Methods for Treating Inflammation

Abstract

The present invention relates to anti-inflammatory pharmaceutical agents, in particular compounds, compositions and methods for the treatment of inflammation and inflammation-related disorders, such as arthritis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) to U.S. Provisional Application No. 63 / 333,281, filed April 21, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Regarding electronically submitted sequence listings Concurrently submitted herein is a computer readable nucleotide / amino acid sequence listing identified as follows, which is hereby incorporated by reference in its entirety as a 16 kilobyte xml file entitled "83864-388942_SL.xml", created on April 17, 2023:

[0003] Field The present invention relates to compounds, compositions and methods as anti-inflammatory pharmaceuticals, particularly for the treatment of inflammation and inflammation-related disorders, such as arthritis. [Background technology]

[0004] background Since its discovery as a biologically active molecule in the late 1980s, nitric oxide (NO) has been found to play important roles in many parts of the organism, both as a signaling molecule and as a cytotoxic or regulatory effector molecule in the innate immune response. NO, synthesized by nitric oxide synthase (NOS), is the smallest known bioactive molecule and is produced by a wide variety of cells. NO plays important roles in neurotransmission, vascular function, host defense, and immune regulation.

[0005] Three isoforms of NO have been identified for its production: neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). The signal molecule NO is synthesized on demand and for a short time (seconds to minutes) after enzymatic activation of constitutively expressed endothelial NO synthase (eNOS) or neuronal NO synthase (nNOS). In contrast, inducible NO synthase (iNOS) is synthesized only after cell activation, after which NO is produced for a relatively long time (hours to days). iNOS can be produced by many types of cells after induction by cytokines or other stimuli. Thus, the difference between quantitative NO production versus controlled short-term pulsatile synthesis distinguishes the physiological and pathophysiological effects of NO.

[0006] NO is an important pro-inflammatory mediator that has an effect on the immune system. In fact, NO has a dual role in immune inflammation. On the one hand, NO can kill microorganisms and has a protective effect on the body, helping it fight various viruses, such as herpes simplex virus (HSV). On the other hand, NO can damage normal tissue cells and produce pathogenic effects, and is widely involved in the development of various diseases, such as 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.

[0007] The iNOS gene is under the transcriptional control of various inflammatory mediators such as cytokines, liposaccharide (LPS) and others. Abnormal expression of iNOS plays an important role in many 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 and psoriasis. For example, iNOS-produced NO is considered to be an important participant in the pathogenesis of OA, contributing to OA pathogenesis by regulating ECM homeostasis and cytokine expression, which causes oxidative damage and chondrocyte apoptosis. Using immunohistochemical staining, 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. Summary of the Invention [Problem to be solved by the invention]

[0008] Numerous studies suggest a similar role for iNOS activity in human diseases. However, it remains to be shown whether iNOS-derived NO inhibition in human patients protects against tissue-destructive processes in OA, RA, MS, type 1 diabetes, etc. Thus, there is a significant unmet need to improve the treatment of a variety of inflammatory diseases, including rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, etc. [Means for solving the problem]

[0009] overview In one embodiment, the present invention provides a method of treating a subject having an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, and the like, comprising administering a therapeutically effective amount of a compound of formula [ka] or a pharma- ceutically acceptable salt thereof.

[0010] In another aspect, the present invention provides a compound of formula [ka] or a pharma- ceutical composition comprising a compound of formula (I) or a pharma- ceutical acceptable salt thereof and optionally a pharma- ceutical acceptable carrier or excipient. In some embodiments, the compound in the pharmaceutical composition is in a therapeutically effective amount for treating inflammatory diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, and Crohn's disease.

[0011] In another aspect, the present invention provides a method for the treatment of a subject having an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, and the like, comprising administering to said subject a therapeutically effective amount of a compound of formula [ka] or a pharma- ceutically acceptable salt thereof.

[0012] Further embodiments, features and advantages of the present invention will become apparent through the following detailed description and practice of the invention. The compounds, methods and compositions of the present invention may be described as embodiments in any of the following numbered paragraphs. It is understood that any of the embodiments described herein may be used in combination with any other embodiment described herein, unless the embodiments are mutually inconsistent.

[0013] 1. A method for treating a subject having an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, comprising administering to said subject a therapeutically effective amount of a compound of the formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0014] 2. The method according to item 1, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0015] 3. The method according to item 1, wherein the inflammatory disease is osteoarthritis (OA).

[0016] 4. The method of any of items 1 to 3, wherein the therapeutically effective amount treats one or more symptoms of an inflammatory disease.

[0017] 5. The method of any of paragraphs 1 to 4, wherein the compound is administered intravenously, orally, subcutaneously, bucally, transdermally, or intranasally.

[0018] 6. The method of any one of items 1 to 5, wherein the compound is administered orally.

[0019] 7. The method according to any one of items 1 to 6, wherein the therapeutically effective amount for an inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0020] 8. The method of any of paragraphs 1 to 7, wherein a 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).

[0021] 9. The method of any of items 1 to 8, wherein the therapeutically effective amount of the compound inhibits NO production in the patient.

[0022] 10. The method of any of paragraphs 1 to 9, wherein a therapeutically effective amount of the compound inhibits iNOS gene expression in the patient.

[0023] 11. The method of any of paragraphs 1 to 10, further comprising administering to the patient one or more additional therapeutic agents.

[0024] 12. The method of paragraph 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.

[0025] 13. The method of paragraph 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 methylprednisone.

[0026] 14. Expression [ka] or a pharma- ceutical composition comprising a compound of formula (I) or a pharma- ceutical acceptable salt thereof and optionally a pharma- ceutical acceptable carrier or excipient, wherein the compound is in a therapeutically effective amount for the treatment of an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, and the like.

[0027] 15. The pharmaceutical composition of item 14, wherein the compound is present in an amount of about 1 mg to about 1000 mg in the composition.

[0028] 16. The pharmaceutical composition of paragraph 14 or 15, wherein a therapeutically effective amount of the compound inhibits NO production in a patient.

[0029] 17. The pharmaceutical composition of any one of items 14 to 16, wherein a therapeutically effective amount of the compound inhibits iNOS gene expression in a patient.

[0030] 18. The pharmaceutical composition of any one of items 14 to 17, wherein the composition is administered intravenously, orally, subcutaneously, bucally, transdermally, or intranasally.

[0031] 19. The pharmaceutical composition of any one of items 14 to 18, wherein the composition is administered orally.

[0032] 20. A compound of formula (I) in the manufacture of a medicament for treating a subject having an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, or Crohn's disease, comprising administering to said subject a compound of formula (I) to said subject a patient having an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, or Crohn's disease. [ka] or a pharma- ceutically acceptable salt thereof.

[0033] 21. The use according to item 20, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0034] 22. The use according to paragraph 20, wherein the inflammatory disease is osteoarthritis (OA).

[0035] 23. The use of any of paragraphs 20 to 22, wherein the therapeutically effective amount treats one or more symptoms of an inflammatory disease.

[0036] 24. The use of any of paragraphs 20 to 23, wherein the compound is administered intravenously, orally, subcutaneously, bucally, transdermally or intranasally.

[0037] 25. The use of any of paragraphs 20 to 24, wherein the compound is administered orally.

[0038] 26. The use according to any one of items 20 to 25, wherein the therapeutically effective amount for an inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0039] 27. The use of any of paragraphs 20 to 26, wherein a 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).

[0040] 28. The use of any of paragraphs 20 to 27, wherein a therapeutically effective amount of the compound inhibits NO production in a patient.

[0041] 29. The use of any of paragraphs 20 to 28, wherein a therapeutically effective amount of the compound inhibits iNOS gene expression in a patient.

[0042] 30. The use of any of paragraphs 20 to 29, wherein the treatment further comprises administering to the patient one or more additional therapeutic agents.

[0043] 31. The use of paragraph 30, wherein the one or more further therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

[0044] 32. The use of paragraph 30, wherein the one or more further 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.

[0045] 33. A compound of the formula: for use in a method for treating an inflammatory disease, such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, in a subject. [ka] or a pharma- ceutically acceptable salt thereof.

[0046] 34. The compound according to item 33, wherein the inflammatory disease is rheumatoid arthritis (RA).

[0047] 35. The compound according to item 33, wherein the inflammatory disease is osteoarthritis (OA).

[0048] 36. The compound of any of paragraphs 33 to 35, wherein a therapeutically effective amount treats one or more symptoms of an inflammatory disease.

[0049] 37. The compound of any of paragraphs 33 to 36, wherein the compound is administered intravenously, orally, subcutaneously, bucally, transdermally or intranasally.

[0050] 38. The compound of any of paragraphs 33 to 37, wherein the compound is administered orally.

[0051] 39. The compound according to any one of items 33 to 38, wherein the therapeutically effective amount for an inflammatory disease is in the range of about 1 mg to about 1000 mg.

[0052] 40. The compound of any of paragraphs 33-39, 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).

[0053] 41. The compound of any of paragraphs 33 to 40, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit NO production in a patient.

[0054] 42. The compound of any of paragraphs 33 to 41, wherein the method comprises administering a therapeutically effective amount of the compound to inhibit iNOS gene expression in the patient.

[0055] 43. The compound of any of paragraphs 33 to 42, wherein the method further comprises administering to the patient one or more additional therapeutic agents.

[0056] 44. The compound of paragraph 43, wherein the one or more further therapeutic agents are corticosteroids, salicylates, acetic acid derivatives, enolic acid (oxicam) derivatives, propionic acid derivatives, anthranilic acid derivatives or Cox-2 inhibitors.

[0057] 45. The compound of paragraph 43, wherein the one or more further 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. [Brief description of the drawings]

[0058] [Figure 1] Figure 1 is a chart showing the differential effects of Compound (I) and Compound (II) on cell proliferation of primary human chondrocytes. Concentrations up to 5 mM were tested for each drug. IL-1β (IL-1B): 5 ng / mL.

[0059] [Diagram 2] Figure 2 is a chart showing a comparison of NDX2 and NAG on 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.

[0060] [Diagram 3] Figures 3A-3B show 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: IL-1β + 2mM NDX2; Lane 4: IL-1β + 2mM NAG). Figure 3B is a chart from semi-quantitative analysis of the gel image. IL-1β: 5ng / mL.

[0061] [Figure 4] Figure 4 is a chart showing the effect of NDX2 on IL-6 mRNA levels in primary human chondrocytes. IL-1β (IL-1B): 5 ng / mL.

[0062] [Diagram 5] 5 is a chart showing the effect of NDX2 on Cox-2 mRNA levels in primary human chondrocytes. IL-1β (IL-1B): 5 ng / mL.

[0063] [Figure 6] Figure 6 is a chart showing the effect of nitrite production in primary human chondrocytes. IL-1β (IL-1B): 5 ng / mL.

[0064] [Figure 7] 7 is a chart showing the effect of NDX1 and NDX2 on cell proliferation of the mouse macrophage cell line RAW264.7 cells. Concentrations up to 5 mM were tested for each drug.

[0065] [Figure 8] Figure 8 is a chart showing a comparison of NDX1, NDX2 and NAG on IL-6 production in RAW264.7 cells. Concentrations up to 5 mM were tested for each drug. LPS: 100 ng / mL.

[0066] [Figure 9] Figure 9 is a gel image showing a comparison of iNOS and expression in mouse RAW264.7 macrophages of NDX1, NDX2 and NAG by Western blot. LPS: 100ng / mL. Lane 1: NT; Lane 2: LPS; Lane 3: LPS+0.1mM NDX1; Lane 4: LPS+1mM NDX1; Lane 5: LPS+0.5mM NDX2; Lane 6: LPS+5mM NDX2; Lane 7: LPS+0.5mM NAG; Lane 8: LPS+5mM NAG.

[0067] [Figure 10]Figure 10 is a gel image showing a comparison of Cox-2 expression in mouse RAW264.7 macrophages of NDX1, NDX2 and NAG by Western blot. LPS: 100ng / mL. Lane 1: NT; Lane 2: LPS; Lane 3: LPS+0.1mM NDX1; Lane 4: LPS+1mM NDX1; Lane 5: LPS+0.5mM NDX2; Lane 6: LPS+5mM NDX2; Lane 7: LPS+0.5mM NAG; Lane 8: LPS+5mM NAG.

[0068] [Figure 11] 11 is a chart showing a comparison of NDX1, NDX2 and NAG on iNOS expression in mouse RAW264.7 macrophages as determined by RT-PCR analysis.LPS: 100 ng / mL.

[0069] [Figure 12] 12 is a chart showing a comparison of NDX1, NDX2 and NAG on IL1β (IL1B) expression in mouse RAW264.7 macrophages as determined by RT-PCR analysis. LPS: 100 ng / mL.

[0070] [Figure 13] 13 is a chart showing a comparison of nitrite production in LPS-activated mouse RAW264.7 macrophages of NDX1, NDX2 and NAG. LPS: 100 ng / mL.

[0071] [Figure 14] Figure 14 is a chart showing a comparison of MPO activity in lung tissue from mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV N=8.

[0072] [Figure 15]Figure 15 is a chart showing a comparison of leukocyte migration into the peritoneal cavity from mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV N=8.

[0073] [Figure 16] 16A-16D show a comparison of leukocyte migration into the peritoneal cavity from mice with various treatments. FIG. 16A shows images of leukocyte migration into the peritoneal cavity from mice treated with LPS: 10 mg / kg, IP; NAG: 200 mg / kg, IV N=8. White arrows: neutrophils; red arrows: monocytes. FIG. 16B shows images of leukocyte migration into the peritoneal cavity from mice treated with LPS: 10 mg / kg, IP; NDX1: 200 mg / kg, IV N=8. White arrows: neutrophils; red arrows: monocytes. FIG. 16C shows images of leukocyte migration into the peritoneal cavity from mice treated with LPS: 10 mg / kg, IP; NDX2: 200 mg / kg, IV N=8. White arrows: neutrophils; red arrows: monocytes. FIG. 16D is a chart showing a comparison of leukocyte migration into the peritoneal cavity from mice with various treatments. LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV N=8.

[0074] [Figure 17] Figure 17 is a chart showing a comparison of serum IL6 levels in mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV N=8.

[0075] [Figure 18] Figure 18 is a chart showing a comparison of serum TNFα levels in mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 300 mg / kg, IV N=8.

[0076] [Figure 19]Figure 19 is a chart showing a comparison of serum IL6 levels in mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV N=8.

[0077] [Figure 20] Figure 20 is a chart showing a comparison of serum TNFα levels in mice with various treatments: LPS: 10 mg / kg, IP; NDX1, NDX2 and NAG: 200 mg / kg, IV N=8.

[0078] [Figure 21] Figure 21 is a chart showing the comparison of the effects of NDX1, NDX2 and NAG on cell proliferation of primary mouse peritoneal macrophages. Concentrations up to 1 mM were tested for each drug. No drug was given to the non-treated group (NT). Each group was repeated 8 times (n=8). *P<0.05 vs. LPS group. The experiment was repeated twice.

[0079] [Figure 22] Figure 22 is a chart showing the comparison of NDX1, NDX2 and NAG on IL-6 production in primary mouse peritoneal macrophages. NDX1 and NDX2 were tested at various concentrations (0.01 mM, 0.1 mM and 1 mM), while 0.1 mM and 1 mM NAG were used as controls. LPS: 100 ng / mL. Each group was repeated 8 times (n=8). *P<0.05 vs. LPS group. The experiment was repeated twice.

[0080] [Figure 23] Figure 23 is a chart showing the comparison of NDX1, NDX2 and NAG on TNFα production in primary mouse peritoneal macrophages. NDX1 and NDX2 were tested at various concentrations (0.01 mM, 0.1 mM and 1 mM), while 0.1 mM and 1 mM NAG were used as controls. LPS: 100 ng / mL. Each group was repeated 8 times (n=8). *P<0.05 vs. LPS group. The experiment was repeated twice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Detailed Description Before the present invention is further described, it is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is used merely for the purpose of describing particular embodiments, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0082] For the sake of brevity, the disclosures of the publications cited herein, including patents, are hereby incorporated by reference. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0083] In this specification and the appended claims, the singular includes plural referents unless the context clearly dictates otherwise. Moreover, it should be noted that the claims may be drafted to exclude all optional elements. That is, this description is intended to presuppose the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the description of the claimed element.

[0084] As used herein, the terms "comprise" and "include" are used in their open, non-limiting sense.

[0085] In order to provide a more precise description, some of the quantitative expressions used herein are not modified by the term "about". Whether or not the term "about" is explicitly used, it should be understood that all amounts shown herein refer to the actual values ​​shown, and also refer to the approximations of such values ​​that are reasonably determined based on the common general knowledge in the art, including the equivalents and approximations of such values ​​according to experimental and / or measurement conditions.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

[0087] It will also be appreciated that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0088] Representative Embodiments In one embodiment, the present invention provides a method of treating a subject having an inflammatory disease, comprising administering to a subject a therapeutically effective amount of a compound of the formula [ka] The method includes administering a compound of formula (I) to a subject.

[0089] In one embodiment, the present invention relates to a method for the treatment of a subject having an inflammatory disease, comprising administering to said subject a compound of formula [ka] or a pharma- ceutically acceptable salt thereof. In one embodiment, the present invention provides the use of a compound of formula [ka] or a pharma- ceutically acceptable salt thereof, and optionally a pharma- ceutically acceptable carrier or excipient.

[0090] As used herein, the term "subject" or "patient" refers to a human or, in the case of veterinary applications, may be a laboratory animal, an agricultural animal, a domestic animal, or a wild animal. The methods described herein may be applied to subjects including, but not limited to, humans, laboratory animals, such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees, domestic animals, such as dogs, cats, and rabbits, and agricultural animals, such as cows, horses, pigs, sheep, and goats.

[0091] The term "therapeutically effective amount" as used herein refers to an amount of a drug or agent that induces a biological or medicinal response in a subject (i.e., a tissue system, animal or human) that is being sought by a researcher, veterinarian, physician or other clinician, including the alleviation of symptoms of the inflammatory disease being treated. In some embodiments, a therapeutically effective amount is an amount of an active agent that can treat or alleviate an inflammatory disease or symptoms of an inflammatory disease with a reasonable benefit / risk ratio applicable to any medical treatment. In some embodiments, a therapeutically effective amount is an amount of an active agent that can act as a disease-modifying drug for the inflammatory disease being treated. In some embodiments, a therapeutically effective amount is an amount of an inactive prodrug that, when converted via normal metabolic processes, produces an amount of an active drug that can induce the biological or medicinal response being sought in the subject. In some embodiments, a therapeutically effective amount is an amount of an active agent that changes the activity of a biological target in a subject to which the active agent is administered. In some embodiments, a therapeutically effective amount is an amount of an active agent that can inhibit NO production in a patient. In some embodiments, a therapeutically effective amount is an amount of an active agent that can inhibit iNOS gene expression in a subject.

[0092] It is recognized that inflammation and inflammatory processes can occur in association with a wide range of diseases and disease states. It is recognized that there are two types of inflammation: acute and chronic. Acute inflammation can be beneficial in that it can indicate that the subject can fight infection and / or accelerate the healing process from injury or disease, whereas chronic inflammation, which can be caused by the immune system sending chemical messengers that drive inflammatory processes for a long period of time, can cause debilitating pain and illness. It is recognized that chronic inflammation is associated with heart disease, diabetes, cancer, arthritis, and intestinal diseases such as Crohn's disease and ulcerative colitis. In some embodiments, the disease treated in association with the present invention can be any disease in which the pro-inflammatory mediator nitric oxide (NO) interacts with tissue in the subject to exert pathogenic effects and / or drive disease. In some embodiments, the disease that the pro-inflammatory mediator nitric oxide (NO) can act on includes 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 treated in conjunction with the present invention can be any disease in which the abnormal expression of iNOS gene plays a role in the progression of the disease.Examples of diseases include inflammatory diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, multiple sclerosis, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaque, ulcerative colitis, Crohn's disease, necrotizing enterocolitis, celiac disease, glomerulonephritis, dilated cardiomyopathy, cutaneous lupus erythematosus, systemic lupus erythematosus, dermatitis, apical periodontitis, psoriasis, etc. In certain embodiments, the inflammatory disease can be rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjogren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaques, ulcerative colitis, Crohn's disease, and the like.

[0093] It is recognized that the methods, uses, compositions or compounds described herein may be administered by any method of administration known in the art. As used herein, "administer" or "administration" includes any means 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, nasal, vaginal, rectal, etc. The methods, uses, compositions or compounds described herein may be administered in unit dosage forms and / or formulations containing conventional non-toxic pharma- ceutically acceptable carriers, adjuvants and / or vehicles.

[0094] 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, particles, capsules containing liquids or powders, lozenges (including liquid-filled), chews, multi- and nanoparticles, gels, solid solutions, liposomes, films, ovules, sprays and liquid formulations.

[0095] Liquid preparations include suspensions, solutions, syrups and elixirs.Such preparations can be used as fillings for soft or hard capsules, and typically contain carriers such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oils, and one or more emulsifiers and / or suspending agents.Liquid preparations can also be produced by reconstitution of a solid, for example, from a sachet.

[0096] Binders are commonly used to impart cohesive qualities to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, 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 dibasic calcium phosphate dihydrate.

[0097] Tablets may also optionally contain surface active agents such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2% to 5% by weight of the tablet, and glidants may comprise from 0.2% to 1% by weight of the tablet.

[0098] Tablets may also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. Lubricants generally comprise from 0.25% to 10% by weight of the tablet, preferably from 0.5% to 3% by weight.

[0099] Other possible ingredients include antioxidants, colorants, flavorings, preservatives and taste masking agents. An example tablet contains up to about 80% drug, about 10% to about 25% to about 90% binder by weight, about 0% to about 85% diluent by weight, about 2% to about 10% disintegrant, and about 0.25% to about 10% lubricant by weight.

[0100] Tablet blends may be directly or roller compressed to form tablets. Tablet blends or portions of blends may be wet-, dry- or melt-granulated, melt congealed or extruded before being compressed into tablets. The final formulation may contain one or more layers and may be coated or uncoated; it may be encapsulated. Tablet formulations are described in Pharmaceutical Dosage Forms: Tablets, Vol. 1, by H. Lieberman and L. Lachman (Marcel Dekker, New York, 1980).

[0101] Solid formulations for oral administration may be formulated as immediate and / or modified release, including delayed-, sustained-, pulsed-, controlled-, targeted and programmed release formulations.

[0102] In some embodiments, the methods, uses, compositions or compounds described herein may be administered directly into the bloodstream, muscle or an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular and subcutaneous means.

[0103] In some embodiments, the method, use, composition or compound described herein can 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, salicylic acid, 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.

[0104] Any effective regimen of administering the compounds and compositions described herein may be used. For example, the compounds and compositions described herein may be administered in a single dose or the dose may be divided and administered as a multiple daily dosing regimen. Additionally, alternating regimens may be used, for example, 1-5 days per week as an alternative to daily treatment. In some embodiments, the subject is administered multiple doses of the methods, uses, compounds or compositions used herein. In some embodiments, the subject is administered multiple doses (preferably from about 2 to up to about 80 doses) of the compounds or compositions described herein, for example, at 8-72 hour intervals or 8-12 hour intervals.

[0105] Any suitable course of treatment with the compound of formula (I) or (II) described herein may be used. In some embodiments, the individual doses and dosing schedules are selected such that a total dose of about 1 mg to about 1000 mg; or about 200 mg to about 1000 mg is administered during a given day. In some embodiments, the compound of formula (I) or (II) is administered in a once-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 twice-daily dose (BID) in a dose of about 300 mg to about 900 mg per dose in the methods or uses described herein. In some embodiments, the compound of formula (I) or (II) is administered in a twice-daily dose (BID) in a dose of about 600 mg to about 800 mg 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 twice a day (BID) in a method or use described herein. In some embodiments, the compound of Formula (I) or (II) is administered in a cycle of days over 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, etc. In some embodiments, the compound of Formula (I) or (II) is administered in a method or use described herein daily for 10-45 days or until the patient's condition as observed by the treating physician indicates the end of treatment. In some embodiments, the compound of Formula (I) or (II) is administered in a method or use described herein daily for 10-20 days or until the patient's condition as observed by the treating physician indicates the end of treatment. In some embodiments, the compound of Formula (I) or (II) is administered in a method or use described herein daily for 25-35 days or until the patient's condition as observed by the treating physician indicates the end of treatment. In certain embodiments, a compound of Formula (I) or (II) is administered daily in the manner or use described herein for about 30 days or until the patient's condition, as observed by the treating physician, indicates the end of treatment.

[0106] It is recognized that the daily dosage unit of a compound of formula (I) or (II), as described herein, can vary significantly depending on the condition of the patient, the inflammatory disease being treated, the route of administration of the compound of formula (I) or (II), and the possibility of co-administration of additional therapeutic agents. The effective amount to be administered to a patient is based on the physician's assessment of body surface area, weight, and the patient's condition.

[0107] The examples and preparations provided below further describe and illustrate certain aspects of the embodiments of the present invention.It should be understood that the scope of the present invention is not limited in any way to 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 method described in WO2022 / 051575, the entire contents of which are incorporated herein by reference.N-acetylglucosamine (also known as NAG) can be purchased from commercial sources such as Sigma-Aldrich (St. Louis, MO).

[0108] Example 1: To determine the non-toxic dose range of NDX for human chondrocyte cultures

[0109] Human chondrocyte primary culture:

[0110] Normal cartilage tissue. Articular cartilage was collected from the femoral condyle and tibial plateau obtained from the UVA Hospital Autopsy Department and approved by the Institutional Review Board. All tissue samples were graded according to the modified Mankin scale, and only cartilage without evidence of osteoarthritis was used as the source of chondrocytes. The time between death and cartilage harvesting from these knee joints in the laboratory was at least 24 hours and ranged up to 96 hours. Cartilage scrapings were collected in the operating room, placed in tissue culture medium (DMEM, 10% FBS, penicillin, streptomycin), and transported to the laboratory at 4°C. Tissues were processed in the laboratory within 24 hours of collection.

[0111] Chondrocytes were isolated from cartilage by collagenase digestion and maintained in continuous monolayer culture in DMEM containing 10% FBS. Cell viability after chondrocyte isolation by collagenase digestion of normal cartilage was >95% as determined by trypan blue staining (Sigma-Aldrich). Experiments were performed on primary or first passage cells. Trypsin was used for cell passaging when necessary.

[0112] Cell proliferation assay

[0113] Human chondrocytes were cultured in a 96-well plate (5 × 10 4 The cells were seeded in 1000 x 1000 cells / well and incubated overnight in culture medium. The cells were then treated as required, and Roche-Cell Proliferation Reagent WST-1 (Fisher Scientific Company, Nazareth, PA) was used in the cell proliferation assay according to the accompanying instructions provided by the manufacturer. IL-1β and other drugs were not added to the non-treated group (NT). Each group was repeated six times (n=6). The experiment was repeated twice. The results are shown in Figure 1.

[0114] WST-1 assay showed that NDX1 was toxic to human chondrocytes in the presence of IL-1β (IL-1B) in a dose-dependent manner. NDX1 showed significant cytotoxicity. NDX2 and NAG could prevent and inhibit cell proliferation induced by IL-1β (n=6).

[0115] Data Analysis:

[0116] The values ​​obtained were expressed as mean ± SD and analyzed using one-way analysis of variance followed by Bonferroni / Dunnett test. The statistical significance level between two groups was set at P < 0.05.

[0117] Example 2: To evaluate the preventive effect of NDX on IL-1β activation in human chondrocyte cultures

[0118] Cell treatment

[0119] Primary cultures of human chondrocytes were cultured in 96-well plates (5 × 10 4 / well) and treated with various drugs for 24 h. Both culture medium and cells were collected for subsequent assays.

[0120] Detection of IL-6 levels

[0121] The amount of IL-6 in the culture medium was measured by a commercial ELISA kit (Sigma-Aldrich) according to the instructions provided by the manufacturer. Briefly, the culture medium was mixed with various assay reagents stepwise. The resulting solution was read at 450 nm using a microplate reader. ELISA test showed that NDX2 significantly inhibited IL-6 production in IL-1B-stimulated human chondrocytes, and its activity was comparable to that of NAG (n=6). IL-1β and other drugs were not added to the non-treated group (NT). The experiment was repeated twice. The results are shown in Figure 2.

[0122] Western blot analysis

[0123] Proteins were prepared from cells growing in 6-well plates (approximately 500,000 / well) and protein concentrations were 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 electrophoretically transferred to a nitrocellulose membrane (Thermo Scientific) overnight at a constant voltage of 10 V. The membrane was blocked with 5% fatty acid-free bovine serum albumin (BSA) fraction V (Roche Diagnostics, Indianapolis, IN, USA) in TBST solution (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 h at RT, washed, and incubated overnight at 4°C in 5% BSA in TBST containing a 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) for 1 h at RT, followed by chemiluminescent substrate and enhancer solution for HRP antibodies mixed in a 1:1 ratio (Thermo Scientific). The membrane was incubated with autoradiography film (Genesee Scientific, San Diego, CA, USA) in the dark, and the film was developed for visualization of the bands. Grayscale images (300–400 dpi) of the film were then scanned using a flatbed scanner. Densitometry was performed in Photoshop. Using the magic wand tool from the tool palette, each band area was selected, and the average histogram was recorded and then charted. IL-1β and other drugs were not added to the non-treated group (NT). The experiment was repeated twice.

[0124] Western blots showed that NDX2 significantly inhibited Cox-2 expression in IL-1B-stimulated human chondrocytes, and its activity was comparable to that of NAG. The results are shown in Figures 3A and 3B.

[0125] Gene expression analysis

[0126] Total RNA was extracted and purified from cells using the RNeasy kit (QIAGEN Sciences, Valencia, CA) according to the protocol provided by the manufacturer. RNase-Free DNase was used for DNA digestion during RNA purification. Synthesis of cDNA from total RNA and quantitative PCR were performed using iscript TM cDNA Synthesis Kit and iQ TM The SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA) was used. Target genes included COX-2 and IL-6. The gene for 18s ribosomal RNA was used as an internal control. Primer sequences were listed as follows: 5'-GTGACCAGTTCACTCTTGGT-3' (forward) (SEQ ID NO: 1), 5'-CATTGGAAGTGAAGCGTTTCG-3' (reverse) (SEQ ID NO: 2) for 18s rRNA, 5'-TGCATTCTTTGCCCAGCACT-3' (forward) (SEQ ID NO: 3), 5'-AAAGGCGCAGTTTACGCTGT-3' (reverse) (SEQ ID NO: 4) for Cox-2, and 5'-GGTACATCCTCGACGGCATCT-3' (forward) (SEQ ID NO: 5), 5'-GTGCCTCTTTGCTGCTTTCAC-3' (reverse) (SEQ ID NO: 6) for IL-6. Amplification times (CT) values ​​were calculated from the amplification plots. 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 fold change. IL-1β and other drugs were not added to the non-treated group (NT). Each group was repeated four times (n=4). The experiment was repeated twice.

[0127] RT-PCR analysis showed that NDX2 could inhibit the increase in the mRNA levels of the pro-inflammatory genes IL-6 and COX-2 induced by IL-1β (n=4). The results are shown in Figures 4 and 5.

[0128] Detection of nitrite production

[0129] The Griess reagent system is based on a chemical reaction using sulfanilamide and N-1-napthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. This system detects nitrite production. In this experiment, nitric oxide detection is 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 including a series of dilutions for the nitrite standard reference curve were mixed with 50 μL of sulfanilamide solution and incubated in a 96-well plate for 10 min at room temperature in the dark. Then, 50 μl of NED solution was added to each well and incubated for 10 min at room temperature in the dark. Optical density (OD) was determined at 530 nm using a microplate reader within 30 min. IL-1β and other drugs were not added to the non-treated group (NT). Each group was repeated six times (n=6). The experiment was repeated twice.

[0130] Using the Griess reagent, NDX2 was found to dose-dependently inhibit nitric oxide production in IL-1β-stimulated human chondrocytes (n=6). The results are shown in Figure 6.

[0131] Data analysis

[0132] The values ​​obtained were expressed as mean ± SD and analyzed using one-way analysis of variance followed by Bonferroni / Dunnett test. The statistical significance level between two groups was set at P < 0.05.

[0133] Example 3: Mouse macrophage assay to determine the non-toxic dose range of NDX for mouse macrophage RAW264.7 cells

[0134] Cell culture and treatments

[0135] Murine RAW264.7 macrophage cell line (ATCC; Manassas, VA, USA) was maintained in DMEM culture medium containing 10% FBS and 1% antibiotic mixture. To treat the cells, various doses of test drugs were added to the cultures, and 0.5 h later, 100 ng / mL LPS (Sigma-Aldrich Co., St Louis, MO, USA) was added, and then the cells were co-treated with LPS for 24 h.

[0136] Cytotoxicity assay

[0137] Control and treated macrophages were seeded in 96-well plates for 24 hours, and the cell numbers were counted using a WST-1 kit (Fisher Scientific). After removing the supernatant, 150 μL of fresh medium was added to each well. Then, 15 μL of Cell Proliferation Reagent WST-1 was added and incubated with the cells for 3 hours in the dark at 37°C in a humidified atmosphere of 100% carbon dioxide. Each sample was determined at 450 nm using a microplate reader. No drug was added to the non-treated group (NT). Each group was repeated 8 times (n=8). The experiment was repeated twice.

[0138] WST-1 assay showed that NDX1 was toxic to mouse macrophage cell line RAW264.7 cells in a dose-dependent manner; NDX2 and NAG were less toxic than NDX1 (n=8). The results are shown in FIG.

[0139] statistical analysis

[0140] Data were expressed as mean ± SD. Statistical evaluation was performed by analysis of variance (ANOVA) using SPSS 15.0 software. Student's t-test (two-tailed) was performed for comparison of differences between two groups, and p<0.05 was considered significant.

[0141] Example 4: To evaluate the effect of NDX on mouse macrophage RAW264.7 cell activation by LPS

[0142] Detection of IL-6 levels

[0143] The amount of IL-6 in the culture medium from the different treatments was measured by a commercial ELISA kit (Sigma-Aldrich) according to the instructions provided by the manufacturer. Briefly, cells were seeded in 96-well plates and different treatments were performed. The culture medium was collected and mixed stepwise with different assay reagents. The resulting solution was read at 450 nm using a microplate reader. Each group was repeated eight times (n=8). *P<0.05 vs. LPS group. The experiment was repeated twice.

[0144] ELISA studies showed that all three tested drugs (NDX1, NDX2 and NAG) could significantly reduce IL-6 production in LPS-activated mouse RAW264.7 macrophages, while NDX1 treatment was the most effective among them (n=8). The results are shown in Figure 8.

[0145] Western blot analysis

[0146] Cellular proteins were prepared from cells growing on 6-well plates (approximately 500,000 / well) using a commercial cell lysis buffer (Thermo Scientific). Protein concentrations were 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 electrophoretically transferred to a nitrocellulose membrane (Thermo Scientific) overnight at a constant voltage of 10 V. The membrane was blocked with 5% fatty acid-free bovine serum albumin (BSA) fraction V (Roche Diagnostics, Indianapolis, IN, USA) in TBST solution (50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Tween 20) for 1 h at RT, washed, and incubated overnight at 4 °C in 5% BSA in TBST solution containing specific primary antibodies against mouse iNOS and Cox-2 (both from Novus Biologicals, LLC). β-actin was used as a loading control. The membrane was then incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) for 1 h at RT, followed by chemiluminescence substrate and enhancer solution for HRP antibodies mixed in a 1:1 ratio (Thermo Scientific). The membrane was incubated with autoradiography film (Genesee Scientific, San Diego, CA, USA) in the dark, and the film was developed for visualization of the bands. Grayscale images (300-400 dpi) of the films were then scanned using a flatbed scanner. Densitometry was performed in Photoshop. Using the magic wand tool from the tool palette, each band area was selected and the average histogram was recorded and then charted. All western blots were performed in duplicate. No LPS or other drugs were added to the non-treated group (NT).

[0147] Western blot assay revealed that for Cox-2 expression inhibition, NAG>NDX2>NDX1 and for iNOS expression inhibition, NDX1>NDX2>NAG, respectively. The results are shown in Figures 9-10.

[0148] Gene expression analysis

[0149] Total RNA was purified using the RNeasy kit (QIAGEN Sciences, Valencia, CA) according to the protocol provided by the manufacturer and stored at -80°C. cDNA synthesis from total RNA and quantitative PCR were performed using iscript TM cDNA Synthesis Kit and iQ TM Analysis was performed using SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA). Target genes included iNOS, IL-6, and IL-1β. The gene for 18s ribosomal RNA was used as an internal control. Amplification times (CT) values ​​were calculated from amplification plots. Data were analyzed using the 2-ΔΔCT method with 18s rRNA serving as the reference. Gene expression was normalized to the control group in each experiment and expressed as fold change. The primer sequences were as follows: 5'-GTGACCAGTTCACTCTTGGT-3' (forward) (SEQ ID NO: 1), 5'-CATTGGAAGTGAAGCGTTTCG-3' (reverse) (SEQ ID NO: 2) for 18s rRNA, 5'-GAGGGATGCCTTCCGCAGCTG-3' (forward) (SEQ ID NO: 7), 5'-GAATCGAACCCTGATTCCCCGTC-3' (reverse) (SEQ ID NO: 8) for iNOS, 5'-CAACCAACAAGTGATATTCTCCATG-3' (forward) (SEQ ID NO: 9), 5'-GATCCACACTCTCCAGCTGCA-3' (reverse) (SEQ ID NO: 10) for IL-1β, and 5'-GAGTCCTTCAGAGAGATACAG-3' (forward) (SEQ ID NO: 11), 5'-TGGTCTTGGTCCTTAGCC-3' (reverse) (SEQ ID NO: 12) for IL-6.

[0150] RT-PCR showed that NDX1 (0.1 mM and 0.5 mM) could inhibit both iNOS and IL1β mRNA expression, while NAG (0.5 mM) could IL1β but not iNOS mRNA expression. NDX2 (0.5 mM) did not inhibit iNOS or IL1β mRNA expression (n=4). The results are shown in Figures 11-12.

[0151] Detection of nitrite production

[0152] The Griess reagent system is based on a chemical reaction using sulfanilamide and N-1-napthylethylenediamine dihydrochloride (NED) under acidic (phosphoric acid) conditions. This system detects nitrite production. In this experiment, nitric oxide detection was performed with a commercial kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's manual. Briefly, all experimental samples and standards including a series of dilutions for the nitrite standard reference curve were mixed with 50 μL of sulfanilamide solution and incubated in a 96-well plate for 10 min at room temperature in the dark. Then, 50 μL of NED solution was added to each well and incubated for 10 min at room temperature in the dark. Optical density (OD) was determined at 530 nm using a microplate reader within 30 min. No LPS or other drugs were added to the non-treated group (NT). The experiment was repeated twice. Each group was repeated six times (n=6). The experiment was repeated twice.

[0153] Nitrite tests showed that NDX1 (0.2 mM, 1 mM and 5 mM) and NDX2 (0.5 mM and 5 mM) could significantly inhibit nitric oxide production (n=6), whereas NAG (0.5-5 mM) could not. The results are shown in Figure 13.

[0154] Example 5: To determine the anti-inflammatory activity of NDX1 and NDX2 against LPS-induced systemic inflammation in mice

[0155] animal

[0156] Male C57BL6 / J mice (8-10 weeks old, 20-25 g) purchased from Charles River were used in this study. Mice were maintained in the University of Virginia (UVa) School of Medicine animal facility. All experimental procedures were approved by the UVa School of Medicine Animal Care and Ethics Committee and followed the National Council on Care and Administration of Animals (CONCEA) guidelines.

[0157] Animal models

[0158] Thirty minutes after intravenous injection of NAG or NDX1 or NDX2 or vehicle (saline), moderate endotoxemia by LPS was induced by a single dose (10mg / Kg, ip) of LPS (E. coli 0111:B4, Sigma Chemical Co., St. Louis, MO, United States). Two doses (200mg / kg body weight and 300mg / kg body weight) were tested for NAG, NDX1 and NDX2. Untreated animals were used to obtain baseline data. Six hours after LPS treatment, inflammation assays were performed as follows. A total of 40 animals were randomly divided into five groups (8 mice / group): no treatment (NT); 10mg / Kg LPS+saline (LPS); 10mg / Kg LPS+NAG (LPS+NAG); 10mg / Kg LPS+NDX1 (LPS+NDX1); and 10mg / Kg LPS+NDX2 (LPS+NDX2). The experiment was repeated twice.

[0159] Leukocyte migration into the peritoneal cavity

[0160] Peritoneal cells were collected by washing with ice-cold Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL, Gaithersburg, MD). Two methods were used: 1) Peritoneal cells were cultured in DMEM supplemented with 10% FBS and antibiotics for 24 hours in a CO2 incubator. Then, WST-1 assay was performed to count the cell number; 2) Peritoneal cells were fixed with 70% ethanol overnight at 4°C for use. The cell suspension (100 μL) was then centrifuged at 1,500 rpm for 10 minutes at 4°C onto cover slips in 96-well plates, stained with fluorescent DNA dye DAPI to indicate cell nuclei, and evaluated with a Cytation 5 image reader and analyzed with Gen5 software.

[0161] Bone marrow peroxidase activity assay

[0162] Leukocyte migration to the lungs was assessed using a bone marrow peroxidase 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 assay. Samples were homogenized using a Tissue-Tearor and the homogenates were centrifuged (13.000 rpm, 2 min, 4°C). Supernatants were assayed spectrophotometrically for bone marrow peroxidase activity at 450 nm.

[0163] Leukocyte migration to the lungs and peritoneal cavity

[0164] Sequestration of leukocytes from the circulation is an event that may impair an appropriate response to infection. To compare the effects of NAG with NDX1 and NDX2 on leukocyte infiltration in the lung, bone marrow peroxidase (MPO) activity was determined. The results shown in Figure 14 indicated 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 pulmonary leukocyte sequestration, WST-1 tests or DAPI staining could show that NDX1, together with LPS, significantly reduced leukocyte migration into the peritoneal cavity compared to LPS alone, whereas NAG or NDX2 did not. The results showed that the sequestered leukocytes were mainly neutrophils (Figures 16A, 16B, 16C, white arrows), with a small number of monocytes (Figures 16A, 16B, 16C, red arrows) that were larger in size than neutrophils. The results are shown in Figures 15, 16A, 16B, 16C, and 16D.

[0165] Determination of cytokine levels

[0166] Serum TNFα and IL-6 concentrations were determined using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Briefly, serum from each animal was collected and mixed stepwise with various assay reagents. The resulting solution was read at 450 nm using a microplate reader.

[0167] In vivo experiments showed that NDX1 was most effective in reducing the inflammatory response to LPS in mice.

[0168] Serum levels of inflammatory cytokines

[0169] The production of inflammatory cytokines coordinates the response to infectious agents through immune cell activation and recruitment. To determine the production of inflammatory mediators, serum concentrations of IL-6 and TNFα were quantified. LPS increased serum levels of IL-6 and TNFα, and all three tested compounds, NDX1, NDX2 and NAG, were able to significantly reduce serum levels of IL-6 and TNFα in LPS-mice at a dose of 300 mg / kg. The results are shown in Figures 17, 18, 19 and 20. There was no significant difference in effect among the three compounds at this dose. However, NDX1 showed significantly higher inhibitory activity against IL-6 and TNFα production than NAG or NDX2 at a low dose of 200 mg / kg. The results are shown in Figures 21, 22, 23 and 24.

[0170] Example 6: To evaluate the anti-inflammatory activity of NDX1 and NDX2 in LPS-activated primary mouse peritoneal macrophages

[0171] Experimental group [Table 1]

[0172] (*Doses 1, 2, and 3 were selected as low, medium, and high doses that were not cytotoxic in the cytotoxicity assay)

[0173] Cell culture and WST-1 assay

[0174] Peritoneal macrophages were isolated from the peritoneal cavity and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, VT) and 100 IU / mL penicillin G and 100 μg / mL streptomycin at 37°C in a humidified atmosphere of 5% carbon dioxide. Cells were grown in 96-well plates and treated with 100 ng / ml LPS (Sigma-Aldrich) 0.5 h after addition of BNAG and NAG. After 24 h, cell numbers were counted using a WST-1 kit (Fisher Scientific). After removing the supernatant, 150 μl fresh medium was added to each cell. Then, 15 μl Cell Proliferation Reagent WST-1 was added and incubated with the cells for 3 h in a humidified atmosphere of 5% carbon dioxide in the dark at 37°C. The concentration of each sample was determined at 450 nm on a microplate reader.

[0175] WST-1 assay showed that 100ng / mL LPS significantly increased cell proliferation of mouse primary peritoneal macrophages. Each of the three tested compounds, NDX1, NDX2 or NAG, at various doses up to 1mM in combination with 100ng / mL LPS had no significant effect on cell number compared to LPS alone. The results are shown in Figure 21. ELISA test showed that 100ng / mL LPS significantly increased IL6 and TNFa production in mouse primary peritoneal macrophages. All three drugs could significantly reduce IL-6 and TNFa production in LPS-activated macrophages, with NDX1 being the most effective among them. The results are shown in Figures 22 and 23.

[0176] Detection of IL-6 and TNFα levels

[0177] The amount of IL-6 and TNFα was measured by commercial ELISA kits (Sigma-Aldrich) according to the instructions provided by the manufacturer. Briefly, cells were seeded in 96-well plates and various treatments were performed. The culture medium was collected and mixed stepwise with various assays. The resulting solution was read at 450 nm using a microplate reader.

[0178] statistical analysis

[0179] All data were reported as mean ± SD. One-way ANOVA test for multiple comparisons was performed. In each figure, p-values ​​were indicated if the differences comparing the selected control group with the rest of the samples were significant [P-value < 0.05 (*)]. Results were obtained using the GraphPad Prims program.

[0180] In vitro experiments showed that NDX1 was effective in reducing the inflammatory response to LPS in mouse primary peritoneal macrophages.

[0181] Example 7

[0182] In Examples 5 and 6, two compounds, NDX1 and NDX2, which are molecules obtained by modification of NAG, were tested. Both were anti-inflammatory against the parent molecule using an in vivo model of LPS-induced mouse systemic inflammation and an in vitro model of mouse primary peritoneal inflammation. Meanwhile, at a test dose of 300 mg / kg, all three compounds showed significant inhibitory effects on the serum level increase of cytokines IL-6 and TNFα production by LPS, and NDX1 showed the highest efficacy among the three compounds at a dose of 200 mg / kg. NDX1 also showed the strongest inhibition on leukocyte migration to the lung and peritoneal cavity induced by LPS. In vitro experiments revealed that NDX1, NDX2 and NAG could significantly inhibit IL-6 and TNFα production in LPS-activated mouse peritoneal macrophages, and NDX1 was the most effective among them. Without being bound by any theory, further studies using human cells / tissues and specific animal disease models will provide the necessary information as to 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 formula for treating inflammatory diseases 【Chemistry 1】 A pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is rheumatoid arthritis (RA), osteoarthritis (OA), colitis, asthma, systemic sclerosis, Sjögren's syndrome, bronchiectasis, idiopathic pulmonary fibrosis, atherosclerotic plaque, ulcerative colitis, or Crohn's disease.

3. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is rheumatoid arthritis (RA).

4. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is osteoarthritis (OA).

5. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is an atheroma.

6. The pharmaceutical composition according to claim 1, which is administered intravenously, orally, subcutaneously, buccally, transdermally, or nasally.

7. The pharmaceutical composition according to claim 1, which is administered orally.

8. The pharmaceutical composition according to claim 1, wherein the therapeutically effective amount of the compound is in the range of about 1 mg to about 1000 mg.

9. The pharmaceutical composition according to claim 1, administered once a week, twice a week, once a day (QD), twice a day (BID), or three times a day (TID).

10. The pharmaceutical composition according to claim 1, wherein the compound inhibits NO production in the patient.

11. The pharmaceutical composition according to claim 1, wherein the compound inhibits iNOS gene expression in a patient.

12. The pharmaceutical composition according to claim 1, which is administered together with one or more further therapeutic agents.

13. The pharmaceutical composition according to claim 12, wherein one or more further therapeutic agents are a corticosteroid, salicylic acid, an acetic acid derivative, an enolic acid (oxicam) derivative, a propionic acid derivative, anthranilic acid derivative, or a Cox-2 inhibitor.

14. The pharmaceutical composition according to claim 12, wherein one or more further therapeutic agents are selected from the group consisting of aspirin, diflunisal, sarsalat, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac, tolmetin, meloxicam, piroxicam, ibuprofen, naproxen, ketoprofen, fenoprofen, flurbiprofen, oxaprozin, mefenamic acid, celecoxib, cortisone, prednisone, and methylprednisone.

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