COMPOSITION FOR PREVENTING OR TREATING LUPUS COMPRISING A RECOMBINANT STABILIZED GALECTIN-9 PROTEIN - Patent application

Abstract

The present invention relates to a composition for preventing or treating lupus or glomerulonephritis, comprising a recombinant stabilized galectin-9 protein. Specifically, the recombinant stabilized galectin-9 protein of the present invention was shown to be safe in an animal model of systemic lupus erythematosus (SLE), and was confirmed to reduce skin lesions, lymphadenopathy, and proteinuria caused by lupus, alleviate lupus nephritis and glomerulonephritis, and reduce plasma anti-dsDNA antibody concentrations. Therefore, the recombinant stabilized galectin-9 protein of the present invention can be effectively used as an active ingredient in a composition for preventing or treating lupus or glomerulonephritis.

Description

[Technical Field]

[0001] The present invention relates to a recombinant stabilized galectin-9 protein and uses thereof, and more particularly to a composition for preventing or treating lupus or glomerulonephritis, which contains said protein as an active ingredient. [Background technology]

[0002] It has been discovered that animal lectins exist in living organisms that specifically recognize sugar chains with β-galactoside structures, and at least 14 genes have been identified to date. Galectins are classified into prototype, chimera, and tandem repeat types based on their structure.

[0003] Galectin-9, a type of tandem repeat galectin, consists of two carbohydrate recognition domains (CRDs) and a linker peptide region connecting them. The N-terminal carbohydrate recognition domain (NCRD) and the C-terminal carbohydrate recognition domain (CCRD) are linked via this linker peptide region, and galectin-9 has been reported to exhibit various biological activities. In T cells, galectin-9 binds to Tim-3, induces apoptosis of Tim-3-positive Th1 cells, and inhibits excessive Th1 responses, thereby suppressing autoimmune inflammation.

[0004] To utilize galectin-9 as a therapeutic agent, research is underway to address the following issues: 1) protease sensitivity, 2) low solubility, and 3) low yield. Evolutionary efforts have been made to enhance protease resistance, such as by creating a galectin-9 variant (G9Null) in which the linker peptide of galectin-9 has been truncated.

[0005] Lupus, on the other hand, is an autoimmune disease associated with antibodies that attack connective tissue and are associated with the production of circulating immune complexes, such as antinuclear antibodies, and activation of the complement system. Generally, the term "lupus" primarily refers to systemic lupus erythematosus (SLE). This disease is systemic in nature, affecting all organ systems and potentially causing severe tissue damage. Lupus patients produce autoantibodies with specificity against anti-DNA, anti-Ro, and antiplatelets, and may develop symptoms such as glomerulonephritis, arthritis, serositis, complete heart block in newborns, or hematological abnormalities.

[0006] Left untreated, lupus not only affects the skin and joints, but also attacks and harms internal organs such as the lungs, heart, and kidneys, potentially leading to life-threatening consequences. Kidney disease is of greatest concern. Kidney damage, measured by the amount of protein in the urine, is one of the acute sites of injury associated with lupus pathogenesis and accounts for more than 50% of lupus-related morbidity and mortality.

[0007] Currently, there is no definitive cure for lupus. From a practical standpoint, physicians typically administer high-dose corticosteroids, prednisone, or various immunosuppressive drugs such as azathioprine or cyclophosphamide. However, many of these drugs have significant drawbacks, including potentially harmful side effects in treated patients.

[0008] Therefore, the present inventors have attempted to develop a safe therapeutic agent for lupus. As a result, they have succeeded in producing a recombinant stabilized galectin-9 protein in which the C-terminal carbohydrate recognition domain (CCRD) and amino acids in the linker peptide of conventional wild-type galectin-9 have been deleted and substituted. The safety of this recombinant stabilized galectin-9 protein was confirmed in an in vivo animal model of systemic lupus erythematosus. Furthermore, in an in vivo animal model of systemic lupus erythematosus, the recombinant stabilized galectin-9 protein was confirmed to reduce lupus-associated skin lesions, lymphadenopathy, and proteinuria, alleviate lupus nephritis and glomerulonephritis, and reduce plasma anti-dsDNA antibody concentrations. These findings demonstrated that the recombinant stabilized galectin-9 protein can be effectively used as an active ingredient in compositions for preventing or treating lupus or glomerulonephritis, leading to the filing of the present application. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korea Patent No. 10-2019-0060550 [Non-patent literature]

[0010] [Non-Patent Document 1] Nishi, N et al. Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins. FEBS Lett. 2005 Apr 11;579(10):2058-64 [Non-patent document 2] Wang, Y., Xiao, S., Xia, Y. et al. The Therapeutic Strategies for SLE by Targeting Anti-dsDNA Antibodies. Clinic Rev Allerg Immunol (2021). https: / / doi.org / 10.1007 / s12016-021-08898-7 Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a composition for preventing or treating lupus or glomerulonephritis, comprising a recombinant stabilized galectin-9 protein. [Means for solving the problem]

[0012] To achieve the objectives of the present invention, the present invention provides: a pharmaceutical composition for preventing or treating lupus or glomerulonephritis, comprising as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a method for preventing or treating lupus or glomerulonephritis, comprising administering to a subject a pharmaceutical composition comprising a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for use in the prevention or treatment of lupus or glomerulonephritis; and use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for the manufacture of a composition for preventing or treating lupus or glomerulonephritis.

[0013] The present invention also provides a functional health food composition for preventing or ameliorating lupus or glomerulonephritis, which comprises as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for use in preventing or ameliorating lupus or glomerulonephritis; and use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for the manufacture of a functional health food composition for preventing or ameliorating lupus or glomerulonephritis. [Effects of the Invention]

[0014] The recombinant stabilized galectin-9 protein of the present invention was shown to be safe in an animal model of systemic lupus erythematosus (SLE), and was confirmed to reduce skin lesions, lymphadenopathy, and proteinuria caused by lupus, alleviate lupus nephritis and glomerulonephritis, and reduce the concentration of anti-dsDNA antibodies in the blood. Therefore, it can be effectively used as an active ingredient in a composition for preventing or treating lupus or glomerulonephritis. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows changes in body weight in an in vivo model of systemic lupus erythematosus (SLE) administered with the recombinant stabilized galectin-9 protein (sGal-9) of the present invention. [Figure 2] FIG. 1 shows the severity of skin lesions scored in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 3] FIG. 1 shows changes in lymphadenopathy scored in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 4]FIG. 1 shows the degree of proteinuria scored in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 5] FIG. 1 shows changes in spleen weight in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 6] FIG. 1 shows histological changes in the kidney in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 7] FIG. 1 shows the degree of IgG deposition in the kidney in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. [Figure 8] FIG. 1 shows the concentration of anti-dsDNA antibodies in plasma in an in vivo model of systemic lupus erythematosus (SLE) administered with sGal-9 of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

[0016] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Therefore, the embodiments of the present invention may be modified in various ways, and the scope of the present invention is not limited to the embodiments described below.

[0017] Throughout the specification of the present invention, when a part is described as "comprising" certain components, this does not mean that other components are excluded, but that other components may additionally be included unless expressly stated otherwise.

[0018] The present invention provides: a pharmaceutical composition for preventing or treating lupus or glomerulonephritis, which comprises as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a method for preventing or treating lupus or glomerulonephritis, which comprises administering to a subject a pharmaceutical composition comprising a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for use in the prevention or treatment of lupus or glomerulonephritis; and use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for the manufacture of a composition for preventing or treating lupus or glomerulonephritis.

[0019] In the present invention, the recombinant stabilized galectin-9 protein can have the effect of preventing or treating lupus or glomerulonephritis without causing side effects such as weight change.

[0020] The term "prevention" as used herein refers to any action of suppressing or delaying the onset of a disease by administering a composition.

[0021] As used herein, the term "treatment" refers to any action in which the symptoms of said disease are ameliorated or beneficially altered by administering a composition.

[0022] In the present invention, the recombinant stabilized galectin-9 protein is a protein that retains the sugar chain recognition activity of wild-type galectin-9 and has a molecular structure that is more stable against proteases.

[0023] Specifically, the recombinant stabilized galectin-9 protein is a recombinant protein produced by modifying a CCRD, which comprises a linker region linking two glycan-recognition domains (CRDs) of wild-type galectin-9 and a C-terminal glycan-recognition domain, having an NCRD-linker-CCRD structure. More specifically, the recombinant stabilized galectin-9 protein is constructed by deleting the entire peptide in the linker region, deleting the amino acid sequence from positions 1 to 10 (SEQ ID NO: 3) in CCRD (SEQ ID NO: 2), and substituting alanine (A) at position 13 with proline (P), resulting in the amino acid sequence set forth in SEQ ID NO: 1. The protein may comprise an amino acid sequence having at least 75%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, the recombinant stabilized galectin-9 protein may further comprise a targeting sequence, a tag, a labeling residue, or an amino acid sequence specifically designed to increase half-life or peptide stability.

[0024] Furthermore, the recombinant stabilized galectin-9 protein may contain a deletion of the first amino acid residue from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1, and specifically, may consist of the amino acid sequence set forth in SEQ ID NO: 4.

[0025] The term "polynucleotide" as used herein refers to a polymer of nucleotides that functions to transmit genetic information. For the purposes of the present invention, a polynucleotide may comprise a sequence that encodes the recombinant protein of SEQ ID NO: 1 and has at least 75%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to the polynucleotide sequence encoding said recombinant protein.

[0026] The term "homology" as used herein is intended to indicate the degree of similarity to a wild-type amino acid sequence or polynucleotide sequence, and such homology comparison can be performed using comparison programs widely known in the art. The homology between two or more sequences can be calculated as a percentage (%).

[0027] The term "lupus," as used herein, refers to an antibody-mediated autoimmune disease that attacks connective tissue and includes chronic inflammatory autoimmune diseases characterized by the presence of autoantibodies, rash, oral ulcers, serositis, neuropathy, low blood counts, and joint pain and swelling. Unless otherwise specified, the term "lupus" as used herein has its conventional meaning as used in the art to which the present invention pertains. In the present invention, lupus includes various additional forms of lupus, such as, but not limited to, systemic lupus erythematosus (SLE), systemic lupus, discoid lupus, drug-induced lupus, or neonatal lupus. Chronic nephritis, such as lupus nephritis or glomerulonephritis, can also be caused by lupus.

[0028] The systemic lupus erythematosus (SLE) referred to in this invention is also known as systemic lupus or systemic lupus erythematosus and has the usual meaning as used in the art to which the invention pertains. SLE is a multi-organ autoimmune disease in which antinuclear antibodies, including anti-dsDNA antibodies, are produced, forming antigen-antibody immune complexes that deposit in small blood vessels. These deposits can cause inflammation and damage to multiple organs, including the skin and kidney basement membrane.

[0029] Discoid lupus is a disease limited to the skin. It usually presents as a rash on the face, neck, hands, and feet. While the affected areas are less widespread than systemic lupus, symptoms are generally severe and can lead to pigmentation and scarring if not treated appropriately. Approximately 10% of patients with discoid lupus may subsequently develop systemic lupus, and 10-20% of patients with systemic lupus may present with discoid skin lesions.

[0030] Drug-induced lupus occurs after the administration of certain drugs and presents with symptoms similar to those seen in systemic lupus erythematosus, such as fever, rash, and joint pain. The most commonly involved drugs are hydralazine, a drug for treating hypertension, and procainamide, an antiarrhythmic drug.

[0031] In the present invention, the pharmaceutical composition can prevent or treat lupus by alleviating skin lesions, lymphadenopathy, or proteinuria caused by lupus, or by reducing the concentration of anti-dsDNA antibodies in the blood.

[0032] In addition, the pharmaceutical composition can prevent or treat lupus nephritis or glomerulonephritis by inhibiting mesangial proliferation, reducing the infiltration of inflammatory cells in the glomeruli, and reducing the infiltration of immune complexes into the kidney.

[0033] In a specific embodiment of the present invention, the present inventors prepared a recombinant stabilized galectin-9 protein (sGal-9) in which the amino acids of the C-terminal glycan recognition domain (CCRD) and the linker peptide of the two glycan recognition sites of wild-type galectin-9 were deleted and substituted.

[0034] Furthermore, the present inventors confirmed that repeated administration of sGal-9 did not affect body weight in an in vivo model of systemic lupus erythematosus.

[0035] Furthermore, the present inventors have confirmed that sGal-9 reduces skin lesions, inhibits lymphadenopathy, and reduces the amount of protein in urine in an in vivo model of systemic lupus erythematosus, thereby alleviating clinical symptoms of lupus.

[0036] Furthermore, the present inventors have confirmed that sGal-9 inhibits mesangial proliferation and reduces inflammatory cell infiltration within and around the glomerulus, as well as immune complex infiltration into the kidney, thereby exhibiting therapeutic effects against lupus nephritis and glomerulonephritis in an in vivo model of systemic lupus erythematosus.

[0037] Furthermore, the present inventors confirmed that sGal-9 reduces the plasma concentration of anti-dsDNA antibodies in an in vivo model of systemic lupus erythematosus.

[0038] Therefore, the present inventors have confirmed that the recombinant stabilized galectin-9 protein according to the present invention is safe in an animal model of systemic lupus erythematosus, alleviates clinical symptoms of lupus, lupus nephritis, and glomerulonephritis, and reduces the concentration of anti-dsDNA antibodies in the blood. Therefore, the recombinant stabilized galectin-9 protein according to the present invention or a polynucleotide encoding it can be advantageously used as an active ingredient in a pharmaceutical composition for preventing or treating lupus or glomerulonephritis.

[0039] In the present invention, the recombinant stabilized galectin-9 protein of the present invention or the polynucleotide encoding it may be carried in a pharmaceutically acceptable carrier such as a colloidal suspension, a powder, saline, a lipid, a liposome, a microsphere, or a nanospherical particle, which may be complexed with or associated with a delivery vehicle and can be delivered in vivo using carrier systems known in the art, including lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0040] Pharmaceutically acceptable carriers include, but are not limited to, those commonly used in pharmaceutical preparations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the formulation may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0041] In the present invention, the pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) depending on the purpose, and the dosage will vary depending on the condition and body weight of the patient, the severity of the disease, the dosage form, the route of administration, the timing of administration, etc., but can be appropriately selected by those skilled in the art.

[0042] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts.

[0043] As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. Effective dosage levels are determined based on the type and severity of the patient's disease, the activity of the drug, the patient's sensitivity to the drug, the time and route of administration, the rate of excretion, the duration of treatment, concurrently used drugs, and other factors well known in the medical arts. The pharmaceutical compositions of the present invention can be used as monotherapy or in combination with surgery, hormonal therapy, drug therapy, or biological response modifiers. They can be administered simultaneously, separately, or sequentially with the aforementioned formulations, and in single or multiple doses. Taking all of these factors into consideration, it is important to administer the minimum amount necessary to achieve maximum efficacy without side effects, which can be easily determined by those skilled in the art.

[0044] Specifically, the effective amount of the pharmaceutical composition of the present invention varies depending on the patient's age, sex, condition, weight, systemic absorption rate, inactivation rate, excretion rate of the active ingredient, type of disease, concomitant medications, etc., and can be increased or decreased depending on the administration route, degree of obesity, sex, weight, age, etc.

[0045] In the present invention, the pharmaceutical composition may be formulated into a dosage form selected from the group consisting of tablets, capsules, injections, lozenges, powders, granules, liquids, suspensions, oral liquids, emulsions, syrups, suppositories, vaginal tablets, and pills, but is not limited thereto, and may be formulated into an appropriate dosage form as needed. Furthermore, when formulating the composition, it is usually prepared using diluents or excipients commonly used in the art, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0046] Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and lozenges. These solid formulations are prepared by mixing one or more recombinant proteins of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups, which may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0047] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories and the like.

[0048] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerol, gelatin, etc.

[0049] The present invention also provides a functional health food composition for preventing or ameliorating lupus or glomerulonephritis, which comprises as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it; a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for use in preventing or ameliorating lupus or glomerulonephritis; and use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding it for the manufacture of a functional health food composition for preventing or ameliorating lupus or glomerulonephritis.

[0050] In the present invention, the details regarding the recombinant stabilized galectin-9 protein and lupus are the same as those described above, and therefore, the detailed description thereof is incorporated herein by reference.

[0051] The term "amelioration" as used in the present invention refers to any action that at least reduces a parameter related to the condition being treated, such as the severity of symptoms. In this case, the health functional food composition can be used for the prevention or amelioration of a disease, either before or after the onset of the disease, simultaneously with or separately from a therapeutic agent for treating the disease.

[0052] Meanwhile, in the present invention, the recombinant stabilized galectin-9 protein according to the present invention was shown to be safe in an animal model of systemic lupus erythematosus, and was confirmed to alleviate clinical symptoms of lupus, such as lupus nephritis and glomerulonephritis, and to reduce the concentration of anti-dsDNA antibodies in the blood. The recombinant stabilized galectin-9 protein according to the present invention or a polynucleotide encoding it can be effectively used as an active ingredient in a health functional food composition for preventing or ameliorating lupus.

[0053] In the functional health food of the present invention, the active ingredient may be added directly to the food, or may be used in combination with other foods or food ingredients, or may be used appropriately according to conventional methods. The amount of the active ingredient to be mixed can be determined appropriately depending on the purpose of use (for prevention or improvement). Generally, in the production of foods or beverages, the functional health food of the present invention can be added in an amount of preferably 15% by weight or less, more preferably 10% by weight or less, based on the raw materials. However, when taking it for long-term health hygiene or health regulation purposes, an amount less than the above range may be used.

[0054] In addition to the active ingredients, the health functional food of the present invention may contain other essential ingredients without limitation. For example, similar to conventional beverages, various flavoring agents or natural carbohydrates may be added as additional ingredients. Examples of natural carbohydrates include conventional sugars such as monosaccharides (e.g., glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, etc.); and polysaccharides (e.g., dextrin, cyclodextrin), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. In addition, thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.), natural flavoring agents, and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents. The proportion of the natural carbohydrates can be determined appropriately at the discretion of those skilled in the art.

[0055] The health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated drinks, etc. These ingredients may be used alone or in combination, and the proportions of such additives can also be appropriately selected by those skilled in the art.

[0056] The present invention will be described in detail below through production examples and experimental examples. However, the following production examples and experimental examples are provided to aid in understanding the present invention and are not intended to limit the scope of the present invention.

[0057] <Production Example 1> Production of recombinant stabilized galectin-9 protein (sGal-9) An expression vector containing a gene encoding a recombinant stabilized galectin-9 protein having the amino acid sequence of SEQ ID NO: 1 was constructed and introduced into Escherichia coli (E. coli) using the heat shock method. Expression of the recombinant protein was induced by culturing the E. coli in LB medium containing 50 μg / mL kanamycin and adding arabinose when the absorbance at 600 nm reached 0.7. The cells in which expression of the recombinant protein was induced were then lysed and filtered, and the target protein was captured using cation exchange chromatography and affinity columns. As a result, highly pure recombinant stabilized galectin-9 protein was obtained in high yield.

[0058] <Experimental Example 1> Safety evaluation of sGal-9 in an in vivo model of systemic lupus erythematosus (SLE) To evaluate the efficacy of sGal-9 in lupus, the safety of sGal-9 was evaluated after administration of the sGal-9 of the present invention to an in vivo model of systemic lupus erythematosus (SLE).

[0059] <1-1> Creation of an in vivo model of systemic lupus erythematosus and administration of sGal-9 To evaluate the efficacy of sGal-9 in lupus, an in vivo model of systemic lupus erythematosus was created and sGal-9 of the present invention was administered.

[0060] Specifically, MRL / FAS lprMice are generally known to be affected by MRL-lpr or lpr mutations. These mice are homozygous for the Faslpr spontaneous lymphoproliferative mutation and exhibit systemic autoimmunity, extensive lymphadenopathy associated with abnormal T cell proliferation, arthritis, and immune complex-mediated glomerulonephritis. In these mice, circulating immune complex levels significantly increase from approximately 3 months of age, and lymph node proliferation in MRL / Mp-Faslpr / Faslpr mice is approximately 75-fold greater than that in control mice. Furthermore, extensive renal pathological differences appear between 4 and 7 months of age, making this model useful for evaluating the therapeutic efficacy of systemic lupus erythematosus (SEL). Therefore, we used 11-week-old male MRL / Fas lpr Mice were purchased from Chuo Experimental Animals Co., Ltd. and used as an in vivo model of systemic lupus erythematosus. lpr Mice were housed and experiments were performed in a specific pathogen-free (SPF) animal facility maintained at a temperature of 21–23°C and a relative humidity of 40–45%. Experimental rodents were provided with Envigo rodent chow ad libitum.

[0061] The 11-week-old male MRL / FAS lpr As shown in Table 1 below, mice were randomly divided into four groups: a vehicle-treated negative control group, a mycophenolate-treated positive control group, an sGal-92 mg / kg treatment group (sGal-92 mg / kg), and an sGal-94 mg / kg treatment group (sGal-94 mg / kg). The negative control group received subcutaneous vehicle once a week. The positive control group received oral administration of mycophenolic acid (Roche), used in the treatment of lupus nephritis, at a dose of 60 mg / kg once daily. The sGal-92 mg / kg and sGal-94 mg / kg treatment groups received subcutaneous administration of sGal-9, prepared as described in Preparation Example 1 above, at doses of 2 mg / kg and 4 mg / kg, respectively, once a week. Each test substance was administered for a total of 9 weeks.

[0062] [Table 1]

[0063] Statistical analyses were also performed using SPSS based on the blinded evaluation data for each evaluation parameter between the negative control group and the test group, or between two test groups. Comparisons between two groups were performed using Student's t-test or Mann-Whitney U test. Repeated-measures ANOVA with Turkey's post hoc decision was performed to compare differences between treatment groups at multiple time points. The significance level was set at p = 0.05 or less.

[0064] <1-2> Body weight assessment in an in vivo model of systemic lupus erythematosus To evaluate the safety of the sGal-9 of the present invention in an in vivo model of systemic lupus erythematosus, the body weight of the experimental animals in Experimental Example <1-1> was measured.

[0065] Specifically, according to the method described in the above Experimental Example <1-1>, the body weight was measured during the 9-week repeated administration of the test substance.

[0066] As a result, as shown in Figure 1, it was confirmed that there were no statistically significant changes in body weight in the negative control group (vehicle), the positive control group (mycophenolic acid), the sGal-92 mg / kg administration group (sGal-92 mg / kg), and the sGal-94 mg / kg administration group (sGal-94 mg / kg).

[0067] Experimental Example 2: Evaluation of clinical parameters after sGal-9 administration in an in vivo model of systemic lupus erythematosus To evaluate the efficacy of sGal-9 of the present invention in an in vivo model of systemic lupus erythematosus, clinical indices were evaluated in the experimental animals of the above Experimental Example <1-1>.

[0068] Specifically, the experimental animals in Experimental Example <1-1> were periodically observed for the occurrence and severity of lupus from the time of grouping until the end of the experiment, and were evaluated according to the criteria shown in Table 2 below.

[0069] [Table 2]

[0070] After the experiment, the experimental animals in Experimental Example <1-1> were sacrificed, and the spleens were removed and weighed. As a result, as shown in Figure 2, it was confirmed that the positive control group (mycophenolic acid) and the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) had reduced skin lesions compared to the negative control group (vehicle). In particular, the reduction in skin lesions was more significant in the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) compared to the positive control group (mycophenolic acid).

[0071] Furthermore, as shown in Figure 3, the positive control group (mycophenolic acid) and the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) showed reduced lymphadenopathy scores compared with the negative control group (vehicle). In particular, the inhibitory effect on lymphadenopathy was more pronounced in the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) compared with the positive control group (mycophenolic acid).

[0072] Furthermore, as shown in Figure 4, it was confirmed that proteinuria was reduced in the positive control group (mycophenolic acid) and the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) compared to the negative control group (vehicle).

[0073] Furthermore, as shown in FIG. 5, it was confirmed that the sGal-92 mg / kg administration group had a decreased spleen weight compared to the negative control group (vehicle).

[0074] <Experimental Example 3> Histomorphological evaluation after sGal-9 administration in an in vivo model of systemic lupus erythematosus To evaluate the efficacy of sGal-9 of the present invention in an in vivo model of systemic lupus erythematosus, hematoxylin and eosin (H&E) staining and IgG immunofluorescence staining were performed to examine histological changes in the kidney tissue of the experimental animals in Experimental Example <1-1>.

[0075] Specifically, after the experiment, the experimental animals in Experimental Example <1-1> were sacrificed and kidney tissues were collected. The collected kidney tissues were fixed in 10% formaldehyde, dehydrated, and then embedded in paraffin. The fixed kidney tissues were sectioned into 2 μm thick sections and stained with hematoxylin and eosin (H&E). The sections were covered with a coverslip and observed under an optical microscope at 400x magnification. Four areas of each tissue were photographed, and two or more researchers scored them based on the items and criteria for histomorphological evaluation listed in Table 3 below. The average score of the researchers' scores was determined, and the average of the scores of the four areas was calculated as the final score for each tissue.

[0076] [Table 3]

[0077] The collected kidney tissue was frozen, sectioned at 4 μm thickness, fixed in cold acetone for 5 minutes, and washed twice in phosphate-buffered saline (PBS) for 5 minutes each. To eliminate nonspecific reactions, the sections were blocked for 30 minutes with a PBS solution containing 1% bovine serum albumin and 0.05% Tween-20. The sections were then incubated with FITC-conjugated goat anti-mouse IgG antibody (1:200, AP308F, Merck Millipore) at room temperature for 1 hour. After three 5-minute washes with PBS, nuclear staining was performed with DAPI, and the sections were mounted on a coverslip using mounting medium. The samples were then observed using a confocal laser scanning microscope (C2 Plus, Nikon). As shown in Figure 6, the negative control group (vehicle) showed clear signs of glomerulonephritis, including mesangial proliferation, infiltration of inflammatory cells within and around the glomerulus, and tubular destruction. In contrast, the positive control group (mycophenolic acid) and the sGal-9-treated groups (sGal-92 mg / kg and sGal-94 mg / kg) showed reduced glomerular inflammatory cell infiltration, and the glomerular lesion score was also reduced in the positive control group (mycophenolic acid) and the sGal-9-treated groups (sGal-92 mg / kg and sGal-94 mg / kg) compared with the negative control group (vehicle).

[0078] Furthermore, as shown in Figure 7, the positive control group (mimicophenolic acid) and the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) showed reduced IgG deposition compared with the negative control group (vehicle).

[0079] From the above results, it was confirmed that sGal-9 of the present invention exhibits therapeutic effects on lupus nephritis and glomerulonephritis by reducing inflammatory cell infiltration within and around the glomerulus and immune complex deposition in the kidney.

[0080] Experimental Example 4: Analysis of plasma anti-dsDNA antibody levels after sGal-9 administration in an in vivo model of systemic lupus erythematosus Anti-dsDNA antibodies, a type of antinuclear antibody (ANA) that targets intracellular nuclear components, are present at high titers in systemic lupus erythematosus (SLE). These antibodies are associated with the persistent progression of renal inflammation and damage. To evaluate the efficacy of sGal-9 of the present invention in an in vivo model of systemic lupus erythematosus, the plasma concentrations of anti-dsDNA antibodies in blood samples from the experimental animals in Experimental Example <1-1> were measured.

[0081] Specifically, after the experiment, cardiac blood was collected from the experimental animals in Experimental Example <1-1>. The collected blood was allowed to clot for 30 minutes using a capillary blood collection tube (Cat. 365967, BD), and then centrifuged at 6000 × g for 10 minutes to obtain plasma. The obtained plasma was diluted 1 / 2,500, and the anti-dsDNA antibody concentration was measured using a mouse anti-dsDNA IgG antibody ELISA kit (Cat. 3031, Chondrex) according to the manufacturer's protocol.

[0082] As a result, as shown in Figure 8, the positive control group (mycophenolic acid) and the sGal-9 administration groups (sGal-92 mg / kg and sGal-94 mg / kg) showed reduced anti-dsDNA antibody concentrations compared to the negative control group (vehicle). In particular, the sGal-94 mg / kg administration group (sGal-94 mg / kg) showed a significant reduction in anti-dsDNA antibody concentrations compared to the positive control group (mycophenolic acid).

[0083] (Possibility of Industrial Applicability) The recombinant stabilized galectin-9 protein of the present invention has been shown to be safe in an animal model of systemic lupus erythematosus (SLE), and has been confirmed to reduce skin lesions, lymphadenopathy, and proteinuria caused by lupus, improve lupus nephritis and glomerulonephritis, and reduce plasma anti-dsDNA antibody concentrations. Therefore, the recombinant stabilized galectin-9 protein can be effectively used as an active ingredient in a composition for preventing or treating lupus or glomerulonephritis.

Claims

1. A pharmaceutical composition for preventing or treating lupus or glomerulonephritis, comprising as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding the same.

2. The pharmaceutical composition according to claim 1, wherein the recombinant stabilized galectin-9 protein is a protein in which the first amino acid residue from the N-terminus of the amino acid sequence set forth in SEQ ID NO: 1 is further deleted.

3. 2. The pharmaceutical composition of claim 1, wherein the lupus is selected from the group consisting of systemic lupus erythematosus (SLE), systemic lupus, discoid lupus, drug-induced lupus, neonatal lupus, and lupus nephritis.

4. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition reduces skin lesions, lymphadenopathy, or proteinuria caused by lupus.

5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is intended to reduce the concentration of anti-dsDNA antibodies in the blood.

6. The pharmaceutical composition of claim 1 , further comprising a pharmaceutically acceptable carrier.

7. 10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is formulated for oral, intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or topical administration.

8. 10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated into a dosage form selected from the group consisting of tablets, capsules, injections, lozenges, powders, granules, liquids, suspensions, oral liquids, emulsions, syrups, suppositories, vaginal tablets, and pills.

9. A functional health food composition for preventing or ameliorating lupus or glomerulonephritis, comprising as an active ingredient a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding the same.

10. A method for preventing or treating lupus or glomerulonephritis, comprising administering to a subject a pharmaceutical composition comprising a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding the same.

11. Use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding the same for the manufacture of a pharmaceutical composition for the prevention or treatment of lupus or glomerulonephritis.

12. Use of a recombinant stabilized galectin-9 protein having the amino acid sequence set forth in SEQ ID NO: 1 or a polynucleotide encoding the same for the manufacture of a health functional food composition for preventing or ameliorating lupus or glomerulonephritis.

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