Use of mm-102 in the manufacture of a product for improving or treating systemic lupus erythematosus

Abstract

The present application relates to the technical field of biological medicine, and more particularly to the use of MM-102 in the preparation of a product for improving or treating systemic lupus erythematosus. The present application proves that MM-102 can reduce facial skin lesions, lymph node enlargement and splenomegaly, reduce the levels of serum anti-double-stranded DNA antibody, anti-nuclear antibody and blood creatinine, reduce kidney IgG deposition, glomerular capillary proliferation and renal interstitial inflammatory cell infiltration, and reduce the proportion of Th17 cells in peripheral blood and spleen. The above results show that MM-102 has the potential to improve systemic lupus erythematosus-related immune abnormalities and organ damage, and provides a new drug intervention scheme for systemic lupus erythematosus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to the use of MM-102 in the preparation of products for improving or treating systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease with complex clinical manifestations and significant individual variability. It can affect multiple organs or tissues, including the skin, mucous membranes, joints, hematopoietic system, kidneys, nervous system, cardiovascular system, and lungs. SLE is more common in women of childbearing age, and the disease course typically involves alternating periods of disease activity and remission. Some patients may experience persistent organ damage due to recurrent disease activity, severely impacting their quality of life and long-term prognosis.

[0003] The occurrence and development of SLE involves multiple factors, including genetic susceptibility, abnormal immune regulation, sex hormones, and environmental factors. Ultraviolet radiation, infection, and certain medications may trigger or worsen the disease. SLE patients may exhibit abnormal immune tolerance, activation of autoreactive lymphocytes, production of autoantibodies, and formation of immune complexes. The deposition of immune complexes in tissues can further induce inflammatory responses and cause corresponding tissue and organ damage.

[0004] The treatment principle for SLE is early diagnosis and treatment, and treatment-to-target (T2T) therapy. This aims to maintain control over the long term, reduce disease relapse and drug-related adverse reactions, ultimately lower the risk of organ damage and death, and improve patients' quality of life. As the core of the T2T strategy, clinical guidelines recommend two internationally validated target states: "clinical remission" and "low disease activity state" in lupus. "Clinical remission" is defined as remission in systemic lupus erythematosus, emphasizing the absence of clinical activity and allowing the use of stable low-dose corticosteroids and antimalarial drugs. Both states can serve as goals for short-term and long-term treatment and are suitable for patients with different risk levels and disease stages.

[0005] Currently, treatment for SLE mainly includes glucocorticoids, hydroxychloroquine, immunosuppressants, biologics, and small molecule targeted drugs. Glucocorticoids can rapidly control acute inflammation, but long-term use may lead to adverse reactions such as osteoporosis, infection, disorders of glucose and lipid metabolism, Cushing's syndrome, and avascular necrosis of the femoral head. Immunosuppressants such as methotrexate, azathioprine, mycophenolate mofetil, and cyclophosphamide may cause bone marrow suppression, liver and kidney damage, and opportunistic infections. Although biologics and small molecule targeted drugs have relatively clear targets, they still have problems such as limited treatment response rates, infection risks, high costs, and relapse in some patients.

[0006] Meanwhile, SLE patients exhibit significant heterogeneity in clinical manifestations, immunological characteristics, and organ involvement. Some patients fail to achieve clinical remission or low disease activity despite existing drug treatments; some patients experience short-term symptom control but still suffer from autoantibody abnormalities, renal immune complex deposition, or immune cell subset imbalances; and some patients struggle to maintain adequate dosage and duration of treatment due to adverse drug reactions. Especially for patients with lupus nephritis, how to control systemic immune abnormalities while mitigating renal tissue damage remains a problem that requires ongoing resolution.

[0007] The molecular formula of MM-102 is C 35 H 49 F2N7O4, with CAS number 1417329-24-8. Studies have shown that MM-102 exhibits potent tumor-suppressive activity in malignant proliferative models such as acute myeloid leukemia. As a tool drug, MM-102 not only downregulates proliferative genes but also possesses the potential to remodel the transcriptional network of inflammatory cytokines. However, the role of MM-102 in autoimmune diseases remains unclear. Therefore, investigating whether MM-102 can improve or treat systemic lupus erythematosus is crucial. Summary of the Invention

[0008] This invention aims to provide a new use for MM-102 in the treatment or improvement of systemic lupus erythematosus (SLE), in order to address the problems of existing SLE treatment regimens, such as relatively nonspecific mechanisms of action, significant adverse reactions with long-term use, difficulty in simultaneously improving autoantibody abnormalities, renal immune complex deposition, and T cell subset imbalance.

[0009] In a first aspect, the present invention provides for the use of compounds in the preparation of products for improving or treating systemic lupus erythematosus, said compounds being of Formula I: .

[0010] In a second aspect, the present invention provides a product for improving or treating systemic lupus erythematosus, comprising: a compound and pharmaceutically acceptable excipients; said compound being of formula I: .

[0011] In one embodiment, the concentration of the compound in the product is 0.5-10 mg / mL, preferably 5 mg / mL.

[0012] In one embodiment, the excipients include, but are not limited to, water-soluble excipients (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble excipients (such as ethyl cellulose, cholesterol stearate, etc.), and enteric excipients (such as cellulose acetate phthalate, carboxymethyl cellulose, carboxyethyl cellulose, etc.).

[0013] In another embodiment, pharmaceutically acceptable excipients also include colorants, preservatives, flavorings, sweeteners, or other materials.

[0014] In one embodiment, the product may be a conventional formulation, a sustained-release formulation, a controlled-release formulation, or a variety of microparticle delivery systems.

[0015] In another embodiment, the dosage form of the product includes an oral formulation or an injectable formulation.

[0016] Specifically, the oral preparations include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, and oral emulsions.

[0017] Preferably, when the dosage form is tablets, a wide variety of excipients known in the art can be used. Examples of excipients include, for instance, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; and disintegrants. Examples of additives include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc.

[0018] Furthermore, the tablets are further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0019] Preferably, when the dosage form is a pill, a wide variety of excipients known in the art can be used. Examples of excipients include, for instance, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0020] Specifically, the injectable preparations include injection solutions and powder injections.

[0021] Preferably, when the dosage form is an injection, such as a solution, emulsion, lyophilized powder for injection, or suspension, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc.

[0022] Furthermore, the injection solution can be further formulated into an isotonic injection solution by adding appropriate amounts of sodium chloride, glucose, or glycerol to the injectable formulation. In addition, conventional solubilizers, buffers, pH adjusters, etc., can also be added.

[0023] Specifically, the administration methods of the injectable preparation include subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection.

[0024] Furthermore, the product also includes dimethyl sulfoxide, polyethylene glycol 300, Tween 80, and water.

[0025] Furthermore, the volume ratio of dimethyl sulfoxide, polyethylene glycol 300, Tween 80, and water is 50:400:50:500.

[0026] In one embodiment, the product further includes at least one of the following: hydroxychloroquine, glucocorticoids, immunosuppressants, biologics, and small molecule targeted drugs.

[0027] Hydroxychloroquine, as a cornerstone drug for the treatment of SLE, can reduce disease activity and lower the risk of recurrence. However, long-term use requires monitoring for retinal toxicity.

[0028] The glucocorticoids are used to control acute inflammation, but long-term use can easily cause side effects such as osteoporosis, diabetes, and infection. Current guidelines emphasize reducing the dosage as much as possible and stopping it as early as possible.

[0029] Specifically, the glucocorticoid is prednisone.

[0030] The immunosuppressants are often used in combination with glucocorticoids to control disease activity, especially for hormone-dependent or refractory patients, but adverse reactions such as liver and kidney function and bone marrow suppression need to be monitored.

[0031] Specifically, the immunosuppressant is at least one of methotrexate, azathioprine, and mycophenolate mofetil.

[0032] Specifically, the biological agent is at least one of belimumab, anilumab, and olibumarab.

[0033] Belimumab targets B-cell activating factor (BAFF), reduces the production of autoantibodies, is suitable for non-renal SLE, and can reduce disease activity and hormone dosage.

[0034] Anifrolumab targets type I interferon receptors and significantly improves symptoms of mucosal, skin, and joint conditions. It is suitable for moderate to severe SLE, but may increase the risk of infection.

[0035] The obinutuzumab, which targets CD20 B cells, can improve the complete remission rate of the kidneys in patients with lupus nephritis.

[0036] Specifically, the small molecule targeted drug is at least one of vorciclosporine, baricitinib, and tofacitinib.

[0037] Voclosporin is a novel calcineurin inhibitor used for lupus nephritis. It can improve renal function, reduce proteinuria, and has a safety profile superior to traditional cyclosporine.

[0038] Baricitinib and tofacitinib are JAK inhibitors that regulate immune responses by inhibiting the JAK-STAT pathway. They are effective for skin and joint symptoms, but risks such as thrombosis and infection should be noted.

[0039] In a third aspect, the present invention proposes the use of the above-described product in the preparation of a medicament for improving or treating systemic lupus erythematosus.

[0040] In one embodiment, the systemic lupus erythematosus (SLE) is any one of active SLE, severe SLE, cutaneous SLE, positive anti-double-stranded DNA antibody, or positive antinuclear antibody; or, the SLE is SLE accompanied by at least one of the following manifestations: skin involvement, kidney involvement, lymphadenopathy, splenomegaly, abnormal autoantibodies, or imbalance of immune cell subsets.

[0041] Specifically, the SLEDAI-2000 score for active systemic lupus erythematosus is ≥6.

[0042] Specifically, the kidney involvement includes at least one of lupus nephritis, renal immune complex deposition, glomerular proliferative lesions, glomerular basement membrane or mesangial region damage, renal interstitial inflammatory cell infiltration, or renal dysfunction.

[0043] Preferably, the lupus nephritis is at least one of the following: immune complex-associated lupus nephritis, proliferative lupus nephritis, renal IgG deposition-associated lupus nephritis, lupus nephritis with glomerular proliferative lesions, lupus nephritis with renal interstitial inflammatory damage, or type III, IV, or V lupus nephritis in the ISN / RPS pathological classification.

[0044] Specifically, the autoantibody abnormalities include elevated levels of anti-double-stranded DNA antibodies and / or antinuclear antibodies; the immune cell subset imbalances include at least one of an increased proportion of Th17 cells, a decreased proportion of Treg cells, or a Th17 / Treg immune imbalance.

[0045] Preferably, the Th17 cells are CD4 cells. + RORγt + Cells and / or CD4 + IL-17A + cell.

[0046] In a fourth aspect, the present invention proposes a method for reducing the proportion of the Th17 subset in peripheral blood mononuclear cells and / or spleen cells of patients with systemic lupus erythematosus (SLE) in vitro, wherein peripheral blood mononuclear cells or spleen cells of SLE patients are cultured or treated with a compound as shown in Formula I: .

[0047] In a fifth aspect, the present invention proposes a method for increasing the proportion of Treg subsets in spleen cells of patients with systemic lupus erythematosus (SLE) in vitro, wherein spleen cells of SLE patients are cultured or treated with a compound as shown in Formula I: .

[0048] In a sixth aspect, the present invention provides a method for improving or treating systemic lupus erythematosus (SLE), comprising administering an effective amount of a compound, or an effective amount of the aforementioned product for improving or treating SLE, to a patient with SLE; said compound being as shown in Formula I: .

[0049] In one embodiment, the effective amount is the dose used to treat systemic lupus erythematosus in patients with systemic lupus erythematosus, preferably 10-20 μg / g body weight, specifically 15 μg / g body weight.

[0050] In another embodiment, the patient is a mammal, such as a rodent or primate.

[0051] Compared with existing technologies, the present invention has the following advantages: (1) Improvement of extrinsic disease phenotype associated with systemic lupus erythematosus: MM-102 can reduce skin lesions and improve the skin phenotype associated with systemic lupus erythematosus disease activity. Moreover, MM-102 can reduce lymphadenopathy and splenomegaly, indicating that it has an ameliorative effect on systemic lupus erythematosus-related immune organ abnormalities.

[0052] (2) Reduce the level of autoantibodies associated with systemic lupus erythematosus: Abnormally elevated levels of anti-double-stranded DNA antibodies and antinuclear antibodies are important serological manifestations of systemic lupus erythematosus. MM-102 can reduce the levels of anti-double-stranded DNA antibodies and antinuclear antibodies in serum, indicating that MM-102 can improve the abnormality of autoantibodies.

[0053] (3) Improvement of renal dysfunction associated with systemic lupus erythematosus: Serum creatinine is one of the indicators for evaluating renal function. MM-102 can reduce the serum creatinine level caused by systemic lupus erythematosus, indicating that MM-102 has an improving effect on renal dysfunction associated with systemic lupus erythematosus.

[0054] (4) Reduced renal histopathological damage associated with systemic lupus erythematosus: MM-102 can reduce glomerular structural abnormalities, glomerular proliferative lesions and renal interstitial inflammatory cell infiltration caused by systemic lupus erythematosus, and can improve pathological changes in the glomerular basement membrane and / or mesangial region, indicating that MM-102 can reduce renal damage associated with systemic lupus erythematosus at the histopathological level.

[0055] (5) Reduce IgG deposition in kidney tissue: Immunoglobulin deposition in the kidney is one of the important pathological manifestations of lupus nephritis. MM-102 can reduce IgG deposition in kidney tissue caused by systemic lupus erythematosus, indicating that MM-102 can improve the pathological changes of the kidney related to immunoglobulin deposition, and thus has the potential to improve lupus nephritis.

[0056] (6) Reducing the proportion of abnormally elevated Th17 cells: MM-102 can reduce the proportion of Th17 cells in peripheral blood mononuclear cells and total spleen cells in patients with systemic lupus erythematosus (SLE). Specifically, the proportion of RORγt-positive cells in peripheral blood mononuclear cells decreased from 5.99-7.94% to 3.75-4.64%, while the proportion of RORγt-positive cells in total spleen cells decreased from 9.93-16.70% to 6.53-7.95%. These results indicate that MM-102 can inhibit the abnormally enhanced Th17 cell response in SLE animal models.

[0057] (7) Regulation of immune cell subset abnormalities associated with systemic lupus erythematosus (SLE): MM-102 reduces the proportion of Th17 cells in peripheral blood mononuclear cells and total spleen cells, while regulating the proportion of Treg cells and the Th17 / Treg balance. Therefore, MM-102 can improve the immune imbalance associated with SLE at the level of immune cell subsets, providing a new immunomodulatory approach for SLE.

[0058] In summary, this invention, through animal experiments using a systemic lupus erythematosus (SLE) model, has for the first time verified the ameliorative effect of MM-102 on the pathological manifestations associated with SLE. MM-102 can simultaneously improve the external disease phenotype, serological abnormalities, renal function, histopathological damage, and abnormal immune cell subsets in the model animals, indicating its potential application value in the preparation of drugs for improving or treating SLE, particularly for improving SLE with lupus nephritis. Of particular note is that MM-102 does not only improve a single disease indicator, but also simultaneously reduces autoantibody levels and the proportion of Th17 cells, decreases renal IgG deposition, and alleviates renal histopathological damage and renal function abnormalities, demonstrating a comprehensive ameliorative effect on the serological abnormalities, cellular immune abnormalities, and organ damage associated with SLE. Attached Figure Description

[0059] Figure 1 A represents the normal control group (Normal) and the model group (MRL / MpJ-Fas). lpr Treatment group (MRL / MpJ-Fas) lpr Comparison photos of skin lesions in +MM-102 mice.

[0060] Figure 1 B shows a comparison of lymph node size in mice from the normal control group, model group, and treatment group.

[0061] Figure 1 C is a comparison of lymph node lengths in mice from the normal control group, model group, and treatment group.

[0062] Figure 1 D is a comparative photograph of the spleen size of mice in the normal control group, model group, and treatment group.

[0063] Figure 1 E is a comparison of spleen length in mice from the normal control group, model group, and treatment group.

[0064] Figure 2 This is a comparison of serum levels of anti-double-stranded DNA antibody, antinuclear antibody, and creatinine in mice from the normal control group, model group, and treatment group; where A represents anti-double-stranded DNA antibody (Anti-dsDNA IgG), B represents antinuclear antibody (Anti-ANA IgG), and C represents creatinine.

[0065] Figure 3 The images show the results of H&E staining, PAS staining, and immunofluorescence staining of kidney tissue from mice in the normal control group, model group, and treatment group; where A represents H&E staining, B represents PAS staining, and C represents immunofluorescence staining.

[0066] Figure 4Flow cytometry results of peripheral blood mononuclear cells from mice in the normal control group, model group, and treatment group; where A and B represent CD4+ cells. + RORγt + Subgroup ratio, C and D are CD4 + IL-17A + Th17 subgroup proportions, E and F are CD4 + CD25 + FoxP3 + Treg subgroup proportions.

[0067] Figure 5 Flow cytometry results of total spleen cells from mice in the normal control group, model group, and treatment group; where A and B represent CD4+. + RORγt + Subgroup ratio, C and D are CD4 + IL-17A + Th17 subgroup proportions, E and F are CD4 + CD25 + FoxP3 + Treg subgroup proportions.

[0068] In the above figure, * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.001, ns represents P >0.05. Detailed Implementation

[0069] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0071] Example 1: Establishment of an SLE model mouse MRL / MpJ-Fas lpr Mice are a disease model of SLE, which is created by introducing Fas into the MRL / MpJ background strain. lprThe homozygous mutant mice exhibited SLE-like phenotypes, including systemic autoimmunity, extensive lymphadenopathy (associated with abnormal T cell proliferation), arthritis, and immune complex glomerulonephritis. From approximately 3 months of age, MRL / MpJ-Fas... lpr Mice showed significantly elevated levels of circulating immune complexes, exhibiting severe proliferative glomerulonephritis. Both sexes were affected, but female mice experienced a faster onset and more severe disease. MRL / MpJ-Fas lpr Mice can be used for organ injury research, as well as immunology, inflammation, and autoimmune studies, such as lupus, Sjögren's syndrome, and rheumatoid arthritis. By inserting an early transposon factor (ETn) into intron 2 of the Fas gene using gene editing technology, the presence of ETn leads to premature transcriptional termination. According to preliminary data provided by Cyagen Biosciences, visual observation reveals that 14-week-old MRL / MpJ-Fas mice... lpr The lymph nodes of the mice initially swelled, becoming severely swollen at 18 weeks of age. H&E staining results showed that, compared to the control group, the MRL / MpJ-Fas ratio at 26 weeks of age was significantly lower. lpr The mice's lymph nodes were noticeably swollen, enlarged in size, and darkened in color.

[0072] Select MRL / MpJ-Fas lpr Mice (Cyagen, C001194) were used as an animal model of systemic lupus erythematosus (SLE). Initially, the mice had normal weight, but lymph node swelling began to appear at 11 weeks of age, reaching 100% incidence by 15 weeks of age. In this example, 13-week-old female MRL / MpJ-Fas mice were used. lpr Mice were used as test animals.

[0073] MM-102 (Selleck, S7265) was used as the test drug. The MM-102 in vivo working solution was prepared fresh each time. For example, to obtain 1 mL of working solution, 50 μL of 100 mg / mL clear DMSO (Solarbio, D8317) stock solution was added to 400 μL of PEG300 (Selleck, S6702), and mixed thoroughly until clear. Then, 50 μL of Tween80 (Selleck, S6704) was added to the above system, and mixed thoroughly until clear. Finally, 500 μL of ddH2O was added to bring the volume to 1 mL.

[0074] Mice were randomly divided into 3 groups (n=6): normal control group (MRL / MpJ mice), MRL / MpJ-Fas mice, and control group (MRL / MpJ-Fas mice). lpr Model group and MRL / MpJ-Fas lpr+MM-102 treatment group. Mice in both the model group and the treatment group developed the disease at 13 weeks of age, with each 13-week-old mouse weighing 35-40g. Subsequently, the 13-week-old mice in both the model group and the treatment group underwent intervention treatment, and were sacrificed 4 weeks after intervention for subsequent experiments.

[0075] The normal control group received no intervention. The treatment group mice received two weekly intravenous injections of MM-102 working solution via tail vein (4 weeks of intervention, 8 injections in total), with a dose of 15 μg MM-102 / g mouse body weight, and were supplemented with physiological saline to a total of 200 μL. The model group mice received two weekly intravenous injections of 200 μL physiological saline via tail vein (4 weeks of intervention, 8 injections in total).

[0076] Example 2: Phenotypic observation of skin lesions, lymph nodes and spleen after MM-102 intervention Four weeks after intervention, facial skin lesions in mice from each group were observed and representative images were taken. After sacrifice, lymph nodes and spleens of mice from each group were removed, surrounding tissues were dissected, and representative images were taken against the same background. The lengths were measured with a ruler and quantitative statistical analysis was performed.

[0077] like Figure 1 As shown in Figure A, the facial skin of mice in the normal control group appeared basically normal, with no obvious skin damage observed; however, the model group mice showed more obvious skin damage on their faces, indicating that MRL / MpJ-Fas lpr Mice exhibited SLE-like extrinsic disease phenotypes at the appropriate age. Compared to the model group, the treated mice showed reduced facial skin lesions and improved skin appearance. These results indicate that MM-102 intervention can alleviate skin lesions in SLE model mice.

[0078] like Figure 1 As shown in Figure B, the lymph nodes in the model group mice were significantly larger than those in the normal control group, indicating lymph node enlargement in the model group mice. In contrast, the lymph nodes in the treatment group mice were smaller than those in the model group, and their appearance was closer to that of the normal control group. Quantitative results of lymph node length are shown below. Figure 1 As shown in Figure C, the lymph node length in the model group was higher than that in the normal control group; compared with the model group, the lymph node length in the MM-102 treatment group was reduced.

[0079] like Figure 1 As shown in Figure D, the spleen of the model group mice was significantly larger than that of the normal control group, indicating splenomegaly in the model group mice. After MM-102 intervention, the spleen size of the treatment group mice was smaller than that of the model group. Quantitative results of spleen length are shown below. Figure 1 As shown in Figure E, the spleen length in the model group was longer than that in the normal control group; compared with the model group, the spleen length in the MM-102 treatment group was shorter.

[0080] Lymph nodes and spleen are both important peripheral immune organs, and their enlargement is related to MRL / MpJ-Fas. lpr One of the phenotypes of systemic autoimmune abnormalities in mice. The above results indicate that MM-102 intervention can not only improve facial skin lesions in model mice, but also reduce lymph node enlargement and spleen enlargement, suggesting that MM-102 can improve the external disease phenotype and immune organ abnormalities in SLE model mice.

[0081] This embodiment demonstrates, from three aspects—skin phenotype, lymph node size, and spleen size—that MM-102 can alleviate related phenotypic abnormalities in SLE model mice, providing experimental evidence at the animal phenotypic level for the use of MM-102 to improve or treat SLE.

[0082] Example 3: Renal tissue morphology after MM-102 intervention H&E staining: Kidney tissue from mice in each group was fixed with 4% paraformaldehyde solution for 24 hours, then dehydrated with a gradient of ethanol (70%, 80%, 95%, 100%), cleared with xylene, and embedded in paraffin. The tissue was then cut into continuous sections with a thickness of 2-3 μm using a microtome. After mounting the sections on glass slides, they were dewaxed with xylene, rehydrated with a gradient of ethanol, and the nuclei were stained with Harris hematoxylin and eosin for 5-10 minutes. After thorough rinsing with tap water, the sections were differentiated with 1% hydrochloric acid ethanol for 3-5 seconds, and then blued again with tap water for 15-20 minutes. Subsequently, the cytoplasm was stained with 0.5% eosin ethanol solution for 30 seconds to 1 minute, dehydrated with a gradient of ethanol, cleared with xylene, and mounted with neutral resin.

[0083] like Figure 3 As shown in Figure A, H&E staining results indicated that the overall structure of the kidney tissue in the normal control group mice was relatively intact, with no obvious abnormalities observed in the glomeruli and tubulointerstitium. Compared with the normal control group, the kidney tissue of the model group mice showed significant pathological changes, including abnormal glomerular structure, increased or proliferative glomerular cells, and inflammatory cell infiltration in the renal interstitium, suggesting that the model group mice had developed SLE-related renal immune-inflammatory damage. Compared with the model group, the glomerular structural abnormalities in the treatment group mice were improved, and the glomerular proliferative lesions and inflammatory cell infiltration in the renal interstitium were reduced. These results indicate that MM-102 intervention can alleviate the pathological damage to the kidney tissue in SLE model mice.

[0084] PAS staining: PAS staining was performed using a PAS staining kit (Solarbio, G1280). Paraffin sections were routinely dewaxed to water, then oxidized in 0.5% periodic acid aqueous solution for 10-15 minutes. After thorough washing with distilled water, they were placed in Schiff's reagent in the dark for 15-30 minutes. The sections were then rinsed three times (2 minutes each time) with 0.5% sodium metabisulfite solution, followed by rinsing with distilled water. Subsequently, the cell nuclei were stained with Harris hematoxylin for 1-2 minutes, differentiated with hydrochloric acid and ethanol, and then treated with a blue reversion process. The sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. PAS-positive substances (glycoproteins, basement membrane, mesangial matrix) appeared purplish-red or magenta, while the cell nuclei appeared light blue.

[0085] like Figure 3 As shown in Figure B, PAS staining results indicated that the glomerular outlines and internal structures of mice in the normal control group were relatively clear, with no obvious abnormalities observed in the glomerular basement membrane and mesangial region. The model group mice exhibited glomerular structural disorder and pathological changes in the basement membrane and / or mesangial region, suggesting glomerular structural damage. Compared to the model group, the treatment group mice showed more intact glomerular structures and reduced abnormalities in the basement membrane and / or mesangial region. These results demonstrate that MM-102 can improve the pathological changes in glomerular-related tissue structures in SLE model mice.

[0086] Immunofluorescence staining: Kidney tissue from each group of mice was collected, and after removing the membrane, it was immediately placed in pre-chilled OCT embedding medium and flash-frozen in a liquid nitrogen-pre-chilled isopentane or dry ice-ethanol bath. The tissues were then transferred to a -80°C freezer for storage. Before sectioning, the embedded blocks were equilibrated for 30 minutes in a cryostat set at -20 to -25°C. After trimming, the tissue blocks were serially sectioned to a thickness of 5-8 μm. Sections were mounted on pre-chilled adhesive slides and allowed to air dry at room temperature for 30 minutes. Sections were fixed in pre-chilled 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes, washed with PBS, and then used for subsequent immunofluorescence staining. During staining, samples were sequentially incubated with Alexa Fluor 488-conjugated anti-mouse IgG antibody (Invitrogen, A11008), Actin primary antibody (Invitrogen, A57245), and secondary antibody. Finally, the slides were mounted with DAPI-containing mounting medium and placed on adhesive slides for imaging observation. The deposition of IgG in the kidneys of mice in each group was assessed by detecting fluorescence intensity.

[0087] like Figure 3As shown in Figure C, immunofluorescence staining results showed that: the IgG fluorescence signal in the kidney tissue of normal control mice was less or weaker; the kidney tissue of model mice showed more granular IgG fluorescence signal, indicating that there was obvious IgG deposition in the kidney tissue of model mice. This result could be corroborated by the pathological changes in the kidney tissue of model mice, suggesting that there was kidney damage related to immunoglobulin deposition; compared with the model group, the granular IgG fluorescence signal in the kidney tissue of treatment group mice was reduced, indicating that MM-102 intervention could reduce IgG deposition in the kidney tissue of SLE model mice.

[0088] H&E staining, PAS staining, and IgG immunofluorescence staining evaluated kidney damage from three aspects: general kidney morphology, changes in the glomerular basement membrane or mesangial region, and immunoglobulin deposition. The results corroborated each other: the model group mice exhibited both pathological changes in the glomerular and interstitial tissues and IgG deposition in the kidneys; after MM-102 intervention, all of these abnormalities were improved to varying degrees.

[0089] Therefore, the results of this embodiment indicate that MM-102 can reduce IgG deposition in the kidney tissue of SLE model mice and alleviate pathological damage to the kidney tissue, such as glomerular proliferative lesions, glomerular structural abnormalities, and interstitial inflammatory cell infiltration, suggesting that MM-102 has an ameliorative effect on SLE-related kidney damage (especially kidney damage accompanied by renal immunoglobulin deposition). In other words, MM-102 has a dual effect of immunosuppression and renal tissue protection in SLE model mice with lupus nephritis.

[0090] Example 4: Serum creatinine, anti-double-stranded DNA antibody (anti-dsDNA-IgG antibody), and antinuclear antibody (anti-ANA-IgG antibody) concentrations after MM-102 intervention The concentrations of anti-double-stranded DNA antibody (Anti-dsDNA IgG), antinuclear antibody (Anti-ANA IgG), and serum creatinine are used as serological indicators for systemic lupus erythematosus; in this embodiment, these three serological indicators were measured.

[0091] Thaw frozen serum samples on ice before the experiment. Prepare standards (concentration of 442 μmol / L or labeled value) and enzyme working solution (containing sarcosine oxidase, peroxidase, 4-aminoantipyrine, etc.) according to the kit instructions (Nanjing Jiancheng, C011-2-1). Set up standard wells, assay wells, and blank control wells in a 96-well plate. Add 6 μL of standard (standard well), serum sample to be tested (assay well), and double-distilled water (blank well) to each well in sequence; then add 180 μL of enzyme working solution and 60 μL of chromogenic reagent provided in the kit, and mix thoroughly. Incubate the reaction system at 37℃ for 10-15 minutes to allow creatinine to be converted to creatine under the catalysis of creatinine oxidase, then hydrolyzed to creatine by creatine oxidase, and finally oxidized by sarcosine oxidase to produce hydrogen peroxide, which reacts with the Trinder reaction system to form a red quinone compound. After incubation, immediately measure the absorbance (OD value) of each well at 546 nm using an ELISA reader and calculate the creatinine content. Each sample has 3 replicates.

[0092] Creatinine content (μmol / L) = (Measured OD value - Blank OD value) ÷ (Standard OD value - Blank OD value) × Standard concentration × Dilution factor.

[0093] Whole blood from each group of mice was collected and left at room temperature for 2 hours, then centrifuged at 2000 rpm for 20 minutes to separate the serum. The serum samples were aliquoted and stored at -80℃. Serum samples were thawed on ice before the experiment, and the anti-dsDNA-IgG and anti-ANA-IgG antibody kits (Beijing Viokai) were warmed to room temperature 1 hour in advance. The samples were diluted 1:3 using the sample diluent provided in the kit to ensure the antibody concentration fell within the linear range of the standard curve. Standard wells, sample wells, and blank wells were prepared. 50 μL of different concentrations of standard were added to each standard well, 50 μL of sample diluent was added to each blank well, and 50 μL of diluted sample was added to each sample well. 100 μL of horseradish peroxidase (HRP)-labeled anti-dsDNA-IgG or anti-ANA-IgG detection antibody was added to each well. The reaction wells were sealed with sealing film and incubated at 37℃ in a water bath or incubator for 45 minutes. Discard the liquid, blot dry on absorbent paper, add 350 μL of washing buffer to each well, let stand for 20 seconds, discard the washing buffer, blot dry on absorbent paper, and repeat the washing process 5 times. Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and measure the OD value of each well at 450 nm within 15 minutes.

[0094] like Figure 2As shown in Figure A, the serum anti-double-stranded DNA antibody level in the model group mice was higher than that in the normal control group, indicating that the model mice exhibited SLE-like serological manifestations with abnormally elevated anti-double-stranded DNA autoantibodies. Compared with the model group, the serum anti-double-stranded DNA antibody level in the MM-102 treatment group mice was lower, indicating that MM-102 intervention can improve the abnormally elevated anti-double-stranded DNA antibody status in SLE model mice.

[0095] like Figure 2 As shown in Figure B, the serum antinuclear antibody (ANA) level in the model group mice was higher than that in the normal control group, further indicating that the model mice had abnormal autoantibodies. After MM-102 intervention, the serum ANA level in the treatment group mice was lower than that in the model group, indicating that MM-102 can reduce the abnormally elevated ANA levels in SLE model mice.

[0096] like Figure 2 As shown in Figure C, the serum creatinine level in the model group mice was higher than that in the normal control group, indicating that the model mice had developed a certain degree of renal function abnormality. Compared with the model group, the serum creatinine level in the MM-102 treatment group mice was reduced. Combined with the results of reduced renal tissue pathological damage and IgG deposition in Example 3, it is shown that MM-102 can not only improve renal tissue morphology, but also improve renal function-related indicators in SLE model mice.

[0097] The decreased levels of anti-double-stranded DNA antibodies and antinuclear antibodies indicate that MM-102 can improve autoantibody abnormalities in model mice. The decreased serum creatinine levels and reduced renal histopathological changes further support the ameliorative effect of MM-102 on SLE-related kidney damage from both functional and histopathological perspectives. The results of this embodiment provide experimental evidence for the use of MM-102 to improve or treat SLE, especially for SLE with renal involvement.

[0098] Example 5: Changes in the proportion of subset cells in PBMCs and total spleen cells after MM-102 intervention Spleen tissue was collected from mice in each group. The tissue was ground and rinsed on a clean bench, then filtered through a 70 μm filter with a 50 mL centrifuge tube placed underneath. During grinding, the tissue was rinsed with PBS containing 0.5% BSA. The resulting tissue fluid was collected, and erythrocytes in the spleen tissue were lysed using erythrocyte lysis buffer (Applygen Technologies, C1311). The mixture was centrifuged at 1500 rpm for 6 minutes, and the supernatant was discarded. The resulting precipitate was the total spleen cells.

[0099] Mouse peripheral blood mononuclear cells were isolated using Lymphoprep (STEMCELL, 18060) density gradient centrifugation buffer. Anticoagulated whole blood (EDTA or heparin anticoagulation) was collected and diluted 1:1 with sterile PBS (pH=7.2) + 2% fetal bovine serum (FBS). In a 15 mL centrifuge tube (STEMCELL, 86415), an equal volume of Lymphoprep separation buffer was added to the diluted blood. The diluted blood was then carefully spread along the tube wall onto the surface of the Lymphoprep buffer, ensuring a clear interface. The centrifuge tube was placed in a horizontal rotor centrifuge and centrifuged at 800×g for 20 minutes at room temperature (centrifuge set to low acceleration and no brake to prevent disruption of the separation interface). After centrifugation, four layers were observed: the upper layer consisted of plasma and diluent; the middle layer contained mononuclear cells (white membrane layer); and the lower layer contained the separation buffer and erythrocyte and granulocyte sediments. Carefully aspirate the white membrane cells using a sterile pipette and transfer them to a new centrifuge tube. Resuspend the cell pellet in complete culture medium (RPMI-1640 containing 10% FBS). The separated PBMCs can be used directly for subsequent flow cytometry.

[0100] PBMCs and total spleen cells were surface-stained using fluorescently labeled antibodies. The Treg antibodies included Zombie Yellow (Biolegend, 423103), FITC anti-mouse CD4 antibody (Biolegend, 100406), PE anti-mouse CD25 antibody (Invitrogen, 12-0251-82), CD45 antibody (Biolegend, 103128), and TCRαβ antibody (Invitrogen, 47-5961-82); Th17 antibodies included Zombie Yellow, CD4, CD45, and TCRαβ.

[0101] Cell membrane and cytoplasmic staining: Five hours before staining, a protein transport inhibitor containing brifidin A (Biolegend, 423303) was added to inhibit cytokine secretion. Cells were then stained with Zombie Yellow, CD4, CD45, and TCRαβ antibodies. Cells were permeabilized and fixed with IC50 fixation buffer (eBioscience, 00-8222-49), stained with IL-17 (Invitrogen, 12-7177-81), and then CD4+ was detected by flow cytometry. + IL-17A + Th17 cell subset.

[0102] Nuclear staining: For nuclear staining, samples were treated with fixation / permeabilization concentrate / dilution buffers (eBioscience, 00-5123-43 and 00-5223-56) for Foxp3 (Invitrogen, 48-5773-82) and RORγt (Invitrogen, 25-6981-82) staining. After permeabilization and fixation, CD4+ was detected by flow cytometry. + RORγt + Th17 subgroup ratio and CD4 + CD25 + FoxP3 + Treg subgroup proportions.

[0103] Following the flow cytometry analysis described above, lymphocyte gating, removal of adherent cells, and removal of dead cells were performed sequentially using Flowjo 10.4.0 software before CD4 analysis. + RORγt + or CD4 + IL-17A + Th17 subgroup ratio and CD4 + CD25 + FoxP3 + Treg subgroup proportions.

[0104] like Figure 4 A and Figure 4 As shown in B, compared with the normal control group, the CD4+ level in the peripheral blood of mice in the model group was significantly lower. + RORγt + The increased proportion of CD4+ cells indicates an abnormal increase in the peripheral blood of model mice associated with Th17 cell differentiation. After MM-102 intervention, the peripheral blood of mice in the treatment group showed an increase in CD4+. + RORγt + The proportion of RORγt-positive cells was lower in the model group than in the treatment group. The proportion of RORγt-positive cells in the peripheral blood of mice in the model group was 5.99-7.94%, while it was 3.75-4.64% in the treatment group. This result indicates that MM-102 can reduce the abnormally elevated CD4+ levels in the peripheral blood of SLE model mice. + RORγt + Cell ratio.

[0105] like Figure 4 C and Figure 4 As shown in Figure D, when IL-17A was used as a detection marker, the level of CD4 in the peripheral blood of mice in the model group was [missing information]. + IL-17A + The proportion of cells was higher in the treatment group than in the normal control group; after MM-102 intervention, the CD4 count in the treatment group was higher. + IL-17A +The proportion of cells was lower than that in the model group. For example... Figure 4 E and Figure 4 As shown in Figure F, there was no significant difference in the proportion of CD4+CD25+Foxp3+Treg cells in the peripheral blood of mice in the normal control group, model group, and MM-102 treatment group. This result indicates that, at the dosage and intervention time used in this embodiment, MM-102 did not significantly affect the proportion of peripheral blood Treg cells.

[0106] like Figure 5 A and Figure 5 As shown in B, compared with the normal control group, CD4 in the spleen of model group mice was significantly lower. + RORγt + The increased proportion of Th17-related cell subsets suggests an abnormal increase in the spleen of model mice. After MM-102 intervention, the CD4+ cell count in the spleen of the treatment group mice was significantly reduced. + RORγt + The proportion of RORγt-positive cells was reduced. In the model group mice, the proportion of RORγt-positive cells in the spleen was approximately 9.93-16.70%, while in the treatment group it was 6.53-7.95%. These results indicate that MM-102 can reduce the abnormally elevated CD4+ levels in the spleen of SLE model mice. + RORγt + Cell ratio.

[0107] like Figure 5 C and Figure 5 As shown in Figure D, when IL-17A was used as the detection marker, CD4 in the spleen of the model group mice was... + IL-17A + The proportion of cells was higher in the treatment group than in the normal control group; while the CD4 count in the treatment group was higher. + IL-17A + The proportion of cells was lower than that in the model group. This result is consistent with the RORγt detection results, reflecting the effect of MM-102 on the Th17-related cell subsets in the spleen from another perspective.

[0108] like Figure 5 E and Figure 5 As shown in F, compared with the normal control group, CD4 in the spleen of model group mice was significantly lower. + CD25 + FoxP3 + The proportion of Treg cells was reduced; after MM-102 intervention, the proportion of Treg cells in the spleen of mice in the treatment group was increased compared with that in the model group. MM-102 can increase the abnormally reduced proportion of Treg cells in the spleen of SLE model mice.

[0109] This embodiment demonstrates that MM-102 can reduce the proportion of Th17-related cells in the peripheral blood and spleen of SLE model mice and can regulate the proportion of Treg cells in the spleen. Combining the results of Examples 2 to 4, the effects of MM-102 on model mice are not only manifested in improving skin lesions, lymphadenopathy, and splenomegaly, but also in reducing autoantibody levels, decreasing renal IgG deposition, alleviating renal tissue pathological damage, and improving abnormal immune cell subsets.

[0110] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of the compound in the preparation of products for improving or treating systemic lupus erythematosus, said compound being of formula I: 。 2. A product for improving or treating systemic lupus erythematosus, characterized in that, include: Compounds and pharmaceutically acceptable excipients; said compounds are shown in Formula I: 。 3. The product according to claim 2, characterized in that, The dosage forms of the product include oral or injectable formulations.

4. The product according to claim 2, characterized in that, The products also include at least one of the following: hydroxychloroquine, glucocorticoids, immunosuppressants, biologics, and small molecule targeted drugs.

5. Use of the product according to any one of claims 2-4 in the preparation of a medicament for improving or treating systemic lupus erythematosus.

6. The use according to claim 1 or 5, characterized in that, The systemic lupus erythematosus (SLE) mentioned refers to any one of the following: active SLE, severe SLE, cutaneous SLE, positive anti-double-stranded DNA antibody, and positive antinuclear antibody. Alternatively, the systemic lupus erythematosus is systemic lupus erythematosus with at least one manifestation of skin involvement, kidney involvement, lymphadenopathy, splenomegaly, abnormal autoantibodies, or imbalance of immune cell subsets.

7. The use according to claim 6, characterized in that, The active phase of systemic lupus erythematosus is defined as a SLEDAI-2000 score ≥ 6.

8. The use according to claim 6, characterized in that, The kidney involvement includes at least one of lupus nephritis, renal immune complex deposition, glomerular proliferative lesions, glomerular basement membrane or mesangial region damage, renal interstitial inflammatory cell infiltration, or renal dysfunction.

9. The use according to claim 6, characterized in that, The autoantibody abnormalities include elevated levels of anti-double-stranded DNA antibodies and / or antinuclear antibodies; the immune cell subset imbalances include at least one of an increased proportion of Th17 cells, a decreased proportion of Treg cells, or a Th17 / Treg immune imbalance.

10. A method for reducing the proportion of the Th17 subset in peripheral blood mononuclear cells and / or spleen cells of patients with systemic lupus erythematosus in vitro, characterized in that, Peripheral blood mononuclear cells or spleen cells from patients with systemic lupus erythematosus were cultured or treated with the compound shown in Formula I. 。

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