The method of any one of claims 1 to 5, wherein the compound is administered orally.

7. The method of any one of claims 1 to 6, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

8. The method of any one of claims 1 to 7, wherein the therapeutically effective amount of the compound is administered once a week, twice a week, once a day (QD), twice a day (BID), or three times a day (TID).

9. The method of any one of claims 1 to 8, wherein the therapeutically effective amount of the compound inhibits NO production in the patient.

10. The method of any one of claims 1 to 9, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

11. The method of any one of claims 1 to 10, further comprising administering one or more additional therapeutic agents to the patient.

12. The method of claim 10, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives, or Cox-2 inhibitors.

13. The method of claim 10, wherein the one or more additional therapeutic agents are selected from the group consisting of aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone, and methylprednisolone.

14. A pharmaceutical composition comprising a compound of the formula or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier or excipient, wherein the compound is taken in a therapeutically effective amount for treating inflammatory diseases, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

15. The pharmaceutical composition of claim 14, wherein the amount of the compound in the composition is from about 1 mg to about 1000 mg.

16. The pharmaceutical composition of claim 14 or 15, wherein the therapeutically effective amount of the compound inhibits NO production in a patient.

17. The pharmaceutical composition of any one of claims 14 to 16, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

18. The pharmaceutical composition of any one of claims 14 to 17, wherein the composition is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

19. The pharmaceutical composition according to any one of claims 14 to 18, wherein the composition is administered orally.

20. Use of a compound of the formula or a pharmaceutically acceptable salt thereof, It is used to prepare a medicament for treating a subject suffering from an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

21. The use according to claim 20, wherein the inflammatory disease is rheumatoid arthritis (RA).

22. The use according to claim 20, wherein the inflammatory disease is osteoarthritis (OA).

23. The use of any one of claims 20 to 22, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

24. The use according to any one of claims 20 to 23, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

25. The use according to any one of claims 20 to 24, wherein the compound is administered orally.

26. The use according to any one of claims 20 to 25, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

27. The use according to any one of claims 20 to 26, wherein the therapeutically effective amount of the compound is administered once a week, twice a week, once a day (QD), twice a day (BID) or three times a day (TID).

28. The use according to any one of claims 20 to 27, wherein the therapeutically effective amount of the compound inhibits NO production in the patient.

29. The use according to any one of claims 20 to 28, wherein the therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

30. The use of any one of claims 20 to 29, further comprising administering one or more additional therapeutic agents to the patient.

31. The use according to claim 30, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

32. The use of claim 30, wherein the one or more additional therapeutic agents are selected from the group consisting of aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone and methylprednisolone.

33. A compound of the formula Or a pharmaceutically acceptable salt thereof, for use in a method for treating an inflammatory disease in a subject, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc.

34. The compound of claim 33, wherein the inflammatory disease is rheumatoid arthritis (RA).

35. The compound of claim 33, wherein the inflammatory disease is osteoarthritis (OA).

36. The compound of any one of claims 33 to 35, wherein the therapeutically effective amount treats one or more symptoms of the inflammatory disease.

37. The compound of any one of claims 33 to 36, wherein the compound is administered intravenously, orally, subcutaneously, buccally, transdermally or nasally.

38. The compound of any one of claims 33 to 37, wherein the compound is administered orally.

39. The compound of any one of claims 33 to 38, wherein the therapeutically effective amount for the inflammatory disease is in the range of about 1 mg to about 1000 mg.

40. The compound of any one of claims 33 to 39, wherein the method comprises administering a therapeutically effective amount of the compound once a week, twice a week, once a day (QD), twice a day (BID), or three times a day (TID).

41. The compound of any one of claims 33 to 40, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit NO production in a patient.

42. The compound of any one of claims 33 to 41, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit iNOS gene expression in the patient.

43. The compound of any one of claims 33 to 42, further comprising administering one or more additional therapeutic agents to the patient.

44. The compound of claim 43, wherein the one or more additional therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives, or Cox-2 inhibitors.

45. The compound of claim 43, wherein the one or more additional therapeutic agents are selected from the group consisting of aspirin, diflunisal, salsalate, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone, and methylprednisolone.

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