Methods of treating an inflammatory-related disease

[0001] The invention relates to pharmaceutical uses and compositions and methods for the treatment of inflammation-related diseases associated with pro-inflammatory cytokine expression and / or reduced anti-inflammatory cytokine expression as defined in the appended claims.

BACKGROUND OF THE INVENTION [0002] Irregular and / or abnormal inflammation is an important component of a number of human diseases. People with multiple degenerative disorders often show elevated levels of pro-inflammatory markers in the blood. One type of such proinflammatory markers are marker inflammatory cytokines, including IL-α, β, IL-2, IL-3, IL-6, IL7, IL-9, IL-12, IL-17, IL-18, TNF -α, LT, LIF, oncostatin and IFNcla, β, γ.

[0003] A non-limiting list of common medical problems that are directly caused by inflammatory cytokines includes: arthritis, in which inflammatory cytokines destroy, leading to changes in the synovium and destruction of articular cartilage and bone; renal failure, in the course of which inflammatory cytokines restrict circulation and damage the nephrons; lupus in which inflammatory cytokines trigger an autoimmune attack; asthma, in which cytokines close the airways; psoriasis in which inflammatory cytokines cause dermatitis; pancreatitis, in which inflammatory cytokines cause damage to pancreatic cells; allergy in which inflammatory cytokines cause autoimmune reactions; fibrosis in which inflammatory cytokines attack damaged tissue; postoperative complications in which inflammatory cytokines prevent healing; anemia in which inflammatory cytokines attack the production of erythropoietin; and fibromyalgia where inflammatory cytokines are elevated in patients with fibromyalgia. Other diseases associated with chronic inflammation include cancer that is caused by chronic inflammation; heart attack, in which chronic inflammation contributes to atherosclerosis; Alzheimer's disease, in which chronic inflammation destroys brain cells; congestive heart failure where chronic inflammation causes myocardial wasting; stroke in which chronic inflammation promotes thromboembolic events and aortic stenosis, in which chronic inflammation damages the heart valves. Arteriosclerosis, osteoporosis, Parkinson's disease, infection, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, and multiple sclerosis (a typical inflammatory autoimmune disease) are also associated with inflammation (1-18). Some diseases at advanced stages can be life threatening. A number of treatments are available for such inflammatory diseases; however, their results are generally unsatisfactory, as indicated by the inefficiency of these methods and drug-related side effects.

Inflammatory bowel disease [0004] Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC); both of these diseases are idiopathic chronic diseases that occur with increasing frequency in many parts of the world. In the United States, there are over 600,000 people every year. IBD can affect the small or large intestine or both. CD can affect any part of the gastrointestinal tract, but most often it covers the distal small intestine and the colon. It saves the rectum or causes inflammation or infection with leakage around the rectum. UC usually causes ulcers in the lower part of the large intestine, often starting in the rectum. Causes various symptoms, which may include diarrhea, fever and pain. Patients with long-term UC have an increased risk of developing colon cancer. There is currently no satisfactory treatment for IBD, as the cause of IBD is still unclear, although infectious and immunological mechanisms have been proposed. Treatment of IBD is directed at controlling inflammatory symptoms, typically with corticosteroids, aminosalicylates and standard immunosuppressants such as azathioprine (6mercaptopurine), methotrexate and cyclosporine. Of these therapies, the only disease-modifying therapies are immunosuppressants, azathioprine and methotrexate, both showing a slow onset of action and only moderate efficacy. Long-term treatment may cause liver damage (fibrosis or cirrhosis) and suppression of bone marrow function. In addition, patients often develop resistance to such treatment. Other treatment regimens only target symptomatic treatment (19, 20).

Psoriasis [0005] Psoriasis is one of the most common chronic skin diseases mediated by the immune system, which occurs in various forms and with varying levels of severity, affecting about 2% or more than 4.5 million people in the United States, of which believes 1.5 million have a moderate or severe form of the disease. Ten to thirty percent of patients with psoriasis also have the form of arthritis - psoriatic arthritis that damages the bones and connective tissue around the joints. Psoriasis is manifested as patches of raised, reddened skin covered with scaly, white build-up. Lesions may also have the appearance of pustules (pustular psoriasis) or burns (erythematous). Psoriasis can also cause intense itching and burning. Patients suffer both psychologically and physically. There are currently several treatments for psoriasis, including topical therapy, phototherapy, and systemic therapy. However, it is generally believed that such therapies merely suppress and modify the course of the disease. None of them cure her. In addition, many therapies are cosmetically undesirable, uncomfortable with long-term use, or associated with significant toxic effects.

[0006] Due to an increasingly better understanding of the biological features of psoriasis in the last two decades, biological therapies aimed at the activity of T lymphocytes and cytokines responsible for the inflammatory nature of this disease have become available. Currently, psoriasis medications include TNF-α inhibitors, initially used to treat rheumatoid arthritis (RA), ENBREL® (etanercept), REMICADE® (infliximab) and HUMIRA® (adalimumab), and the AMEVIVE® T cell inhibitor (alefacept) with Biogen registered in 2002 and RAPTIVA® (efalizumab) with Genentech / Xoma registered in 2003 (21). AMEVIVE ALEFACEPT® is an immunoglobulin fusion protein, consisting of the first extracellular domain of human LFA-3 fused to the hinge region, C (H) 2 and C (H) 3 of human IgG (1). It inhibits the proliferation of T cells by NK cells (22). RAPTIVA® is also known as anti-CD11a antibody, a humanized monoclonal antibody that targets a T cell adhesion molecule, an antigen associated with leukocyte-1 (LFA-1) activity. Preventing binding of LFA-1 to its ligand (ICAM-1, a molecule of intercellular adhesion-1) inhibits lymphocyte activation and migration, leading to reduced lymphocyte infiltration and thus limiting the cascade of events ultimately leading to signs and symptoms of psoriasis (23). However, the potential side effects of the TNF-α inhibitor are severe and include the development of lymphoma (24), exacerbation of congestive heart failure, lead to severe infection and sepsis, and exacerbations of multiple sclerosis and central nervous system problems (25, 26). Although the side effects of the T cell inhibitor AMEVIVE® / RAPTIVA® may be better tolerated in the treatment of psoriasis, RAPTIVA® is an immunosuppressant. Immunosuppressants may increase the risk of infection, reactivate latent, chronic infections, or increase the risk of developing cancer.

[0007] Although in the last two decades significant progress has been made in understanding the biological features of psoriasis and, as described above, unconventional treatment of psoriasis has become available, there is still no way to deal with the great suffering it causes this disease. A survey of 40,000 US patients with psoriasis by the National Psoriasis Foundation in 1998 found that 79% of younger patients were frustrated by the ineffectiveness of their treatment. 32% of patients with severe disease believed that their treatment was not sufficiently aggressive (27, 28).

Rheumatoid Arthritis [0008] Rheumatoid arthritis (RA) is another example of a troublesome inflammatory disorder. It is a common, chronic inflammation-related disease characterized by chronic inflammation of the lining of the joints (synovium) and / or other internal organs. Inflammatory cells can also attack and damage bone and cartilage. There may be a loss of shape and axiality of the affected joint, which leads to loss of mobility. RA patients experience pain, stiffness, warmth, redness and swelling of the joint, and other systemic symptoms such as fever, fatigue and anemia. About 1% of the population or 2.1 million residents of the United States are currently affected, including more women (1.5 million) than men (0.6 million). The pathology of RA is not fully understood, although a cascade of abnormal immune responses has been postulated as a mechanism for developing the disease. Unfortunately, regular treatment is ineffective in RA (29). The disease does not respond completely to symptomatic drugs, including corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs) used since the 1950s. In addition, these drugs carry the risk of serious side effects. The therapeutic effect of disease-modifying drugs (DMARDs) such as methotrexate (MTX) is often inconsistent and short-lived.

[0009] Recently, a new class of biological DMARDs (disease-modifying drugs) for the treatment of RA has been developed, based on understanding the role of cytokines, TNF-α and IL-1 in the inflammatory process. The FDA has registered a number of such DMARDs, including ENBREL® (etanercept) from Immunex / Amgen Inc. in 1998, REMICADE® (infliximab) from Centocor / Johnson & Johnson, HUMIRA® (adalimumab) from Abbott Laboratories Inc. in 2002 and KINERET® (anakinra) from Amgen in 2001. ENBREL® is a soluble recombinant TNF receptor (TNFR) protein. REMICADE® is a humanized mouse (chimeric) anti-TNF-α monoclonal antibody. HUMIRAL® is a fully human monoclonal anti-TNF antibody, produced using phage display technology, resulting in antibody with heavy and light chain variable regions of human origin and human IgG1: k variable regions. All 3 protein-based drugs are directed against TNF-α and bind to it, blocking the action of TNF-α. KINERET® is a recombinant IL-1 receptor antagonist that is similar to natural human IL-1Ra, except for the addition of a single methionine residue at the amino terminus. KINERET® blocks the biological activity of IL-1 by competitive inhibition of IL-1 binding to the type I IL-1 receptor (IL-1RI) and, as a result, the reduction of the pro-inflammatory effects of IL-1.

[0010] Treatment with these biological DMARDs alleviates the symptoms, inhibits the progression of structural damage and improves the physical fitness of patients with moderate or severe RA. These 3 commercially available TNF-α blocking agents show similar efficacy to MTX, the widely used DMARD, in the treatment of RA patients (30). Although they provide significant efficacy and show a good overall safety profile in the short to medium term in many RA patients, these biological drugs can pose serious problems and cause long-term adverse effects such as on the liver and still require evaluation. An alarming relationship has been found between the use of ENBREL® or REMICADE® and the development of lymphoma (24). As described above, several reports indicate that patients treated with ENBREL® or REMICADE® exacerbate congestive heart failure and develop serious infection and sepsis, and increase exacerbations of multiple sclerosis and other central nervous system problems (26, 27).

Multiple sclerosis [0011] Multiple sclerosis (MS) is an autoimmune disease diagnosed in 350,000 to 500,000 people in the United States. A sign of this disease are numerous areas of myelin inflammation and scarring in the brain and spinal cord. Patients with MS show neurological disorders of varying degrees depending on the location and severity of myelin scarring. Common symptoms of MS include fatigue, weakness, spasticity, imbalance, bladder and bowel problems, numbness, blindness, tremors and depression. Current MS treatment only alleviates symptoms or delays the progression of disability, and a number of new MS therapies, including stem cell transplantation and gene therapy, are conservative (31, 32). Although TNF antibodies have been shown to be protective in experimental autoimmune encephalomyelitis (EAE), these agents exacerbate the disease in MS patients, suggesting that TNF-α inhibition alone is not sufficient (33).

Neurodegenerative diseases [0012] Alzheimer's disease (AD) and Parkinson's disease (PK) are the two most common neurodegenerative diseases. AD is a brain disorder. It seriously impairs the patient's ability to perform daily activities. It occupies parts of the brain that control thinking, memory and speech. An estimated 4 million Americans, usually over 60, suffer from AD.

[0013] PK is a progressive central nervous system disorder affecting over 1.5 million people in the United States. Clinically, this disease is characterized by a decrease in spontaneous movements, difficulty in walking, postural instability, stiffness and tremor. PK is caused by the degeneration of pigmented neurons of the black matter of the brain, which leads to a decrease in dopamine availability. The causes of these neurodegenerative disorders are unknown and there is currently no cure for this disease. [0014] Thus, there is a need for new approaches for the treatment of the above and other inflammation-related diseases. Although the mechanisms that cause inflammatory diseases are unclear and often differ from each other, it has been shown that immune dysfunction caused by cytokine regulation disorders play an important role in the formation and progression of inflammation (Table 1) (27, 34, 35).

[0015] Cytokines can be broadly divided into 3 types: proinflammatory (IL-Ια, β, IL-2, IL-3, IL-6, IL-7, IL-9, IL-12, IL-17, 1L-18 , TNF-α, LT, LIF, oncostatin and IFNc1a, β, γ); anti-inflammatory (IL-4, IL-10, IL-11, W-13 and TGFe) and chemokines (IL-8, Groa, MIP-1, MCP-1, ENA-78 and RANTES).

[0016] In many inflammations, it seems that proinflammatory cytokines, especially TNF-α, IL-1e, and IL-6, and the anti-inflammatory cytokine IL-10 play an important role in the pathogenesis of various inflammation-related diseases and can therefore serve as potential therapeutic goals. For example, elevated levels of some proinflammatory cytokines (TNF-α, IFNy, IL-1, IL-2, IL-6 and IL-12) and chemokines (IL-8, MCP-1 and RANTES) have been found in a number of inflammation-related diseases, such as CD, psoriasis, RA, Graves disease and Hashimoto's thyroiditis (34), which are simultaneously accompanied by an increase in soluble TNF receptors, IL-1 receptor antagonists and anti-inflammatory cytokine IL-10 (35, 37). IL-10 has been shown to inhibit the increased production of proinflammatory cytokines both in vitro, in LPMC cultures, and in vivo in patients (38). The positive response of patients with CDs treated with IL-10 indicates that there may also be an imbalance between the production of pro-inflammatory and anti-inflammatory cytokines in CD. [0017] In summary, the strategy for treating inflammation-related diseases has evolved in recent years, partly as a result of growing concern about the severity of these diseases, and partly due to significant progress in understanding the important role cytokines play in their immune pathogenesis. Most efforts focused on targeting TNF-α and IL-1 (39); and a number of products (TNF-α inhibitors: infliximab, anti-TNF-α monoclonal antibody, and etanercept, TNF-α p75 receptor) are currently commercially available or undergoing clinical trials for the treatment of RA, psoriasis and IBD as mentioned above. A number of other potential drugs or strategies targeting EL-1 (40), IL-6 or IL-10 (40-42) are under development. These biological therapies provide significant short- and long-term efficacy in many RA patients (43-46). Although these drugs are well tolerated and have a good overall safety profile, they require active pharmacovigilance. Based on their mechanism of action and prior notifications of a wide range of side effects, there is a need to investigate the long-term risk of side effects, including hematological, infectious, neurological, oncological and immunological effects.

[0018] Strategies targeting a single pro-inflammatory cytokine as anti-inflammatory therapy ignore the very important fact that inflammation-related diseases involve the extensive "system" of the cytokine network. For example, chemokines, a family of IL-8-related immune system molecules, contain about 50 ligands and 20 receptors, often acting with redundancy, so the selection of appropriate specific antagonists is not only difficult but does not provide long-term efficacy. In addition, currently commercially available products or products being developed are mainly protein-based agents, the production of which is expensive and the administration (i.e. infusion) inconvenient. Thus, since the activity of the immune system is precisely balanced by the activity of mediators or pro-inflammatory and anti-inflammatory cytokines, modulation of numerous pro / anti-inflammatory cytokines instead of blocking only one specific cytokine by small molecules should not only lead to better therapeutic efficacy with less side effects, but will have also have many benefits associated with small molecule drugs.

[0019] Based on this concept, we have tested a number of types of small molecules to examine their ability to regulate many cytokines and investigated their potential clinical applications in the treatment of various inflammation-related diseases.

[0020] Meisoindigo has been used to treat chronic myeloid leukemia (CML) in China with minor side effects (47). In our earlier patent (US Patent No. 6,566,341) we have shown that Meisoindigo and similar compounds have activity against solid tumors due to their ability to inhibit cyclin-dependent kinases, induce cell differentiation and promote apoptosis. In the present invention, we present new therapeutic activities for this class of molecules in the treatment of various inflammation-related diseases, including inflammatory bowel diseases and psoriasis in rodents as well as in humans. We demonstrate that agents of this type inhibit the secretion and expression of numerous proinflammatory cytokines, including IL-β, IL-6 and TNF-α in cell lines, and promote the production of anti-inflammatory cytokine IL-10. In one human case, Meisoindigo also proved to be very effective in IBD, with no significant side effects.

[0021] EP 1 079 826, Eisenbrand et al., Entitled "Use of Indigoid Bisindole Derivatives for the Manufacture of a Medicament to Inhibit Cyclin Dependent Kinases", relates to the use of indigoid bisindole derivatives in the manufacture of a medicament for the treatment of diseases associated with loss of reproduction control. According to EP 1 079 826, psoriasis, cardiovascular diseases, infectious diseases, nephrological disorders, neurodegenerative disorders and viral infections are all diseases associated with loss of control of cell proliferation. EP 1 079 826 states that this drug is effective in the treatment of these diseases associated with the loss of proliferation control by inhibition of cyclin-dependent kinases (CDKs).

[0022] On the contrary, the Applicants have found that isoindigo, indigo, inddyrubine and their derivatives can be used to suppress or inhibit the expression of proinflammatory cytokines, e.g. TNF-α, IL-1 and IL-6, for the treatment of inflammation-related diseases. associated with cytokine expression.

[0023] Although some of the diseases mentioned in EP 1 079 826 as associated with loss of cell proliferation control are also associated with cytokine expression, applicants have found that the amount of therapeutic agent required to treat these respective diseases by inhibiting cytokine levels is significantly less than required for CDK inhibition as given in EP 1 079 826.

SUMMARY OF THE INVENTION [0024] The present invention is defined in the appended claims and enables the treatment of various inflammation-related diseases with minimal side effects. One of the most important advantages of the present invention is that the therapeutic compounds not only target the symptoms of various inflammation-related diseases, but also modify the diseases by suppressing the expression / secretion of numerous pro-inflammatory cytokines (IL-1a, β, IL-2, IL- 3, IL-6, IL-7, IL-9, IL-12, IL-17, IL-18, TNF-α, LT, LIF, oncostatin or IFNc1a, β, γ) and / or by stimulating anti-inflammatory cytokine expression (IL-4, IL-10, IL-11, W-13 or TGFe).

In a similar way, documents DE 100 53 474, WO 02/074742, WO 03/100401,

WO 02/092079, WO 03/051900 and US 2002/132792 relate to all CDK inhibitory activities of individual compounds.

[0026] The present invention often leads to a cure instead of just a temporary remission of disease symptoms. In contrast, existing therapies for inflammation-related diseases in most cases only alleviate the symptoms for a short period of time. In addition, the therapeutic compounds of the present invention are small molecules that are simple, chemically stable, and that their preparation and administration is generally easy. In addition, applicants have found that relatively low doses / concentrations of the compounds are usually sufficient to substantially inhibit pro-inflammatory cytokines in the patient's body, reducing the risk of treatment-related side effects.

[0027] The pharmaceutical compositions described herein preferably contain at least one compound selected from compounds of formula (I) or (II) as defined in the claims, an anti-inflammatory agent and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS [0028]

Figure 1 schematically shows the role of pro / anti-inflammatory cytokines and growth factors and the site of action of Meisoindigo in the pathological process of chronic inflammatory diseases.

Figure 2 shows the effect of Meisoindigo on IL-1 β secretion in LPS stimulated human monocytic THP-1 cells. Inhibitory effect of Meisoindigo on the production of IL-1 β in LPS-stimulated human monocytic THP-1 cells. THP-1 cells were treated / stimulated with 1 μg lipopolysaccharide (LPS, Sigma) or not and exposed for 24 h to a series of Meisoindigo concentrations (from 31.25 nM to 16000 nM). Cell viability was examined under a microscope after triptan blue staining. Levels of IL-1 β protein secreted into the culture medium were measured by ELISA and calculated from its calibration curve (panel A) using the R&D Systems test kit as described in Materials and Methods in Example 1 below. The t-test was used to determine statistical significance. student. *** indicates P <0.001. As shown in panel B, Meisoindigo significantly inhibits IL1 β production already at a concentration of 31 nM.

Figure 3 shows the effect of Meisoindigo on IL-6 secretion and expression in LPS stimulated human monocytic THP-1 cells. Effect of Meisoindigo on IL-6 production (panel B) and transcription (panel C) in LPS-stimulated THP-1 cells. THP-1 cells were treated / stimulated with 1.0 μg / ml LPS or not and exposed for 24 h to a series of Meisoindigo concentrations (from 0.031 to 16 μΜ). IL-1e secreted into the culture medium was measured by ELISA and IL-6 transcription in cells was measured by real-time PCR as described in Materials and Methods in Example 2 below. Panel A: Standard curve plotted using pure IL-1 protein 6 and used to calculate protein production on panel B; Panel C: real-time PCR test for IL-6 transcription assay. ***: P <0.001. As shown in panels B and C, Meisoindigo significantly inhibits both IL-6 secretion and transcription.

Figure 4 shows the effect of Meisoindigo on TNF-α secretion and expression in THP-1 human monocyte cells. Effect of Meisoindigo on protein production (panel B) and gene transcription (panel C) TNF-α in LPS stimulated THP-1 cells: THP-1 cells were treated / stimulated with 1.0 μg / ml LPS or not and exposed for 24 h to Meisoindigo concentration series (from 0.031 to 16 μΜ). TNF-α protein in the medium was measured by ELISA and its transcription in cells was measured using real-time PCR technology as described in Materials and Methods in Example 3 below. Panel A: Standard curve plotted using pure TNF-α protein used for calculating protein production on panel B. Concentration dependent inhibition of Meisoindigo TNF-α secretion (panel B). Panel C: real-time PCR test for determination of TNF-α transcription. There was no effect of the agent on TNF-α transcription. ***: P <0.001. Figure 5 shows Meisoindigo's stimulation of IL-10 production in THP-1 cells. Stimulation of IL-10 production by Meisoindigo in THP-1 cells whether or not treated with LPS: THP-1 cells were exposed or not with 1.0 μg / ml LPS and exposed for 24 h to a series of Meisoindigo concentrations (from 0.031 to 16 μΜ). IL-10 protein in the medium was measured by ELISA, as described in the Materials and methods of Example 4 below. Panel A: Standard curve drawn using pure IL-10 protein and used to calculate protein production on panel B. While the inflammatory LPS stimulant reduced the level of IL-10 protein, Meisoindigo significantly increased protein production, and maximal stimulant effect occurred at 62 , 5 nM, which showed an approximately two-fold increase in IL-10 secretion (panel B). **: P <0.01.

Figure 6 shows the effect of Meisoindigo and NATURA on expression of proinflammatory cytokines and cyclin dependent kinases in THP-1 cells: THP-1 cells growing exponentially were stimulated with 1 μg LPS (panel A and B) or not (panel C) and exposed for 24 h to Meisoindigo or NATURA concentrations indicated. Cell viability was examined by trypan blue exclusion. The levels of IL-1e, IL-6 and IL-10 proteins secreted into the culture medium were measured by ELISA as described in the above examples, using the R&D Systems test kit as described in Materials and Methods in Example 5 below. To determine the significance statistical t-Student test was used. * indicates P <0.001. Meisoindigo and NATURA significantly inhibit IL-1e and IL-6 production and promote IL-10 production at concentrations of 31.25 and

62.5 nM. However, no inhibitory effect of the compounds on CDK2 was found at low concentrations (31.25 and 62.5 nM) under the same experimental conditions.

FIG. 7 shows that Meisoindigo is effective in acute ulcerative DSS-induced colitis in mice. Examples of histochemical examination of colon walls from Balb / c mice with acute ulcerative colitis caused by 5% DSS, treated with or without Meisoindigo (H&E staining, original magnification x 100). Induction of DSS and treatment of Meisoindigo were performed as described in the Materials and Methods in Example 7 below. Panel A shows the normal morphology of the colon wall from animals from the normal control group that were given drinking water without DSS. Panel B shows the wall of the colon from mice with acute colitis caused by 5% DSS, which shows the presence of increased inflammatory cell infiltration (lymphatic papules, red arrows) and focal loss of crypts in the mucosa (erosive lesions, blue arrows). Panel C shows the colon wall from mice with acute ulcerative colitis induced by 5% DSS treated with Meisoindigo. The morphology is similar to that seen in normal controls (panel A), indicating that Meisoindigo is effective in acute ulcerative DSS-induced colitis in mice.

FIG. 8 shows a photo from a flexible speculum sigmoidoscopy performed on a patient with inflammatory bowel disease, before and after Meisoindigo treatment. The upper panels show photos from two places of inflammation (A and B), which clearly show inflammation with severe edema. A pathomorphological study by a pathologist at Mount Sinai Medical Center in New York found "severe, active, chronic proctitis and colitis with erosions and features suggestive of idiopathic inflammatory bowel disease." The lower panels show photos from a sigmoidoscopy with a flexible speculum of the same sites 9 weeks after treatment of the patient Meisoindigo. After treatment, the colon surface became normal, the edema subsided and the blood vessels became clearly visible, although at B (lower panel) there were scars during the remission period. The pathomorphological examination revealed "inactive, chronic proctitis that suggests idiopathic inflammatory bowel disease".

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0029] The present invention is defined in the appended claims. A preferred use of the present invention includes administering to a live animal in need of such treatment one or more compounds as defined in the claims.

[0030] It should be remembered that the present methods include, but are not limited to, treating an inflammation-related disease by preventing disease-related inflammation by regulating the cytokines involved in the progression of the pathology and thereby preventing the occurrence of inflammation-related disease.

[0031] The inflammation-related disease is selected from the group consisting of: inflammatory bowel disease; rheumatoid arthritis and lupus. Preferably, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0032] Other inflammation-related diseases disclosed herein but not claimed include: psoriasis; multiple sclerosis; neurodegenerative disorder; congestive heart failure; stroke; aortic stenosis; renal failure; inflammation of the pancreas; allergies; fibrosis; anemia; atherosclerosis; metabolic disease; bone disease; cardiovascular disease; chemotherapy / radiation related complication; type I diabetes; type II diabetes; liver disease; disorder of the gastrointestinal tract; eye disease; allergic conjunctivitis; diabetic retinopathy; Sjogren's syndrome; uveitis; pulmonary disorder; kidney disease; skin infection; HIV-related cachexia; brain malaria; ankylosing spondylitis; leprosy; anemia; and fibromyalgia.

[0033] The neurodegenerative disorders described herein include: Alzheimer's disease and Parkinson's disease; a complication on the part of the gastrointestinal tract is diarrhea; the liver disease is selected from the group consisting of: autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis or fulminant hepatic failure; the gastrointestinal tract disorder is selected from the group consisting of: celiac disease and inflammatory colitis; bone disease is osteoporosis; the pulmonary disorder is selected from the group consisting of: hay fever, asthma, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis and sarcoidosis; the cardiovascular disease is selected from the group consisting of atherosclerotic heart disease, congestive heart failure and restenosis; and the kidney disease is selected from the group consisting of glomerulonephritis and vasculitis.

[0034] The compound is in an amount that inhibits the expression of proinflammatory cytokines and / or stimulates the expression of anti-inflammatory cytokines. In one embodiment, the compound is preferably in an amount that inhibits at least 30% of the expression of one or more pro-inflammatory cytokines selected from the group consisting of: IL-1a, β, IL-2, IL-3, IL-6, IL-7, IL- 9, IL-12, IL-17, IL5 18, TNF-α, LT, LIF, oncostatin and IFNc1a, β, γ; more preferably cytokine expression is at least 40%, and most preferably it is inhibited by 50% or more. In another embodiment, the compound is preferably in an amount that stimulates the expression of the anti-inflammatory cytokine. In this embodiment, the compound is preferably in an amount that increases expression of the anti-inflammatory cytokine selected from the group consisting of: IL-4, IL-10, IL-11, W-13 or TGFe by at least 25%, more preferably at least 50%, and most preferably at least 75%.

Chemical structures:

[0035] The present invention relates to the use of specific groups of compounds of formulas (I) or (II)

<img file="PL1706112T3_D0001.tif" />

wherein R3, R4, R5, R6, R7, R8, R9 and R10 are the same or different and represent a hydrogen atom;

hydroxyl group; nitroso group; nitro group; monosaccharide; disaccharide; halogen; a hydrocarbyl group; or a hydrocarbyl functional group unsubstituted or substituted by one or more hydroxyl moieties, carboxyl moieties, nitroxyl moieties, monosaccharides, disaccharides, amines, amides, thiols, sulfates, sulfonates, sulfonamides or halogen atoms, where the hydrocarbyl has 1 to 8 carbon atoms; a group -R11R12 wherein R11 and R12 may be the same or different and represent a hydrogen atom, a straight-chain or branched-chain alkyl group having 1 to 18 carbon atoms, which may additionally have one or more hydroxyl and / or amino groups, substituted or unsubstituted aryl groups that may contain one or more heteroatoms, or an acyl group, or R11 and R12 together form a ring containing 2 to 6, optionally unsubstituted, CH2 groups; the azo group -N = N-R13 wherein R13 is an aromatic system which may be substituted by one or more carboxyl groups and / or phosphoryl groups, or a group selected from the group consisting of sugars, amino acids, peptides or steroid hormones; or R1 and R6, respectively, and R2 and R7 together form, independently of each other, a ring containing 1 to 4, optionally substituted, CH2 groups; [0036] The groups R1 and R2 are the same or different and represent a hydrogen atom; halogen; hydroxyl group; a hydrocarbyl group or a functional hydrocarbyl group unsubstituted or substituted by one or more hydroxyl moieties, carboxyl moieties, nitroxyl moieties, monosaccharides, disaccharides, amines, amides, thiols, sulfates, sulfonates, sulfonamides or halogen atoms, where the hydrocarbyl has 1 to 8 carbon atoms; a mono-, di- or trialkylsilyl group having 1 to 6 carbon atoms in each case independently of each other in a straight or branched chain alkyl group; a mono-, di- or triarylsilyl group with substituted or unsubstituted aryl groups in each case independently of each other; a NR17R18 group in which R17 and R18 may be the same or different and represent a hydrogen atom, a straight or branched chain alkyl group having 1 to 18 carbon atoms, which may additionally have one or more hydroxyl and / or amino groups, substituted or unsubstituted aryl which may contain one or more heteroatoms or an acyl group; methyleneamino -CH17R18 wherein R17 and R18 are as defined above; physiological amino acid residue associated with the nitrogen atom as an amide, a substituted or unsubstituted monosaccharide, disaccharide or oligosaccharide group; or a sugar, amino acid, peptide or steroid hormone moiety.

[0037] Preferred compounds are those in which at least one R1, R2, R3, R4, R5, R6, R7, R8, R9 or R10 is independently a monosaccharide, disaccharide or hydrocarbyl group, or a hydrocarbyl functional group substituted with one or more the number of hydroxyl moieties, carboxyl moieties, nitroxyl moieties, monosaccharides, disaccharides, amines, amides, thiols or halogen atoms, where the hydrocarbyl has 1 to 8 carbon atoms; and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9 or R10 increases the biological activity or bioavailability of the compound.

[0038] It is preferred that R1, R2, R3, R4, R5, R6, R7, R8, R9 or R10 increase the biological activity or bioavailability of the compound by increasing the solubility of the compound. It is more preferred that both bioactivity and bioavailability are increased by one or more of R1, R2, R3, R4, R5, R6, R7,

R8, R9 or R10.

[0039] Preferred compounds are those wherein at least R1 or R2 is a monosaccharide moiety; a disaccharide moiety unsubstituted or substituted by one or more hydroxyl moieties or carboxyl moieties; halogen; a hydrocarbyl group or a hydrocarbyl functional group unsubstituted or substituted by one or more hydroxyl moieties, carboxyl moieties, nitroxyl moieties, monosaccharides, disaccharides, amines, amides, thiols, sulfates, sulfonates, sulfonamides or halogen atoms, where hydrocarbyl has 8 coal. In many cases, only one of R1 or R2 must be one of the mentioned moieties, and one of the most preferred substituents is -CH2CH2OH.

[0040] More preferred compounds of formulas (I) or (II) are those wherein R1 or R2 is a glycoside molecule, more preferably a monosaccharide, and most preferably an acetylated monosaccharide.

[0041] In one embodiment of the invention, the uses include Meisoindigo, triacetylated glyco-Meisoindigo (prodrug) (shown as formula (V)) and NATURE (shown as formula (VI)).

<img file="PL1706112T3_D0002.tif" />

[0042] The term "hydrocarbyl" in the context of the present invention and in the formulas above broadly refers to a monovalent hydrocarbon group in which valence results from the cleavage of a hydrogen atom from a carbon atom. Hydrocarbyl includes, for example, aliphatic (straight or branched chain), cycloaliphatic, aromatic and mixed (e.g. aralkyl and alkaryl) groups. Hydrocarbyl also includes those groups with internal unsaturation and activated unsaturation. Even more particularly, hydrocarbyl includes (but is not limited to) groups such as alkyl, cycloalkyl, aryl, aralkyl, alkaryl, alkenyl, cycloalkenyl and alkynyl, preferably having up to 12 carbon atoms. Preferred forms include those in which the hydrocarbyl group has 1 to 8 carbon atoms. These and other hydrocarbyl groups may optionally include a carbonyl group or carbonyl groups (which are / are included in the number of carbon atoms) and / or heteroatom or heteroatoms (such as at least one oxygen, sulfur, nitrogen or silicon), in a chain or ring.

[0043] The term "functional hydrocarbyl" in the context of the present invention and in the above formulas broadly refers to a hydrocarbyl containing "reactive" side and / or end groups and / or "latent reactive" functional groups and / or leaving groups a. Reactive functional groups refer to functional groups that are reactive to ordinary monomer / polymer functional groups under normal conditions that are well understood by those of ordinary skill in the art. Examples of reactive functional groups are active hydrogen-containing groups such as hydroxyl, amino, carboxy, thio, amide, carbamoyl and activated methylene; isocyanate, cyano and epoxy groups; ethylenically unsaturated groups such as allyl and metallyl; and activated unsaturated groups such as acryloyl and methacryloyl, and maleate and maleimide (including their Diels-Alder adducts with dienes such as butadiene). Latent reactive functional groups in the sense of the present invention, and as will be well understood by those of ordinary skill in the art, refer to reactive functional groups that are blocked or masked to prevent premature reactions. Examples of latent reactive functional groups are ketimines and aldimines (amine blocked with ketones and aldehydes, respectively); amine carboxylate salts; and blocked isocyanates, such as varieties protected with alcohol (carbamates), oxime and caprolactam. A "leaving group" in the sense of the present invention, and as will be well understood by those of ordinary skill in the art, is a substituent attached to a hydrocarbyl chain or ring that is displaced during the reaction to form valence on a carbon atom or heteroatom in a hydrocarbyl chain or ring . Examples of leaving groups are halogen atoms such as chlorine, bromine and iodine; quaternary ammonium salts; sulfonium salts and sulfonate moieties.

[0044] The monosaccharide or disaccharide of the present invention is preferably glucose, fructose, ribulose, galactose, mannose, cellobiose, allose, altrose, ribose, xylose, arabinose, sucrose or lactose. More preferably it is D-glucose, D-ribose, D-galactose,

D-lactose, D-xylose or D-sucrose.

[0045] In one preferred embodiment, the monosaccharide or disaccharide is acetylated, preferably at least diacetylated, and more preferably triacetylated.

[0046] The term "halogen" refers to a fluorine atom, chlorine atom, bromine atom or iodine atom. Preferably it is fluorine or chlorine.

[0047] The term amino acid as used herein means L- or D-amino acid (or a residue thereof), preferably L-, selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine , isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. The term peptide means two or more amino acids linked by a peptide bond, preferably it contains 2 to 8 amino acids, and more preferably contains 2 to 6 amino acids.

Pharmaceutical preparations and administration:

[0048] The invention can be used to treat an animal with an inflammatory disease as defined in the claims, wherein the preferred animal is a mammal, and more preferably the animal is a human.

[0049] It should also be noted that therapeutic benefits are generally achieved by administering at least 1, 2, 3 or more compounds simultaneously or sequentially. Compounds as defined in the claims are also combined with other therapies to provide combined, therapeutically effective amounts. The compound can be administered, for example, in combination with additional agents, preferably anti-inflammatory agents.

[0050] In a preferred embodiment, the pharmaceutical composition for the treatment of an inflammation-related disease associated with pro-inflammatory cytokine expression as defined in the claims comprises one or more compounds as described above; an anti-inflammatory agent and a pharmaceutically acceptable carrier, wherein the anti-inflammatory agent is selected from the group consisting of: an analgesic; anti-rheumatic agent; agent acting on the gastrointestinal tract; gout preparation; glucocorticoids; ophthalmic preparation; respiratory agent; nasal preparation and mucosal agent. [0051] Preferably the analgesic is selected from the group consisting of: naproxen, indomethacin, ibuprofen, ketorolac tromethamine, magnesium choline trisalicylate and rofecoxib; the anti-rheumatic agent is selected from the group consisting of: cyclosporin, sulfasalazine, valdecoxib, penicillamine and dexamethasone; the gastrointestinal tract agent is selected from the group consisting of: mesalamine, basalide disodium and olsalazine sodium; the gout preparation is sulindac; glucocorticoid is selected from the group consisting of: dexamethasone, dexamethasone phosphate, methylprednisolone acetate, hydrocortisone and sodium hydrocortisone phosphate; the nasal preparation is selected from the group consisting of beclometasone dipropionate monohydrate, fluticasone propionate, triamcinolone acetonide, flunizolide, mometasone furoate monohydrate and budesonide; the ophthalmic preparation is ketorolac tromethamine; the respiratory agent is nedocromil sodium; and the mucosal agent is selected from the group consisting of: alclometasone dipropionate, hydrocortisone butyrate, flurandrenolide, betamethasone valerate and clobetazole propionate.

[0052] In a further preferred embodiment, the pharmaceutical composition comprises Meisoindigo and / or NATURA. In general, the pharmaceutically acceptable carrier is an inert diluent.

[0053] The pharmaceutical compositions may be in a variety of forms adapted to the chosen route of administration, as discussed above. Those skilled in the art know various synthetic methodologies that can be used to prepare the non-toxic pharmaceutically acceptable compositions of the compounds described herein. Those skilled in the art know a wide range of non-toxic, pharmaceutically acceptable solvents that can be used to prepare solvates of the compounds of the invention, such as water, ethanol, mineral oil, vegetable oil and dimethyl sulfoxide.

[0054] The compositions may be administered orally, topically, parenterally, by inhalation or spraying, or rectally in unit dose preparations containing standard non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. It should further be understood that the best method of administration can be a combination of methods. Oral administration in the form of a pill, capsule, elixir, syrup, lozenge, troche or the like is particularly preferred. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g. intravenous), intramuscular, spinal, intrathecal injection or similar injection or infusion techniques.

[0055] The pharmaceutical compositions are preferably in a form suitable for oral use, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.

[0056] Compositions intended for oral use may be prepared according to any method known in the art for the production of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavors, coloring agents and preservatives to provide pharmaceutically elegant preparations that are pleasant to taste. The tablets may contain the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients which are suitable for the production of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricants, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing prolonged action. For example, a time retardant such as glycerin monostearate or glycerin distearate may be used.

[0057] Formulations for oral use may also be hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules in which the active ingredient is mixed with water or medium oily, for example peanut oil, liquid paraffin or olive oil.

[0058] Aqueous suspensions contain the active substances in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia; and dispersing or wetting agents that can be a naturally occurring phospholipid, for example lecithin or condensation products of alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example heptadeethylene ethoxylation ethoxylate partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0059] Oily suspensions may be formulated by suspending the active ingredients in vegetable oil, for example peanut oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweeteners such as those mentioned above and flavors may be added to provide palatable oral preparations. These compositions can be protected by the addition of an antioxidant such as ascorbic acid.

[0060] Dispersible powders and granules suitable for the preparation of aqueous suspensions by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are illustrated by examples by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0061] The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or peanut oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be naturally occurring gums, for example acacia or tragacanth; naturally occurring phospholipids, for example soybean, lecithin and esters or partial esters derived from fatty acids and hexitol; anhydrides, for example sorbitan monooleate; and condensation products of these partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

[0062] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such preparations may also contain a soothing agent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of sterile, aqueous or oily injection suspensions. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol. Acceptable substrates and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile vegetable oils are normally used as a solvent or suspending medium. For this purpose, any neutral non-volatile vegetable oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in injectables.

[0063] The composition may also be administered in the form of suppositories, e.g. for rectal drug administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Cocoa butter and polyethylene glycols are such substances.

[0064] Alternatively, the compositions may be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can be suspended or dissolved in the vehicle. Preferably, adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.

[0065] For administration to non-human beings, the composition containing the therapeutic compound may be added to animal food or drinking water. It will also be convenient to formulate food for animals and drinking water so that animals receive the right amount of compound in their diet. In addition, it will be convenient for the compound to be present in the composition as a premix to be added to food or drinking water. The composition can also be added as a food or drink supplement for humans.

[0066] Dosage levels of the order of from about 5 mg to about 250 mg per kilogram body weight per day, and more preferably from about 25 mg to about 150 mg per kilogram body weight per day, are useful in the treatment of the conditions mentioned above. The amount of active ingredient that can be combined with carrier substances to produce a single dosage form will vary depending on the condition being treated and the particular type of administration. Unit dosage forms will generally contain between from about 1 mg to about 500 mg of active ingredient. For the treatment of inflammatory diseases by cytokine modulation, the dose will preferably be at least three times less than for the treatment of proliferative disorders by CDK inhibition. For example, the dose of Meisoindigo for the treatment of CML is generally about 125 mg per day, while the dose of Meisoindigo for the treatment of IBD is usually only 25 mg per day. This is due to the much lower amount needed to regulate cytokines by this class of molecules than is required to regulate CDK.

[0067] The frequency of dosing may also vary depending on the compound used and the particular disease being treated. However, a dosage regimen of 4 times a day or less is preferred for the treatment of most disorders. However, it will be understood that specific dose levels for any particular patient will depend on a variety of factors, including the activity of the particular compound employed, age, body weight, overall health, sex, diet, administration time, route of administration and excretion rate, drug combination and the severity of the specific diseases being treated.

[0068] Preferred compounds will have desirable pharmacological properties that include, but are not limited to, oral bioavailability, low toxicity, low plasma protein binding, and desirable in vitro and in vivo half-lives. Penetration of the blood-brain barrier is necessary for compounds used to treat CNS disorders, while low brain levels of compounds used to treat peripheral disorders are often preferred.

[0069] Studies can be used to predict these desirable pharmacological properties. Studies used to predict bioavailability include transport across human intestinal cell monolayers, including Caco-2 cell monolayers. Toxicity to cultured hepatocytes can be used to predict compound toxicity. Penetration of the compound across the blood-brain barrier in humans can be predicted based on brain levels in laboratory animals that have received the compound intravenously.

[0070] Serum protein binding can be predicted from albumin binding studies. Such studies are described in the article by Oravcova, et al. (Journal of Chromatography B (1996) volume 677, pages 1-27).

[0071] The half-life of a compound is inversely proportional to the frequency of dosing of the compound. In vitro half-lives of compounds can be predicted based on microsomal half-life studies as described by Kuhnz and Gieschen (Drug Metabolism and Disposition, (1998) Vol. 26, pages 1120-1127).

[0072] The amount of composition required for use in treatment will vary not only with the particular compound selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately remain at the discretion of the attending physician or clinician.

Therapeutic indications for the use of Meisoindigo and other derivatives of isoindigo, indigo and indigorubine:

[0073] The invention is defined in the appended claims.

[0074] Over the past 10 years, the development of TNF-α inhibitors and other cytokines has become one of the most active drug development areas for the treatment of various inflammation-related diseases. Although the clinical results of the short-term use of these specific inhibitors in the treatment of various inflammation-related diseases are beneficial and even exciting, and the future development of anti-TNF-α and anti-cytokine therapy in general will be of interest (34), the long-term efficacy of these therapies is questioned due to the fact that not only a single pathway but a complicated cytokine network system is involved in the pathological process of inflammation-related diseases.

[0075] Proinflammatory cytokines are involved in a number of pathological inflammatory processes, as well as reduced expression of anti-inflammatory cytokines.

[0076] FIG. 1 schematically presents pathological processes in human inflammation-related diseases in which various cytokines and growth factors are involved, schematically showing the role of pro / anti-inflammatory cytokines and growth factors and the sites of action of such derivatives as Meisoindigo in the pathological process of chronic inflammation-related diseases: Pathological stimulation of inflammation triggers inflammatory responsive cells (lymphocytes, monocytes, neutrophils, endothelial cells, tissue macrophages and mast cells) to release proinflammatory cytokines and growth factors. In turn, these proinflammatory cytokines and growth factors lead to the release of immune cells, neutrophils and blood monocytes from the blood vessels, followed by their accumulation at sites of inflammation. As a result, they cause various inflammation-related diseases and / or autoimmune disorders. These diseases include inflammatory bowel disease (IBD), psoriasis, rheumatoid arthritis, neurodegeneration and others. Meisoindigo at low concentrations (e.g. 30 nM) inhibits the production of numerous proinflammatory cytokines, including IL-1e, IL-6 and TNF-α, and stimulates the anti-inflammatory cytokine IL-10. [0077] Table 1 summarizes the contribution of various cytokines to the pathological process of autoimmune disorders. Although there is a significant increase in the concentration of various proinflammatory cytokines in tissues / organs in the course of autoimmune diseases, there is a moderate increase in the concentration of some regulatory cytokines, which balances the excessive activation of proinflammatory cytokines. It is also believed that autoimmune disorders are caused by an imbalance between pro-inflammatory and regulatory cytokines (48).

[0078] Our observations show that Meisoindigo down regulates the secretion / expression of a number of important pro-inflammatory cytokines: TNF-α, IL-1 and IL-6 and upregulates the anti-inflammatory cytokine IL-10. The term "consequences of the release of proinflammatory cytokines and growth factors" refers to T cell epidermotropism, induction of excessive K6 / 16 proliferation, macrophage lining, activated neutrophil and T cell infiltration, mast cell number and activation, ICAM-1 and MHC class II induction, angiogenesis , changes in vascular permeability, apoptosis, brain and central nervous system damage, regulation of synovial cell proliferation, cartilage breakdown and bone resorption.

TABLE 1. List of cytokines and growth factors involved in the pathological process of various inflammatory disorders

<td>Proinflammatory regulatory growth</td><td>Regulatory cytokines</td><td>chemokines</td><td>Growth factors</td>

<td>TNF-α</td><td>IL-4</td><td>IL-8</td><td>FGF</td>

<td>LT</td><td>IL-10</td><td>Groa</td><td>PDGF</td>

<td>LIF</td><td>IL-11</td><td>MIP-1</td><td>VEGF</td>

<td>Oncostatin M.</td><td>W-13</td><td>MCP-1</td><td>GM-CSF</td>

<td>W15</td><td></td><td>ENA-78</td><td>M-CSF</td>

<td>IFNcło / β</td><td></td><td>RANTES</td><td>TGF-β</td>

<td>IFN-</td><td></td><td></td><td></td>

<td>IL-1 α, β</td><td></td><td></td><td></td>

<td>IL-2</td><td></td><td></td><td></td>

<td>IL-3</td><td></td><td></td><td></td>

<td>IL-6</td><td></td><td></td><td></td>

<td>IL-7</td><td></td><td></td><td></td>

<td>IL-9</td><td></td><td></td><td></td>

<td>IL-12</td><td></td><td></td><td></td>

<td>IL-17</td><td></td><td></td><td></td>

<td>IL-18</td><td></td><td></td><td></td>

[0079] Inflammatory bowel disease (IBD): IBD includes Crohn's disease (CD) and ulcerative colitis (UC), which are 2 overlapping, chronic gastrointestinal tract diseases associated with inflammation caused by immune regulation disorders (20) . IBD patients have defective intestinal epithelial barrier function, which allows bacterial colonization of the epithelium. As a result, bacterial products and pro-inflammatory cytokines (TNF-α, IL-1 and IL-6) cause long-lasting stimulation of inflammation. Bacterial antigens are introduced into the immune system by mucosal dendritic cells and macrophages. In response, intestinal phagocytes (mainly monocytes and neutrophils) multiply and increase the expression and secretion of proinflammatory cytokines. Like the cell growth inhibitors and inducers of cell differentiation shown in our previous patent, Meisoindigo and compounds of formula (I) or (II) will effectively inhibit the excessive proliferation of these inflammatory cells, while suppressing the expression / secretion of pro-inflammatory cytokines by these cells, as shown in Examples 1 to

4. This conclusion was confirmed in animal models and in an IBD patient as shown in Examples 7-8.

[0080] Psoriasis: Cytokines are intracellular messengers that play an important role in the development and maintenance of dermatitis. A number of cytokines have been reported to play a key role in the pathogenesis of inflammatory skin disorders. IL-1, TNF-α and IFN-γ induce the expression of ICAM-1 and class II major histocompatibility complex (MHC) (48, 49). IL-1, TNF-α and granulocyte-macrophage colony stimulating factor are capable of causing the activation, maturation and migration of dendritic cells, and IL-1 activates mast cells (50). IL-6 and TGF-α increase the multiplication of keratinocytes. IL-1, TNF-α, TGF-α and VEGF induce angiogenesis and attract inflammatory cells (51-53). The priority of cytokines in the induction of cutaneous immune responses makes them a highly attractive target for new biological response modifiers (18). Thus, many cytokine regulators, small molecules as claimed in this invention, Meisoindigo and compounds of formula (I) or (II) will be effective against psoriasis. As shown in Example 6, we showed in the rodent model that Meisoindigo was actually effective in a dose-dependent manner against psoriasis and that the effect was better than in the MTX positive control. [0081] Rheumatoid Arthritis (RA): The role of cytokine networks in mediating RA inflammation and destruction has been extensively studied in recent years. In addition to TNF-α, IL-1 plays a key role in the pathogenesis of clinical manifestations of RA (54). The ability of IL-1 to cause inflammation and erosion of the joints and inhibit tissue repair processes has been unambiguously demonstrated in in vitro systems and animal models, and inflammation symptoms in RA patients have been improved by blocking IL-1 (55). IL-6 is a multifunctional cytokine that regulates the immune response, hemorrhage, acute phase response and inflammation. Disturbances in the regulation of IL-6 production are involved in the pathology of several diseases, including RA. A therapeutic strategy for blocking the IL-6 signal has been used by the use of a humanized anti-IL-6R antibody in diseases, including RA. (11, 56). IL-10 is an anti-inflammatory cytokine. Expression of IL-10 has been shown to prevent arthritis or ameliorate disease in animal models (57, 58). While it is obvious that cytokines such as TNF-α, IL-1, IL-6 and IL-10 play independent roles, they act jointly, mediating some pathophysiological processes in RA. Determining the class of molecules described in this invention that are capable of modulating these different cytokines will lead to dramatic progress in the treatment of RA.

[0082] Multiple sclerosis (MS): MS is an autoimmune inflammatory disorder. Although the reason why the body attacks its own myelin in patients with MS is still unknown, disturbed cytokine regulation is undoubtedly involved in the development of this disease. Using experimental autoimmune encephalomyelitis (EAE), a widely used model for MS studies based on autoimmune, histopathological, genetic and clinical similarities, it was shown that in the early active stages of the disease, both EAE and MS are characterized by the presence of perivascular infiltrates inflammatory disease spread in the CNS, a process in which chamotactic cytokines (chemokines) play an important role. There is evidence that the expression of chemokines (from the IL-8 family) during autoimmune CNS inflammation is regulated by some proinflammatory cytokines such as TNF (59). The role of other pro / anti-inflammatory cytokines such as IL-1 β, IL-6 and IL-10 has also been confirmed in EAE animal models (60-62) and in humans (63). IL-1e occurs in MS lesions. The IL-1 receptor antagonist (IL-1Ra) inhibits the induction of experimental autoimmune encephalomyelitis (EAE). People with a high IL-1 production ratio (3 versus IL-Ra and a high TNF production ratio versus IL-10 have an increased risk of developing MS (63).

[0083] Neurodegenerative Disorders: Alzheimer's Disease (AD) and Parkinson's Disease (PK) are the two most common neurodegenerative disorders associated with neuritis. Neuritis is characteristic of diseased tissue in a number of neurodegenerative disorders. These changes are particularly observed in the affected areas of the brain in cases of AD (64). The role of cytokines in the pathogenesis of AD has been suggested, although the mechanism by which cytokines contribute to its pathogenesis is not fully understood. And AD, microglia, especially associated with amyloid deposits, exhibits a phenotype that is consistent with the activation state, including immunoreactivity with anti-class II antibodies of the major histocompatibility complex antigens and anti-inflammatory cytokines IL-1e and TNF-α (65). One of the important neuropathological features of AD are senile plaque deposits in the brain, which are mainly composed of the toxic beta amyloid peptide (Abeta), which is produced from the family of precursor proteins containing Abeta (AbetaPP). Cytokines have been shown to stimulate the expression of AbetaPP transcription genes (66). The analysis of genetic connections of sites controlling the age of AD and PK revealed a significant relationship between AD and the glutathione S-transferase, omega-1 and 2 genes (GSTO1, GSTO2) (7). GSTO1 activity appears to be associated with post-translational processing of the IL-1 β pro-inflammatory cytokine (67).

[0084] Inflammation associated with radiation therapy: Inflammatory rectal and sigmoid diseases associated with radiation damage are the most common complications of radiation therapy for pelvic tumors, including cervical, endometrial, prostate, bladder and testicular cancer. Radiation proctitis is the most common clinical manifestation of colon damage after pelvic irradiation and occurs with a frequency of 5% to 20%. Patients typically have symptoms of painful pressure, bleeding, diarrhea with low stool volume and rectal pain. In rare cases, there may be a small degree of obstruction or fistulae to adjacent organs.

[0085] The mechanism of radiation therapy is thought to be to damage DNA in actively proliferating cells. Pathological damage after local radiation therapy in the intestinal / colon area can be divided into acute and chronic. Initial pathological changes include loss of lymphocytes in the dermis and microscopic damage to mucosal epithelial cells and vascular endothelial cells. These changes are manifested by the blunting of villi and a decrease in the number of crypt regenerative cells, followed by significant submucosal edema with increased vascular permeability.

[0086] Progressive endometritis appears to be the main mechanism by which chronic effects occur, which later manifest themselves as progressive fibrosis, leading to muscle wasting, the formation of strictures and thrombosis, resulting in secondary ischemic changes. In the course of radiation colitis in the chronic phase, there is a very significant crypt distortion, vascular telangiectasia and fibrosis of the lamina propria. Interestingly, some of these pathological changes also occur in long-term IBD (68).

[0087] Thus, cytokines may play a key role in various gastrointestinal diseases in which inflammation is important. Recent studies have focused on the key role of cytokines in chronic IBD (69-74). To clarify the role of cytokines in radiation proctitis, Indaram et al. (75) examined the levels of cytokines in the colon mucosa in patients with radiation proctitis and compared them with those found in normal controls and IBD patients. They found that the levels of IL-2, IL-6 and IL-8 in the mucosa were significantly higher and statistically significantly different (p &lt;0.05) from both the diseased (5.62 ± 0.13, 1.60 ± 0 , 31, 21.45 ± 4.03 pg / mg) and normal-looking mucosa (3.83 ± 0.78, 1.36 ± 0.34, 13.45 ± 3.18 pg / mg) in group of radiation proctitis, compared to normal controls (1.74 ± 0.23, 0.67 ± 0.05, 4.99 ± 1.39 pg / mg).

[0088] Thus, these observations show similar cytokine activation in patients with radiation proctitis and IBD. It was demonstrated that in patients with radiation proctitis, IL-2, IL-6 and IL-8 concentrations in the mucosa were significantly higher compared to normal controls. For comparison, patients with IBD (UC and CD) had significantly higher levels of cytokines, including IL-1, IL-2, IL-6 and IL-8 compared to normal controls.

[0089] The similarity of cytokine expression in the mucosa in these 2 diseases is probably directly related to the intense inflammatory nature of these diseases. It has been postulated that this similarity of cytokine activation in these 2 diseases may translate into similar pathological changes observed in chronic IBD and radiation proctitis. This hypothesis is supported by the fact that treatment of radiation proctitis, although rather unsatisfactory, involves treatment with various amino salicylic acid derivatives and oral or topical corticosteroids. These treatment options are identical to those used for IBD.

[0090] As shown in the present invention, Meisoindigo and the class of small molecules to which this agent belongs are capable of downregulating IL-β, IL-6 and TNF-α and upregulating the IL-10 regulatory cytokine; high effectiveness and low severity of side effects should be expected.

[0091] Other diseases associated with cytokine regulation disorders: Cardiovascular diseases (CVD), atherosclerosis and metabolic diseases (metabolic syndrome) have also been associated with abnormal secretion / expression of pro / anti-inflammatory cytokines (10, 1214, 76).

[0092] Diabetes: The primary disadvantage in type 2 diabetes is insulin resistance, in which insulin does not inhibit hepatic glucose production and does not stimulate its use by peripheral tissues, leading to hyperglycaemia. Pancreatic β cells respond to excessive plasma glucose by more insulin secreted to overcome the effects of insulin resistance. As insulin resistance progresses, when the β cells are no longer able to meet the demand for increased insulin secretion, plasma glucose increases and type diabetes develops.

2.

[0093] Many factors can contribute to the onset of type 2 diabetes. Because 80% of patients with type 2 diabetes are obese and obesity is always associated with insulin resistance, molecular mediators that combine obesity with insulin resistance have been extensively studied. Various factors have been found to contribute to insulin resistance in obesity and type 2 diabetes associated with obesity, namely those produced by adipose tissue, FFA (free fatty acids), TNo., IL-6, leptin, adiponectin and resist. Both mRNA and TNFα protein concentrations are strongly increased in fat tissues of obese animals (77) and humans (78). All different cell types in adipose tissue are capable of producing cytokines. Adipocytes express TNFα receptors and are also an important source of TNFα, which is thought to act in adipose tissue mainly in an autocrine / paracrine manner.

[0094] Prolonged exposure of cultured cells (79) or animals (80) to TNFα induces insulin resistance, whereas neutralization of TNFα increases insulin sensitivity and reduces hyperglycemia in the animal model of type 2 diabetes (81). The lack of TNFα or TNFα receptors as a result of gene elimination leads to a significant improvement in insulin sensitivity in animal models of obesity (82).

[0095] Mechanisms of insulin resistance induced by TNF? In adipocytes as well as systemic have been proposed (83). TNFα inhibits phosphorylation of the insulin receptor and insulin-1 receptor substrate (IRS-1) by the kB kinase-β (IKK-β) inhibitor. TNFα activation of NF-kB is necessary to suppress the genes intensively expressed in adipocytes, necessary for adipocyte function, and is also sufficient to inhibit gene transcription mediated by PPAR-gamma. TNFa also stimulates lipolysis and expression of other cytokines in adipose tissue and triggers the release of FFA. In fact, FFV levels rise before overt hyperglycaemia occurs in some animal models of insulin resistance (83). There is ample evidence to suggest that excess FFA in plasma may induce and lead to progress of systemic insulin resistance. In hepatocytes, FFA contribute to excessive glucose and VLDL production. In muscle cells, high concentrations of FFA interfere with insulin signal transduction and promote FFA oxidation, leading to a significant reduction in glucose oxidation.

[0096] Currently available insulin sensitizers, which belong to PPAR-gamma agonists, inhibit TNFα-induced gene expression profile in adipocytes via the NF-kB pathway (84). Since adipocyte-derived TNF? Plays the role of an autocrine or paracrine factor, systemic delivery of anti-TNF? Antibody may not be effective in blocking the biological activity of locally expressed TNF? In adipose tissue (85). NATURE, which represents a new type of small molecule TNFα inhibitor, distributed by simple diffusion, can therefore be an effective agent for blocking the function of locally expressed TNFα and potentially useful in the treatment of type 2 diabetes.

[0097] Type 1 diabetes is an autoimmune disease characterized by the infiltration of mononuclear cells in the Langerhans islands and the selective destruction of insulin-producing beta cells. While CD8 + T cells can be important initiators, CD4 + T cells (86) and macrophages (87, 88) are important cellular effectors of the immune process leading to beta cell death. Activated macrophages directly secrete IL-1beta, IL-6, IL-12, TNFalpha, indirectly trigger the production of INF-gamma by activated T cells. The contribution of cytokines such as TNFalpha, INFgamma, IL-1beta, IL-6 and IL- 10, in the pathogenesis of type 1 diabetes, it has been well explained thanks to studies correlating the expression of cytokines and the development of type 1 diabetes, studies on increasing the concentration of cytokines and studies with cytokine deficiency (89). In addition to cytokine neutralizing antibodies and soluble cytokine receptors, anti-inflammatory compounds also exhibit a delaying or preventing effect of type 1 diabetes in animal models.

[0098] The pathogenesis of diabetes, from pancreatitis to complete beta cell destruction, is a relatively chronic process. Meisoindigo, NATURE and other derivatives inhibit pro-inflammatory cytokines and stimulate anti-inflammatory cytokines. Therefore, they can be used as a means to prevent or delay the onset of the disease, as well as for its treatment.

[0099] In summary, cytokine regulation disorders are involved in a variety of diseases, including inflammation-related diseases and diseases that are not normally considered inflammation-related diseases. A molecule that is capable of modulating both pro and anti-inflammatory cytokines should provide therapeutic benefits with minimal side effects in all types of diseases associated with impaired function of these inflammatory components. As demonstrated in the present invention, the nature of the effect of Meisoindigo, representative small molecule derivatives of isoindigo, indigo and inddyrubine, on the regulation of expression / secretion of numerous pro / anti-inflammatory cytokines, allows the effective use of the compounds defined in the appended claims in the treatment of inflammation-related disorders associated with expression proinflammatory cytokines as defined in the appended claims.

EXAMPLES

Example 1: Meisoindigo reduces IL- [beta] secretion in a human monocytic cell line in THP-1 cells

Materials and methods [0100] Materials: Meisoindigo and NATURA were synthesized by Natrogen Therapeutics, Inc., purified using high-performance liquid chromatography (HPLC) with a purity of 98.5%, and their structures were confirmed by mass spectrometry and nuclear magnetic resonance (NMR). Meisoindigo is a dark reddish crystal with a molecular weight of 376. It was prepared in a solution of dimethyl sulfoxide (DMSO) and stored at -20 ° C for in vitro experiments. A human monocytic cell line, THP-1 (90), was purchased from ATCC. Cells were maintained according to the supplier's instructions. About 1x10⁵ cells / ml were cultured at 37 ° C, in 5% CO2 for 24 hours in modified RPMI-1640 medium (Invitrogen) supplemented with 10% FBS.

[0101] Methods: Cells were stimulated with 1 μΜ lipopolysaccharide (LPS, Sigma) or not and exposed for 24 hours to various concentrations of Meisoindigo (from 31.25 nM to 16.000 nM). Cell viability was examined under a microscope after trypan blue staining. The levels of IL-1 β protein secreted into the culture medium by the cells were then measured by ELISA and calculated from a standard curve using the test kit from R&amp;D Systems according to the instructions provided by the supplier. The method was developed and validated on the basis of a good standard curve obtained. An example of the calibration curve is shown in Fig. 2, panel A.

[0102] Statistical analysis: All data were expressed as mean ± standard deviation (SD). The statistical significance of all differences between control (LPS) and experimental groups was determined using Student's t-test. For the result to be considered statistically significant, the p-value between 2 groups had to be less than 0.05.

Results and discussion [0103] IL-1 β is a pleiotropic pro-inflammatory cytokine involved in the pathological process of various inflammation-related diseases. To clarify the activity of Meisoindigo, a small molecule representative of compounds of formula (I) or (II), against inflammation, we examined the effect of Meisoindigo on IL-1 β secretion in human monocytic THP-1 cells. As shown in Fig. 2, panel B, it was found that basal IL-1 β in human monocytic THP-1 cells was undetectable. It has previously been shown that the increase in IL-1 β and mRNA levels in response to lipopolysaccharide (LPS) is mainly the result of increased gene transcription (91, 92). In this invention, we also found that after LPS stimulation, THP-1 cells secreted large amounts of IL-β into the medium (92.38 ± 3.667 pg / ml, Fig. 2, panel B). Interestingly, the stimulated secretion of IL-β was significantly inhibited by the simultaneous exposure of cells to Meisoindigo. Most importantly, we found that Meisoindigo was a potent but also a moderate inhibitor of IL-1 β.

[0104] These characteristics will be an advantage for patients due to their high efficiency with less side effects if used for the treatment of inflammatory disorders. Strong because over 50% decrease in secretion was repeatedly achieved

LPS-mediated IL-1e when cells were exposed to Meisoindigo at a concentration of 31.25 nM; moderate because increasing the concentration of Meisoindigo to 8 μΜ did not lead to a further reduction in secretion, indicating that maximum activity was achieved. This is different from the effect of Meisoindigo or NATURA on the inhibition of cyclin-dependent kinases (CDKs), in which a much higher concentration is necessary for 50% inhibition of CDK activity (about 1.6 μM) in LNCaP prostate cancer cells, as demonstrated in our previous patent description.

[0105] This last point is important because in the prior art, EP 1 079 826, only CDK and not cytokine inhibition was found. As a result, the present invention uses significantly lower drug concentrations compared to earlier literature. In addition, specific compounds may also be more suitable for cytokine inhibition as compared to CDK inhibition.

Example 2: Meisoindigo inhibits IL-6 secretion and expression in human monocytic cell line, THP-1 cells

Materials and methods [0106] Materials: A representative Meisoindigo compound was used. The cell line and ELISA procedure were the same as described in Example 1. The IL-protein protein standard was used to plot the standard curve for calculating IL-6 in the medium secreted by the cells (LPS stimulated or non-stimulated cells in the presence or absence of Meisoindigo). 6. A typical calibration curve is shown in Fig. 3, panel A. Statistical analysis was also consistent with the method described in Example 1.

methods:

[0107] Real-time PCR: The effect of Meisoindigo on IL-6 transcription (RNA levels) was determined by real-time polymerase chain reaction (real-time PCR) technique. Total RNA was extracted using the Qiagen Rneasy mini kit and the HPRT gene was used as internal control.

[0108] Human monocytic THP-1 cells in the exponential growth phase were exposed to 1 μg / ml LPS, 1 μM Meisoindigo or 1 μg / ml LPS plus 1 μM Meisoindigo for 24 hours. The cells were then harvested, washed and extracted from total RNA for real-time PCR. Total RNA (300 ng) was treated with DNase I (Promega, Madison, WI) and SuperScript II (Invitrogen, Carlsbad, CA) and oligo (dT) was used for reverse transcription according to the manufacturers instructions. Real-time PCR was performed in a volume of 25 μl containing diluted cDNA, Sybr Green PCR Master Mix (Applied Biosystems) and 2.5 μM of each of the IL-6: R: 5'-TCAATTCGTTCTGAAGAGG gene specific primers (SEQ ID NO. 1 ) and F: 5'- CCCCCA33

GGAGAAGATTCC (SEQ. ID NO. 2). The ABI SDS7700 analyzer (Applied Biosystems) was used at 50 ° C for 2 minutes and at 95 ° C for 10 minutes, followed by 40 cycles at 95 ° C for 15 seconds and at 60 ° C for 1 minute. Test cDNA results were normalized to the HPRT internal control measured on the same plate. After completion of the cycles, the specificity of the amplification was verified by creating a melting curve by slow denaturation of the PCR products followed by gel electrophoresis.

Results and discussion [0109] IL-6 is another key pro-inflammatory cytokine that is involved in inflammation. For this reason, the effect of Meisoindigo on its secretion / expression was investigated. As with IL-1 β, baseline IL-6 was undetectable in human monocytic THP-1 cells. After stimulation with 1 μg / ml LPS, the cells secreted a moderate amount of IL-6 into the medium (33.64 ± 3.29 pg / ml). Meisoindigo was found to strongly inhibit IL-6 secretion in LPS-stimulated THP-1 cells. An approximately 85% reduction in secretion was observed when stimulated cells were exposed to Meisoindigo at the lowest concentration of 31.25 nM used in the experiment (P &lt;0.001) (Fig. 3, panel B). [0110] To investigate whether the decrease in Meisoindigo-mediated IL-6 secretion was due to its inhibition of LPS-induced IL-6 expression, real-time PCR was used to measure the effect of Meisoindigo on IL-6 mRNA transcription. As shown in Fig. 3, panel C, when THP-1 cells were exposed to 1 μg / ml LPS, significant induction of IL-6 transcription was found, which is in line with previous reports (93). Interestingly, it was found that LPS-induced IL-6 transcription could be completely inhibited by displaying LPS-stimulated THP-1 cells on Meisoindigo (P &lt;0.001). This observation therefore indicates that Meisoindigo's inhibition of LPS-stimulated IL-6 secretion is probably the result of an agent inhibiting IL-6 production in LPS-mediated THP-1 cells.

[0111] LPS stimulation of human monocytes activates IL-6 transcriptional signaling pathways. LPS can bind to a protein called LPS binding protein (LBP). It was demonstrated that after LPS was transferred by LBP to the CD 14 receptor, it interacted with the TLR4 signaling receptor and MD-2 helper protein. This interaction led to the activation of NF-κΒ and 3 MAP kinases, thereby increasing IL-6 transcription (94, 95). Whether Meisoindigo's suppression of LPS-mediated transcription of L-6 is due to a disorder of signal transduction pathways requires further study.

Example 3: Meisoindigo suppresses TNF-α secretion in human monocytic cells

THP-1

Materials and methods [0112] A representative Meisoindigo compound was used. The cell line and ELISA procedure for measuring TNF-α secretion were the same as described in Example 1, except that for plotting a standard curve for calculating TNF-α in cell secreted medium (LPS stimulated or non-stimulated, in the presence or presence of in the absence of Meisoindigo), the TNF-α protein standard was used. A typical standard curve is shown in Fig. 4, panel A.

[0113] The effect of Meisoindigo on TNF-α transcription (RNA levels) was determined by real-time PCR using the same procedures as described in Example 2, with the exception of the following TNF-α 5'-TGCCCAGACTCGGCAAAG specific primers (SEQ. ID NO. 3) and 5'GGAGAAGGGTGACCGACT (SEQ. ID NO. 4). Total RNA was extracted using the Qiagen Rneasy mini kit and the HPRT gene was used as internal control.

[0114] Human monocytic THP-1 cells in the exponential growth phase were exposed to 0.1 μg / ml LPS, 4 μM Meisoindigo or 1 μg / ml LPS plus 4 μM Meisoindigo for 24 hours. The cells were then harvested, washed and extracted with total RNA for real-time PCR as described in Example 2.

Results and discussion [0115] TNF-α is a key pro-inflammatory cytokine intensively studied over the past ten years because of its important biological functions against cancer and the pathological role in inflammatory disorders. A number of TNF-α inhibitors are commercially available for the treatment of various inflammation related diseases. As part of this invention, we examined the role of the potential anti-inflammatory agent, Meisoindigo, in the regulation of TNF-α. [0116] As a recognized model system, stimulation of THP-1 cells by LPS led to massive TNF-α secretion (Fig. 4, panel B). In contrast, Meisoindigo effectively inhibited TNF-α secretion in LPS stimulated THP-1 cells in a concentration-dependent manner (Fig. 4, panel B). After exposure of the activated cells to 2.0 μM Meisoindigo for 24 hours, an approximate 50% reduction in secretion (P &lt;0.001 for comparison of LPS plus Meisoindigo with LPS alone) was obtained in which no apoptotic cells were found using trypan blue staining. Increasing the concentration of Meisoindigo to 8 μM did not further reduce TNF-α secretion, but no cell death was observed. In contrast, complete inhibition was achieved when stimulated cells were treated with 16 μM Meisoindigo, at which about 20% of apoptotic cells appeared.

[0117] Real-time PCR showed no effect of Meisoindigo (4 μM) on TNF-α mRNA levels (FIG. 4, panel C), indicating that Meisoindigo reduced TNF-α production in LPS stimulated THP-1 cells occurs on post-transcript level. It is well known that AU rich (ARE) elements in TNF-α 3 'UTR mRNA are associated with mRNA stability and translation efficiency (96). ARE TNF-α is the target of the mRNA stabilizing factor, HuR (97). The maturation of TNF-α mRNA is affected by the cis element (2-APRE) in the 3'UTR, which makes the assembly of TNF-α precursor transcripts dependent on the activation of RNA-activated protein kinase (PKR) (98). [0118] Although the mechanism by which Meisoindigo inhibits TNF-α secretion in LPS-stimulated THP-1 cells requires clarification, Meisoindigo is a new small TNF-α inhibitory molecule without cytotoxic effects, which may make it an ideal drug for the treatment of inflammation-related diseases as defined in the appended claims.

Example 4: Meisoindigo stimulates IL-10 secretion in THP-1 human monocyte cells

Materials and methods [0119] Meisoindigo and the THP-1 cell line used in this Example were the same as described in Example 1. ELISA assay procedures for measuring IL-10 secretion also followed the procedures described in Example 1, with the exception of that the IL-10 protein standard was used to plot the standard curve for calculating the IL-10 protein (FIG. 5, panel A) in cell secreted medium (LPS stimulated or non-stimulated, in the presence or absence of Meisoindigo).

Results and discussion [0120] The functioning of the immune system is precisely regulated by the activity of pro-inflammatory and regulatory mediators or cytokines. Inflammation-related diseases are thought to be the result of an imbalance between these types of molecules (41, 46). In order to understand whether the anti-inflammatory effect of small molecules as defined in the appended claims is able to induce regulatory cytokines, the effect of Meisoindigo on IL-10 secretion was investigated. As shown in Fig. 5, panel B, there was a moderate but significant stimulation of IL-10 secretion in THP-1 cells. An approximately 60% increase in IL-10 secretion was obtained when THP-1 cells were treated with 0.0625 μM Meisoindigo (P &lt;0.05). In contrast, the inflammation-promoting factor, LPS, slightly reduced the secretion of this cytokine.

Example 5: Meisoindigo and NATURA select cytokines rather than CDK as primary molecular targets at low concentrations

Materials and methods [0121] Materials: Meisoindigo and NATURA were synthesized by Natrogen Therapeutics, Inc. as described in the above examples.

[0122] A human monocytic cell line, THP-1 (90), was purchased from ATCC. Cells were maintained according to the supplier's instructions. About 1x10⁵ cells / ml were cultured at 37 ° C, in 5% CO2 for 24 hours in modified RPMI-1640 medium (Invitrogen) supplemented with 10% FBS.

methods:

[0123]

1) Effect of Meisoindigo and NATURA on the expression / secretion of IL-β, IL-6, IL10 cytokines: THP-1 cells growing exponentially were stimulated with 1 μg lipopolysaccharide (LPS, Sigma) or not and exposed for 24 hours to different concentrations of Meisoindigo and NATURA, respectively (from 31.25 nM and 62.5 nM). Cell viability was tested by trypan blue exclusion. The levels of IL-1 β protein secreted into the culture medium were measured by ELISA and calculated from a standard curve using the R&amp;D Systems test kit according to the instructions provided by the supplier, as described in Examples 1 to 4.

2) Effect of Meisoindigo and / or NATURA on cyclin-dependent kinase (CDK) activity in THP-1 cells (99): Exponentially growing THP-1 cells were exposed to 31.25, 62.5 and 1500 nM Meisoindigo or NATURA, respectively 24 h. Cells were harvested, washed and extracted with total protein as previously described (100). One hundred μg of proteins were immunoprecipitated using anti-cdk2, cdk4 / 6 or cyclin D1 antibodies overnight at 4 ° C in the presence of a cocktail of protease inhibitors. Immunoprecipitates were washed four times with protein extraction buffer and once with kinase assay buffer and reacted with 75 μg / ml histone H1 in the presence of [y-³²P] -ATP (10 μΟ / 10 μM). Phosphorylated histone H1 (representing cdk activity) was determined by scintillation counting or SDS polyacrylamide gel electrophoresis (101, 102).

3) Statistical analysis: All data were expressed as mean ± SD. The statistical significance of all differences between control (LPS) and experimental groups was determined using Student's t-test. For the result to be considered statistically significant, P values ​​between 2 groups had to be minimum less than 0.05.

Results and discussion [0124] Since indigorub and its derivatives have been shown to inhibit cy37 wedge dependent kinases, it can therefore be an effective drug for the treatment of diseases associated with loss of proliferation control by CDK inhibition. In order to investigate which molecules in the cell are the main targets associated with anti-inflammatory properties of this class of compounds, we compared how Meisoindigo and NATURA modulate CDK and cytokine activity under the same experimental conditions of low concentration of compounds tested.

[0125] As shown in FIG. 6, similar to the observations presented in Examples 1, 2 and 4, the LPS-stimulated increase in IL-1 β and IL-6 production was significantly inhibited by cell exposure to Meisoindigo and NATURA at concentrations as low as 31.25, while suppression IL-10 in THP-1 cells mediated by LPS was increased almost twice by Meisoindigo and NATURA at similar concentrations (FIG. 6A). [0126] In contrast, at the same exposure, Meisoindigo and NATURA did not inhibit cyclin 2, 4 and 6 dependent kinases or cyclin D1 levels (data not shown). Partial inhibition (23%) was only obtained when the cells were treated with 1.5 μΜ (48 times more) of Meisoindigo or NATURA (FIG. 6B).

[0127] In addition, the effect of NATURA on glycogen synthase 3β (GSK-3e) kinase was also investigated in the context of the present invention, as CDK inhibitors are usually also GSK-3 [beta] inhibitors. However, no effect was found when the cells were exposed to NATURA at concentrations as high as 50 βΜ (data not shown). [0128] Thus, the data in this example clearly show that Meisoindigo and a class of similar molecules are able to significantly modulate different cytokines (inhibits pro-inflammatory cytokines and stimulates anti-inflammatory cytokines) at a noticeably low concentration where there is no inhibitory effect on CDK. This shows that at low concentrations compared to those necessary for CDK inhibition, Meisoindigo and similar compounds are mainly targeted at cytokines and not at cyclin dependent kinases. This conclusion is confirmed by recent observations that inddyrubine is not a truly biological inhibitor of CDK, as CDK inhibition by these compounds occurs through physical aggregation rather than biological interaction (103). In addition, our villages are also confirmed by clinical observations that a total dose of 8696 mg Meisoindigo is necessary to achieve maximum remission of chronic myelogenous leukemia (CML) (104), while only 525 mg of this drug are necessary to completely cure inflammatory bowel disease.

Summary [0129] THP-1 cells secreted ^ -1β, IL-6, IL-8 and TNF-α, but not IL-2, IL-4, IL-10 and IL-12 after 24 hours of LPS stimulation, whereas basal the levels of these cytokines were not detectable by ELISA, which is in line with previous reports (93, 105).

To evaluate the potential clinical applications of the small molecule class I or II in the treatment of various inflammation-related diseases, we examined, for example, the regulatory effect of Meisoindigo on the secretion and expression of pro- and anti-inflammatory cytokines in the model of human monocytic THP-1 cells. The data are summarized in Table 2.

Meisoindigo significantly inhibited IL-Ιβ, IL-6 and TNF-α proinflammatory cytokine secretion in LPS stimulated THP-1 cells and stimulated IL10 regulatory cytokine secretion, but it was not found to affect IL-2 simply because the cells could not be stimulated for the secretion of these proinflammatory cytokines. Table 2 shows the maximum reduction or stimulation of secretion of these cytokines.

Table 2. Meisoindigo modulation of proinflammatory and regulatory cytokine secretion in LPS stimulated THP-1 cells

<td rowspan="2">Treatment</td><td colspan="4">Percentage of maximum response without cytotoxicity</td>

<td>TNF-α (braking)</td><td>IL-1e (braking)</td><td>IL-6 (braking)</td><td>IL-10 (Stimulation)</td>

<td>LPS</td><td> 100,00±4,85</td><td> 100,00±3,43</td><td> 100,00±9,78</td><td> -18,27±10,15</td>

<td>LPS / Meisoindigo</td><td> 49,20±3,37</td><td> 48,76±3,69</td><td> 83,51±5,41</td><td> 201,97±11,2</td>

[0130] Meisoindigo's reduction of IL-6 secretion in LPS stimulated THP-1 cells may be the result of down-regulation of transcription of the cytokine gene determined using real-time PCR. Real-time PCR showed a moderate inhibitory effect of Meisoindigo on IL-15 in LPS stimulated THP-1 cells (data not shown). No such down regulation of the TNF-α gene was found using the same technique. Although the mechanism by which Meisoindigo and this class of molecules regulate pro- and anti-inflammatory cytokines requires further research, our data in the present invention show that this class of small molecules is capable of modulating important cytokines associated with various inflammation-related diseases.

[0131] In the past few years, strategies targeting pro-inflammatory cytokines have been developed using a range of protein-based agents to treat various inflammatory disorders, including TNF-α inhibitors: etanercept (ENBREL®), infliximab (REMICADE®; Centocor), adalimumab (HUMIRA®; Abbott) and IL-1 receptor antagonist KINERET®. The early stages of clinical use of these agents indicate that these revolutionary therapeutic agents represent advances in the treatment of autoimmune diseases such as IBD, RA and psoriasis. However, currently available protein-based therapies for injections are associated with risks including the potential for exacerbation of malignancies, infections, and congestive heart failure (42). In addition, these strategies also have limitations and the challenge is an extensive cytokine network system. Although a number of small molecules have been shown to be specific inhibitors of proinflammatory cytokines such as TNF-α and NF-κΒ inhibitors, and they have different advantages over protein-based agents, the targeting of a single pro-inflammatory cytokine may not be strong enough to terminate pathologies inflammatory pathways, which limits their clinical efficacy.

On the contrary, in addition to all the advantages of small molecules in clinical use, such as the fact that they are easy to produce and convenient to administer, most importantly, the molecules claimed in this invention not only simultaneously suppress various pro-inflammatory cytokines, i.e. IL-1e, IL-6 and TNF-α, but also stimulate the anti-inflammatory cytokine IL-10. In addition, we have shown in our earlier patent specification that at higher concentrations these molecules induce cell differentiation and inhibit cell proliferation. Thus, they provide greater clinical activity. This conclusion confirms the extraordinary effectiveness achieved when using Meisoindigo in treating an IBD patient without any side effects. This observation is presented in Example 7 of this invention.

Example 6: Meisoindigo increases epidermal cell differentiation and inhibits rodent hyperplasia and hyperkeratosis

Materials and methods [0133] Materials: Meisoindigo has been synthesized, purified and its structure has been characterized by Natrogen Therapeutics, Inc. The purity of the compound was 98.5% as described in the above Examples. A fresh suspension of Meisoindigo in 0.5% methylcellulose sodium was prepared and administered orally in the animal studies described. Other chemicals used in the following experiments were purchased from Sigma.

[0134] Pathogen-free female Kunming mice, 12 weeks old, weighing 22 to 25 grams, were housed 5 cages and had free access to fresh tap water and commercially available granulated rodent feed. The conditions in the animal housing were controlled at 24 ± 2 ° C with a relative humidity of 60 ± 5% and a 12-hour light / dark cycle was used (from 07:00 to 19:00).

methods:

[0135] Effect of Meisoindigo on differentiation of epidermal cells of murine tails: Sixty female Kunming mice were randomly divided into 5 groups, 20 in one control group and 10 in each of the 4 drug groups. A fresh suspension of Meisoindigo in 0.5% methylcellulose sodium was prepared. The drug was administered orally in doses of 50, 100 and

200 mg / kg, once daily for 13 days. Methotrexate (MTX) was used as a positive control and was administered at a dose of 1 mg / kg, intraperitoneally, every other day, for the same period of time. The same volume and excipient used to prepare the Meisoindigo suspension was used as a negative control. Twenty-four hours after the last administration, the animals were sacrificed and their tails cut off 1.5 cm from the base. Preparations for histological examination were prepared as described by Bosman et al., (106). The preparations were prepared by cutting the tails into oblong sections and stained with hematoxylin and eosin (H&amp;E). The preparations were then examined under a light microscope to assess orthokeratosis (OK) and epidermal growth. The first was made by measuring the length in the horizontal plane of a fully developed granular layer (stratum granulosum) on a single scale in relation to its total length. Drug activity was determined using an increase in the number of positive cells in the cuticle containing a granular layer (stratum granulosum) between two hair follicles.

[0136] Meisoindigo anti-mitotic effect on mouse vaginal epithelium: Sixty female Kunming mice were randomly divided into 5 groups, 20 in one control group and 10 in each of the 4 drug groups. All mice were given estrogen at a dose of 0.2 mg / animal for 3 days to allow vaginal epithelial cells to develop under estrogen hormone stimulation. A fresh suspension of Meisoindigo in 0.5% sodium methylcellulose was prepared. The drug was administered orally at doses of 50, 100 and 200 mg / kg, once a day, for 3 days. Methotrexate was used as a positive control and was administered at a dose of 1 mg / kg, intraperitoneally, once a day, for the same period of time. The same volume and excipient used to prepare the Meisoindigo suspension was used as a negative control. One hour after the last administration, animals were administered colchicine, intraperitoneally, at a dose of 2 mg / kg, to arrest cells in phase M. Five hours later, the animals were sacrificed and the vaginal tissue fixed in 10% formalin, embedded in paraffin and preparations prepared. Slides were stained with H&amp;E and at least 500 gout cells were evaluated under a microscope. Mitotic index (percentage of mitotic cells) was calculated.

Results and Discussion [0137] In this Example, a commonly used mouse tail model was used to quantify if the compounds claimed in this invention are capable of enhancing epidermal cell differentiation and thereby reducing their proliferation and hyperkeratosis. This model was first described by Jarrett A (107) and modified by Bosman et al. (106) and is also currently used by others (108-110).

[0138] Table 3 shows the effect of Meisoindigo on the formation of the granular layer by epithelial cells in the mouse tail scale. A significant increase in granular layer formation in the tail epithelium of mice was found when the mice were given Meisoindigo for 13 days in a dose-dependent manner. The therapeutic effect of all 3 doses was better than the effect of positive control, in which mice were given clinically available drug,

MTX, for the treatment of psoriasis.

Table 3. The effect of Meisoindigo on the formation of the granular layer by epithelial cells in the tail scales of mice

<td>group</td><td>Dose^(mg^{/ k}g)</td><td>No animal</td><td>Grainy scales (X ± SD)</td><td>Value p</td>

<td>Control</td><td>not applicable</td><td> 20</td><td> 14,81 ± 4,61</td><td></td>

<td>MTX</td><td> 1,0</td><td> 10</td><td> 17,22 ± 4,92</td><td> &lt;0,05</td>

<td>Meisoindigo</td><td> 50,0</td><td> 10</td><td> 22,37 ± 6,20</td><td> &lt; 0,01</td>

<td>Meisoindigo</td><td> 100,0</td><td> 10</td><td> 25,98 ± 4,12</td><td> &lt; 0,001</td>

<td>Meisoindigo</td><td> 200,0</td><td> 10</td><td> 31,30 ± 7,92</td><td> &lt; 0,001</td>

[0139] MTX is a chemotherapeutic agent with immunosuppressive activity that significantly reduces cell mitosis. To investigate whether Meisoindigo enhances epidermal cell differentiation and thus reduces hyperplasia and hyperkeratosis in a similar mechanism to MTX, the effect of Meisoindigo on the mitotic index (MI) of mouse vaginal epithelium was investigated. As shown in Table 4, administration of MTX mice for 3 days led to a significant reduction in MI compared to the control without MTX administration (14.76 ± 4.29 in the MTX group compared to 20.04 ± 3.71 in the control group, P &lt;0.001). In contrast, as shown in Table 4, no significant effect of Meisoindigo on MI at lower doses (50 and 100 mg / kg, P&gt; 0.05) was found under the same experimental conditions. At the higher dose (200 mg / kg), only a slight decrease was found

MI (16.06 ± 2.66 in the Meisoindigo group compared to 20.04 ± 3.71 in the control group, P &lt;0.05).

[0140] Our data show that Meisoindigo significantly stimulated the formation of the granular layer in the mouse tail epithelium, while no effect was found on MI, suggesting that Meisoindigo induced epithelial cell differentiation and promoted the maturation of incompletely differentiated epithelial cells in the husk. This in vivo observation further confirms our earlier report that Meisoindigo was able to induce ML-1 cell differentiation in vitro (111).

TABLE 4. Effect of Meisoindigo on the mitotic index in estrogen-stimulated vaginal epithelium in female mice

<td>group</td><td>Dose^(mg^{/ k}g)</td><td>No animal</td><td>Mitotic indicator (X ± SD)</td><td>Value p</td>

<td>Control</td><td>not applicable</td><td> 20</td><td> 20,04 ± 3,71</td><td></td>

<td>MTX</td><td> 1,0</td><td> 10</td><td> 14,76 ± 4,29</td><td> &lt;0,001</td>

<td>Meisoindigo</td><td> 50,0</td><td> 10</td><td> 20,01 ± 3,62</td><td> &gt;0,05</td>

<td>Meisoindigo</td><td> 100,0</td><td> 10</td><td> 17,92 ± 4,75</td><td> &gt;0,05</td>

<td>Meisoindigo</td><td> 200,0</td><td> 10</td><td> 16,06 ± 2,66</td><td> &lt;0,05</td>

[0141] The mouse tail model used in this Example is a frequently used in vivo model to evaluate the therapeutic value of agents for the treatment of psoriasis. The present invention is defined in the claims and does not apply to the treatment of psoriasis - however, our observations above strongly suggest that the small molecules defined herein are capable of treating psoriasis, a disease associated with inflammation. However, the mechanism of anti-psoriatic action of these small molecules is different from the mechanism of action of the immunosuppressant and chemotherapeutic agent, MTX, as no direct effect of Meisoindigo on cell mitosis was found. Instead, the modulation of the secretion / expression of different types of cytokines demonstrated in Examples 1-4 in this invention along with the differentiation of cells under the influence of these molecules probably form the molecular basis for anti-psoriatic activity.

Example 7: Meisoindigo suppresses acute ulcerative colitis in mice

Balb / c

Materials and methods [0142] Materials: Meisoindigo has been synthesized, purified and its structure has been characterized by Natrogen Therapeutics, Inc. as described in the above Examples. A fresh suspension of Meisoindigo in 0.5% methylcellulose sodium was prepared and administered orally in the animal studies described below. DSS (sodium dextran sulfate, molecular weight: 36000-44000) was purchased from ICN Biomedicals. Other chemicals used in the following experiments were purchased from Sigma.

[0143] Pathogen-free female Balb / c mice, 12 weeks old, 22 to 25 gram body weight, housed 5 individuals in a cage and had free access to fresh tap water and commercially available granular rodent feed. The conditions in the animal housing were controlled at 24 ± 2 ° C with a relative humidity of 60 ± 5% and a 12 hour light / dark cycle was used (from 07:00 to 19:00).

[0144] Induction of acute ulcerative colitis, DSS-induced colitis in Balb / c mice: Colitis was induced with DSS in drinking water (molecular weight 36,000 - 50,000, ICN Biochemicals) as previously described (112) . In brief, the mice were randomly divided into 3 groups of 10 mice in each. In the negative control group (Group 1), the mice had free access to fresh tap water and granulated MF food, changed to fresh twice a week, for 7 days. In the positive control group (DSS group or group 2), 5% DSS was administered in tap water for 7 days to induce colitis; mice were fed MF in pellets. In the DSS-Meisoindigo group (study group or group 3), mice were given 5% DSS in drinking water and given Meisoindigo orally, once a day, at a dose of 50 mg / kg for 7 consecutive days. The characteristics of colitis on a daily basis were recorded on the basis of feces, including body weight and the nature of the feces (loose and / or with blood or occult blood). The mice were then sacrificed. Colon tissue was removed, fixed in 10% formalin / PBS and embedded in paraffin. To reduce physical artifacts, the removed colon was placed on thick qualitative filter paper, without stretching. The colon was then exposed from the inside, cutting it along. The preparations were stained with H&amp;E and evaluated, using a blank method, by 3 technicians / pathomorphologists.

Statistical analysis [0145] All data were expressed as mean SD. The statistical significance of all differences between the control group and the experimental groups was determined using the Student's t test, with a P value &lt;0.05.

Results and discussion [0146] Loose stools, occult blood, and weight loss after drinking 5% DSS on day 4 gradually developed in all animals in the positive control group. In several severe cases (7/10) in addition to the symptoms mentioned above, macroscopic blood was found. adhered to the anus. Although the procedures for causing ulcerative colitis in this experiment were very aggressive and the emerging symptoms were very acute (occurred within 4 days), occult blood in the Meisoindigo group only occurred in 40% (4/10) of the animals. Other symptoms, such as loose stools, were also less severe than in the control group. There were no signs of colitis in animals from the negative control group (normal mice), Fig. 7, panel A.

[0147] Histological examination showed that tissues from all animals from the positive control group showed severe lesions reminiscent of those found in ulcerative colitis, as indicated by inflammatory cell infiltration, including polynuclear leukocytes and multiple erosive lesions (score 1- 3). Crypt abscesses and epithelial regeneration were also found in the colonic mucosa (Fig. 7, panel Β). To avoid subjective evaluation, ulcerative colon damage in positive control and Meisoindigo receiving mice was quantified and blinded by 3 separate technicians / pathologists under a microscope. A 55% reduction in ulceration was found in the animals in the Meisoindigo group compared to the positive control group (2.89 ± 1.46 in the group receiving

Meisoindigo compared to 6.38 ± 2.20 in the positive control group). Fig. 7, panel C shows the colon wall of a mouse receiving Meisoindigo with acute ulcerative colitis caused by 5% DSS. Its morphology is similar to that found in a normal control (panel A), indicating that Meisoindigo is effective against acute ulcerative colitis caused by DSS in mice.

Example 8: Meisoindigo completely stopped idiopathic inflammatory bowel disease in a patient [0148] Patient: A 43-year-old woman was diagnosed as having active chronic proctitis and colitis for more than four years with erosions and features suggesting idiopathic inflammatory bowel disease . The first diagnosis was made at North Shore University Hospital Manhasset, Long Island, New York in 1999 based on colonoscopy. The main symptoms included diarrhea, loose stools and bleeding, while the patient's overall health was excellent. The clinical activity index of the disease (Table 5) (113) was between 7 and 8. The patient's doctor prescribed her hydrocortisone in the form of foam, which she used for 10 days as prescribed by the doctor. However, no therapeutic effect of this agent was obtained. In February 2000, the patient returned to her home in China and repeatedly reported to Chinese doctors and tried various Chinese herbal remedies suggested to her, but found no therapeutic effect. In early 2002, she reported to the well-known and respected Chinese Medical Hospital in Beijing, where sigmoidoscopy was performed with a flexible sight glass. She was again diagnosed with active inflammatory bowel disease.

Table 5. Clinical activity index for assessing patients with ulcerative colitis (113)

<td>Symptom</td><td>Standard punctation</td><td colspan="2">Patient scoring</td>

<td></td><td></td><td>Before treatment</td><td>After treatment</td>

<td></td><td></td><td>not M</td><td></td>

<td>Diarrhea (stools daily)</td><td></td><td></td><td></td>

<td> 0-2</td><td> 0</td><td></td><td></td>

<td>3 or 4</td><td> 1</td><td> 2</td><td></td>

<td>5 or 6</td><td> 2</td><td></td><td> 0</td>

<td> 7-9</td><td> 3</td><td></td><td></td>

<td>10 or more</td><td> 4</td><td></td><td></td>

<td>Diarrhea at night</td><td></td><td></td><td></td>

<td>No</td><td> 0</td><td> 0</td><td> 0</td>

<td>Yes</td><td> 1</td><td></td><td></td>

<td>Visible blood in stool (% of bowel movements)</td><td></td><td></td><td></td>

<td> 0</td><td> 0</td><td></td><td></td>

<td> &lt;50</td><td> 1</td><td></td><td></td>

<td> &gt;50</td><td> 2</td><td> 2</td><td> 0</td>

<td> 100</td><td> 3</td><td></td><td></td>

<td>Stool incontinence</td><td></td><td></td><td></td>

<td>No</td><td> 0</td><td></td><td></td>

<td>Yes</td><td> 1</td><td> 1</td><td> 0</td>

<td>Pain or stomach cramps</td><td></td><td></td><td></td>

<td>Lack</td><td> 0</td><td></td><td></td>

<td>mild</td><td> 1</td><td> 1</td><td> 0</td>

<td>moderate</td><td> 2</td><td></td><td></td>

<td>Heavy</td><td> 3</td><td></td><td></td>

<td>General well-being</td><td></td><td></td><td></td>

<td>Perfectly</td><td> 0</td><td></td><td></td>

<td>Very good</td><td> 1</td><td></td><td></td>

<td>good</td><td> 2</td><td> 2</td><td> 0</td>

<td>Medium</td><td> 3</td><td></td><td></td>

<td>Wrong</td><td> 4</td><td></td><td></td>

<td>horrible</td><td> 5</td><td></td><td></td>

<td>Abdominal tenderness</td><td></td><td></td><td></td>

<td>Lack Gentle and positioned Mild or moderate and diffuse Severe or when releasing pressure</td><td> 0 1 2 3</td><td> 0</td><td> 0</td>

<td>The need for antidiarrheal drugs</td><td></td><td></td><td></td>

<td>No</td><td> 0</td><td> 1</td><td> 0</td>

<td>Yes</td><td> 1</td><td></td><td></td>

<td>Maximum score</td><td> 21</td><td> 9</td><td> 0</td>

[0149] At that time, as previous therapies had no effect, the patient's physician commissioned her with a number of traditional Chinese drugs. She used them for several months, but there was no symptomatic improvement. In the period between February 2000 and summer 2002, the patient also tried various other traditional Chinese medicines due to the persistent and very embarrassing nature of the disease, but none of them was effective. After returning to the United States in 2002, she reported to the Mount Sinai Medical Center, a well-known American center for the treatment of inflammatory bowel diseases. In October 2002 her new doctor again performed a colonoscopy along with histological examination of two biopsies (A and B) (FIG. 8, panel A, before treatment). The conclusions of the study were as follows: in two places (A and B) the inflammation was "severe, active, chronic proctitis and colitis with erosions and features suggestive of idiopathic inflammatory bowel disease". The doctor prescribed CANASA® (mesalamine suppository), a drug recently approved by the FDA, considered the best drug available in this state.

[0150] After 7 days of CANASA® administration, it seemed that the patient's symptoms had alleviated, but at the same time had significant side effects. These include itching, abdominal cramps, pain and bleeding. The patient then turned to her doctor for advice and was advised to use this medicine intermittently. However, the side effects actually increased after each use of the drug. Then the patient stopped using this medicine.

[0151] It has been suggested that Meisoindigo is a potential drug based on the finding that it can lead to suppression of a number of post-inflammatory cytokines and stimulation of anti-inflammatory cytokines such as IL-10. Due to the very low side effects found at 150 mg daily of this drug in China in the treatment of chronic myelogenous leukemia, the use of Meisoindigo has been suggested.

[0152] The patient herself took Meisoindigo at the recommended dose of 25 mg once daily for a designated period of three weeks of treatment. After the first three doses, complete remission occurred (three days after treatment) and all inflammatory symptoms resolved. As a result, a clinical activity score of zero was obtained (Table 5). After nine weeks of remission (three weeks of taking the medicine, three weeks of taking a break from taking the medicine and again three weeks of taking the medicine), the patient again reported to her doctor and asked for a flexible sight sigmoidoscopy to check whether her remission was subjective or objective . Sigmoidoscopy with a flexible sight glass was performed on November 4, 2003. After reviewing its results, the patient's attending physician stated that the inflammation had stopped completely. Pathomorphological studies confirmed the validity of this conclusion and showed that in samples A and B "currently inactive, chronic proctitis and colitis with features suggestive of idiopathic inflammatory bowel disease were found." FIG. 7 presents the photo before (panel A, October 2002) and after treatment (panel B, November 4, 2003) of the same inflammation site in a colonoscopy performed by the same doctor.

Example 9: Therapeutic effect of Meisoindigo on chronic ulcerative colitis in Balb / c mice [0153] Meisoindigo has been shown to be a regulator of numerous cytokines, IL-1e, IL-6, IL-10 and TNF-α, which are known to be involved in pathological processes and the maintenance of various inflammatory diseases. It has also been shown that Meisoindigo is a cytostatic agent that inhibits the rapidly growing cell proliferation and promotes cell differentiation and maturation.

[0154] Our previous experiments have shown that Meisoindigo exerts a healing effect on sodium-induced dextran sulfate (DSS) in acute, aggressive ulcerative colitis in Balb / c mice. These results show that Meisoindigo is an effective agent against various chronic inflammatory diseases as defined in the claims, including inflammatory bowel disease (IBD).

[0155] In this experiment, a DSS-induced chronic ulcerative colitis model was used to verify molecular targets in vivo and to assess Meisoindigo activity in IBD.

Materials and methods [0156] Materials: Meisoindigo has been synthesized, purified and its structure has been characterized by Natrogen Therapeutics, Inc. A fresh suspension of Meisoindigo in 0.5% sodium methylcellulose was prepared and stored at 4 ° C. The drug suspension was orally administered for the purposes of the animal studies described below.

5-aminosalicylic acid (5-ASA) was purchased in Sigma / DSS (sodium dextran sulfate, molecular weight: 36000-44000) was purchased from ICN Biomedicals. Other chemicals used in the following experiments were purchased from Sigma.

[0157] Pathogen-free Balb / c mice, 12 weeks of age, body weight from 22 to 25 grams, were housed five cages each and had free access to fresh tap water and commercially available granular rodent feed. The conditions in the animal housing were controlled at 24 ± 2 ° C with a relative humidity of 60 ± 5% and a 12-hour light / dark cycle was used (from 07:00 to 19:00).

[0158] Methods: Induction of chronic ulcerative colitis using DSS in Balb / c mice: Chronic ulcerative colitis was induced using DSS in drinking water (molecular weight 36000-44000, ICN Biochemicals) as previously described [4]. Briefly, 60 mice were randomly divided into 5 groups of 12 mice each. Group 1 was used as a negative control (without causing the disease) and had free access to tap water and granulated fresh food changed twice a week from the beginning of the experiment. Groups 2-5 were used to chronically induce ulcerative colitis and to investigate the activity of Meisoindigo. The animals in groups 2-5 were fed normally, as were the animals in the negative control group, and they also received drinking water containing 5% DSS (molecular weight 36,000 - 44,000) for 7 days, followed by distilled water for the next 10 days. The animals were again given drinking water containing 5% DSS for 7 days, followed by distilled water for another 10 days. This procedure was repeated for a total of 3 cycles.

[0159] After 3 cycles, chronic ulcerative colitis developed and was stabilized as described. Group 2 (positive control group) was given saline, and group 3 was given 5-ASA at a dose of 50 mg / kg / day by gavage as a positive drug control. Meisoindigo was administered in groups 4 and 5 at doses of 25 and 75 mg / kg, once daily for 12 days.

[0160] During the experimental period, an independent researcher determined and recorded disease activity indices (DAI) daily, including body weight, fecal consistency, and occult blood or macroscopic rectal blood in accordance with the methods previously described [114, 116]. These clinical parameters are comprehensive, functional measures similar to subjective clinical symptoms found in people with ulcerative colitis (UC) and correlate well with the degree of histological healing measured as crypt scores [114-116]. The mice were then sacrificed. Tissues / organs: spleen, colon and other were collected, their appearance was examined and their mass was recorded. To reduce physical artifacts, the removed colon was placed on thick qualitative filter paper, without stretching. The colon was then exposed from the inside, cutting it along. All colon tissues were fixed in 10% formal / PBS, embedded in paraffin, and section preparations were made. The preparations were stained with H&amp;E for histological examination. The preparations were evaluated using a blinded method by a technician / pathomorphologists and photographed. The severity of ulcerative colitis was rated on a scale of 0 to 3 and expressed as a pathological indicator according to a standard scoring system: 0, normal; 1, focal lesions of inflammatory cells, including polynuclear leukocytes; 2, inflammatory cell infiltration, loss of glands and crypt abscesses; 3, mucosal ulceration. Numbers of lymphatic papules, ulcerative lesions were counted in longitudinal sections of the colon under a light microscope.

Statistical analysis [0161] All data are expressed as mean SD. The statistical significance of all differences between the control group and the experimental groups was determined using the Student's t-test with a P value &lt;0.05.

Results and discussion:

[0162] Disease Activity Index (DAI): After drinking 5% DSS for 7 days, all mice initially had diarrhea and fecal occult blood accompanied by significant weight loss, but no such symptoms were seen in animals receiving distilled water. However, these signs disappeared (DAI reached a minimum in 8-9 days) after giving the mice distilled water for the next 10 days. In contrast, during subsequent DSS administrations (3 administration cycles) these clinical symptoms did not improve, but rather exacerbated during 10 days of drinking distilled water.

[0163] After the third DSS administration cycle, the animals were fed by gavage with vehicle, 50 mg / kg / day 5-ASA, 25 or 75 mg / kg / day Meisoindigo for 12 days. Twenty-four hours after the last administration, DAI scores in all five groups were determined. As shown in Table 6 below, after discontinuing DSS for 12 days, the DAI score decreased by 50% in the vehicle group, which was most likely associated with self-healing [117]. In contrast, the degree of decrease in DAI in the 5-ASA group and in the two groups receiving Meisoindigo was significantly greater, up to 70%, which reflects the therapeutic response to treatment. The effectiveness of Meisoindigo at both doses was equal to that of 5-ASA.

Table 6 - Disease activity index (weight loss, stool consistency and bleeding / 3)

<td colspan="2">Center</td><td>Average ± SD before treatment</td><td>After treatment, mean ± SD</td><td>Improvement (%)</td>

<td rowspan="3">Brine</td><td>Females (n = 5)</td><td> 2,40±0,2</td><td> 1,07±1,0</td><td></td>

<td>Males (n = 6)</td><td> 2,40±0,2</td><td> 1,33±1,15</td><td></td>

<td>Average (n = 11)</td><td> 2,40±0,2</td><td> 1,2±1,2</td><td> 50,6</td>

<td rowspan="3">ASA 50 mg / kg</td><td>Females (n = 0)</td><td>not applicable</td><td>not applicable</td><td></td>

<td>Males (n = 10)</td><td> 3,27±0,3</td><td> 0,86±1,0</td><td></td>

<td>Average (n = 10)</td><td> 3,27±0,3</td><td> 0,86±1,0</td><td> 73,1 0=0,031)</td>

<td rowspan="3">Meisoindigo 25 mg / kg</td><td>Females (n = 5)</td><td> 3,20±0,2</td><td> 0,67±1,15</td><td></td>

<td>Males (n = 7)</td><td> 2,73±0,35</td><td> 0,93±1,0</td><td></td>

<td>Average (n = 12)</td><td> 3,00±0,1</td><td> 0,80±1,06</td><td> 73,3 0=0,023)</td>

<td rowspan="3">Meisoindigo 75 mg / kg</td><td>Females (n = 6)</td><td> 3,11±0,68</td><td> 0,93±1,0</td><td></td>

<td>Males (n = 6)</td><td> 3,27±0,75</td><td> 0,97±1,0</td><td></td>

<td>Average (n = 12)</td><td> 3,22±0,71</td><td> 0,95±1,0</td><td>70.3 (p = 0.033)</td>

<td rowspan="3">Lack</td><td>Females (n = 6)</td><td> 0</td><td> 0</td><td>not applicable</td>

<td>Males (n = 6)</td><td> 0</td><td> 0</td><td>not applicable</td>

<td>Average (n = 12)</td><td> 0</td><td> 0</td><td>not applicable</td>

Latent blood score:

[0164] Table 7 below shows the occult blood score in all treated and untreated animal groups. Twelve days after discontinuation of DSS, a slight improvement (27.2%) was found in the animals receiving the vehicle, which was consistent with the chronic nature of colitis caused by 3 consecutive DSS cycles, as previously described [4]. In contrast, significant improvement was found in the 5-SAS and Meisoindigo groups. The best therapeutic response (87%) was found in the low-dose Meisoindigo group in animals compared to 75.7% in the high-dose group. The low dose was also better than the 5-ASA positive control showing 80% improvement. The treatment of DSD-induced bleeding in DSD by Meisoindigo in animals is also consistent with previous observation that administration of Meisoindigo induces a rapid end to bleeding in a patient with ulcerative colitis.

Qualitative histological examination:

[0165] Transverse (middle) sections of the mouse colon were prepared in formalin for embedding in paraffin. The preparations were stained with H&amp;E. Two mice from each group were used to obtain formulations and to study the quality of formulations and staining.

[0166] Histological examination of the animals receiving the vehicle showed loss of surface epithelium, loss of glands and the presence of chronic inflammation. In preparations from animals in both groups receiving Meisoindigo, as in the 5-ASA groups, less erosions, milder inflammatory infiltrates were found compared to IBD mice.

Table 7 - Latent blood scoring

<td colspan="2">Center</td><td>Average ± SD before treatment</td><td>After treatment, mean ± SD</td><td>Improvement (%)</td>

<td rowspan="3">Brine</td><td>Females (n = 5)</td><td> 2,4+0,89</td><td> 1,2+1,09</td><td> 50</td>

<td>Males (n = 6)</td><td> 2,0+0</td><td> 2,0+0</td><td> 0</td>

<td>Average (n = 11)</td><td> 2,2+0,6</td><td> 1,6+0,8</td><td> 27,2</td>

<td rowspan="3">ASA 50 mg / kg</td><td>Females (n = 0)</td><td>not applicable</td><td>not applicable</td><td>not applicable</td>

<td>Males (n = 10)</td><td> 3,0+1,05</td><td> 0,6+0,96</td><td> 80,0</td>

<td>Average (n = 10)</td><td> 3,0+1,05</td><td> 0,6+0,96</td><td>80.0 (p &lt;0.01)</td>

<td rowspan="3">Meisoindigo 25 mg / kg</td><td>Females (n = 5)</td><td> 2,8+1,09</td><td> 0,8+1,0</td><td> 71,4</td>

<td>Males (n = 7)</td><td> 3,1+1,06</td><td> 0+0</td><td> 100</td>

<td>Average (n = 12)</td><td> 3,0+1,04</td><td> 0,4+0,77</td><td>86.7 (p &lt;0.01)</td>

<td rowspan="3">Meisoindigo 75 mg / kg</td><td>Females (n = 6)</td><td> 3,3+1,03</td><td> 0,6+1,0</td><td> 81,8</td>

<td>Males (n = 6)</td><td> 3,6+0,81</td><td> 1,0+1,0</td><td> 72,2</td>

<td>Average (n = 12)</td><td> 3,5+0,9</td><td> 0,83+1,0</td><td>76.3 (p &lt;0.01)</td>

<td>Lack</td><td>Females (n = 6)</td><td> 0</td><td> 0</td><td>not applicable</td>

<td></td><td>Males (n = 6)</td><td> 0</td><td> 0</td><td>not applicable</td>

<td></td><td>Average (n = 12)</td><td> 0</td><td> 0</td><td>not applicable</td>

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