Composition for preventing or treating lupus comprising recombinant stable galectin 9 protein

Abstract

The present invention relates to a composition for the prevention or treatment of lupus or glomerulonephritis, comprising a recombinant stable galectin 9 protein, and specifically, the recombinant stable galectin 9 protein of the present invention can be used in the prevention or treatment of systemic lupus erythematosus; the present invention shows safety in an SLE animal model, reduces skin lesions, lymphadenectasis and proteinuria caused by lupus, alleviates lupus nephritis and glomerulonephritis, reduces the concentration of anti-dsDNA antibodies in plasma, and provides a new means for treating lupus nephritis and glomerulonephritis. Therefore, the recombinant stable galectin 9 protein of the present invention can be effectively used as an active ingredient of a composition for preventing or treating lupus or glomerulonephritis.

Description

Technical Field

[0001] The present invention relates to a recombinant stable galectin-9 protein and uses thereof, and in particular to a composition for preventing or treating lupus or glomerulonephritis, comprising the protein as an effective ingredient. Background Art

[0002] Animal lectins have been found in vivo, which specifically recognize sugar chains with β-galactoside structures, and at least 14 genes have been identified so far. Galectins are divided into prototype, chimera, and tandem repeat types based on their structures.

[0003] Galectin-9 is a tandem repeat galectin, which consists of two sugar chain recognition domains (carbohydrate recognition domains; CRD) and a connecting peptide region connecting them. Its N-terminal domain (N-terminal Carbohydrate Recognition Domain; NCRD) and C-terminal domain (C-terminal Carbohydrate Recognition Domain; CCRD) are connected by the connecting peptide region. Currently, there are reports indicating its activity in various aspects. For T cells, galectin-9 induces apoptosis of Tim-3-positive Th1 cells by binding to Tim-3, and inhibits autoimmune inflammation by inhibiting excessive Th1 response.

[0004] In order to use galectin-9 as a practical therapeutic agent, research is being conducted to produce a protease-resistant galectin-9 variant (G9Null) by cleaving the linker peptide of galectin-9 to solve the problems of 1) protease sensitivity; 2) low solubility; and 3) low yield of galectin-9.

[0005] In addition, lupus is an autoimmune disease associated with antibodies that attack connective tissue, which is related to the production of antinuclear antibodies as circulating immune complexes and the activation of the complement system. The lupus commonly mentioned generally refers to systemic lupus erythematosus (SLE), which is a systemic lupus. The disease is systemic, can occur in any organ system, and causes severe tissue damage. Lupus patients may produce autoantibodies with anti-DNA, anti-Ro, and anti-platelet specificity, and may also cause the occurrence of diseases such as glomerulonephritis, arthritis, serositis, complete heart block in newborns, or hematological abnormalities.

[0006] Without treatment, lupus can spread from attacking the skin and joints to attacking internal organs such as the lungs, heart and kidneys, making it potentially fatal, with kidney disease being the most worrisome. Kidney damage, as measured by the amount of protein in the urine, is one of the acute sites of damage associated with the pathogenicity of lupus, accounting for 50% or more of the mortality and morbidity from lupus disease.

[0007] Currently, there is no clear cure for lupus patients. From a practical perspective, doctors usually use a variety of immunosuppressants, such as high doses of corticosteroids, prednisone, azathioprine or cyclophosphamide, most of which have potentially harmful side effects for the patients being treated.

[0008] To this end, the inventors have devoted themselves to developing a safe lupus therapeutic agent, and finally produced a recombinant stable galectin-9 protein, and confirmed the safety of the recombinant stable galectin-9 protein in an in vivo model of systemic lupus erythematosus, wherein the recombinant stable galectin-9 protein is a protein in which the amino acids of the C-terminal domain (CCRD) and the connecting peptide in the two sugar chain recognition regions of the existing wild-type galectin 9 are deleted and replaced. In addition, it was confirmed that the recombinant stable galectin-9 protein reduces skin lesions, lymphadenopathy and proteinuria caused by lupus, alleviates lupus nephritis and glomerulonephritis, and reduces the concentration of anti-dsDNA antibodies in plasma in an in vivo model of systemic lupus erythematosus, and therefore can be effectively used as an active ingredient of a composition for preventing or treating lupus or glomerulonephritis, and finally the present application was completed.

[0009] Prior art literature

[0010] Patent Literature

[0011] Korean Patent Publication No. 10-2019-0060550

[0012] Non-patent literature

[0013] Nishi,N et al.Development of highly stable galectins:truncation of the linker peptide confers protease-resistance on tandem-repeat typegalectins.FEBS Lett.2005Apr 11;579(10):2058-64

[0014] Wang,Y.,Xiao,S.,Xia,Y.et al.The Therapeutic Strategies for SLE byTargeting Anti-dsDNAAntibodies.Clinic Rev Allerg Immunol(2021).https: / / doi.org / 10.1007 / s12016-021-08898-7 Summary of the invention

[0015] Technical issues

[0016] The present invention aims to provide a composition for preventing or treating lupus or glomerulonephritis, which comprises a recombinant stable galectin-9 protein.

[0017] Technical Solution

[0018] To achieve the purpose of the present invention, the present invention provides the following: a pharmaceutical composition for preventing or treating lupus or glomerulonephritis, comprising a recombinant stable galectin-9 protein or a polynucleotide encoding the same as an active ingredient, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; a method for preventing or treating lupus or glomerulonephritis, comprising administering the pharmaceutical composition to an individual, wherein the pharmaceutical composition comprises a recombinant stable galectin-9 protein or a polynucleotide encoding the same, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in preventing or treating lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; and use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in preparing a composition for preventing or treating lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0019] The present invention provides as follows: a health functional food composition for preventing or improving lupus or glomerulonephritis, comprising a recombinant stable galectin-9 protein or a polynucleotide encoding the same as an effective ingredient, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; a use of the recombinant stable galectin-9 protein or a polynucleotide encoding the same in preventing or improving lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; and a use of the recombinant stable galectin-9 protein or a polynucleotide encoding the same in preparing a health functional food composition for preventing or improving lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0020] Beneficial Effects

[0021] The recombinant stable galectin-9 protein of the present invention is safe in a systemic lupus erythematosus (SLE) animal model, reduces skin lesions, lymphadenopathy and proteinuria caused by lupus, alleviates lupus nephritis and glomerulonephritis, and reduces the concentration of anti-dsDNA antibodies in the blood, and can therefore be effectively used as an active ingredient of a composition for preventing or treating lupus or glomerulonephritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the weight change of the in vivo systemic lupus erythematosus (SLE) model administered with the recombinant stable galectin-9 protein (sGal-9) of the present invention.

[0023] Figure 2 FIG. 1 is a schematic diagram showing the scoring of the severity of skin lesions in the in vivo systemic lupus erythematosus model to which sGal-9 of the present invention is administered.

[0024] Figure 3 It is a schematic diagram showing the scoring of lymphadenopathy changes in the in vivo model of systemic lupus erythematosus to which sGal-9 of the present invention is administered.

[0025] Figure 4 FIG. 1 is a schematic diagram showing the scoring of the degree of proteinuria in the in vivo systemic lupus erythematosus model to which sGal-9 of the present invention is administered.

[0026] Figure 5 It is a graph showing the change of spleen weight in the in vivo model of systemic lupus erythematosus to which sGal-9 of the present invention is administered.

[0027] Figure 6 FIG. 1 is a schematic diagram showing the histological changes of kidneys in the in vivo model of systemic lupus erythematosus to which sGal-9 of the present invention is administered.

[0028] Figure 7 FIG. 1 is a graph showing the extent of IgG deposition in the kidney of an in vivo systemic lupus erythematosus model to which sGal-9 of the present invention is administered.

[0029] Figure 8 FIG. 5 is a graph showing the anti-dsDNA antibody concentration in plasma in an in vivo systemic lupus erythematosus model to which sGal-9 of the present invention is administered. DETAILED DESCRIPTION

[0030] Hereinafter, the embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. The embodiments of the present invention are used to further fully describe the present invention to those of ordinary skill in the art. Therefore, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

[0031] Throughout the specification of the present invention, when it is described that a part “includes” a certain constituent element, it means further including another constituent element, rather than excluding another constituent element, unless explicitly stated otherwise.

[0032] The present invention provides the following: a pharmaceutical composition for preventing or treating lupus or glomerulonephritis, comprising a recombinant stable galectin-9 protein or a polynucleotide encoding the same as an active ingredient, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; a method for preventing or treating lupus or glomerulonephritis, comprising administering the pharmaceutical composition to an individual, wherein the pharmaceutical composition comprises a recombinant stable galectin-9 protein or a polynucleotide encoding the same, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in preventing or treating lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; and use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in preparing a composition for preventing or treating lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0033] In the present invention, the recombinant stable galectin-9 protein can prevent or treat lupus or glomerulonephritis without causing side effects such as weight change.

[0034] The term "prevention" used in the present invention refers to all actions to inhibit or delay the occurrence of a disease by administering a composition.

[0035] The term "treatment" used in the present invention refers to all actions that improve or benefit the symptoms of the disease by administering a composition.

[0036] In the present invention, the recombinant stable Galectin-9 protein is a protein that maintains the sugar chain recognition activity of wild type Galectin-9 and has a more stable molecular structure to proteases.

[0037] Specifically, the recombinant stable galectin-9 protein is a protein produced by modifying the connecting region of two CRDs (Carbohydrate Recognition Domains) and CCRD (C-terminal Carbohydrate Recognition Domain) of wild-type galectin-9 (which has NCRD-linker-CCRD). More specifically, the recombinant stable galectin-9 protein may consist of the amino acid sequence shown in SEQ ID NO:, and all peptides in the linker region of the amino acid sequence are completely deleted, the amino acid sequence (SEQ ID NO: 3) at positions 1 to 10 in CCRD (SEQ ID NO: 2) is deleted, and Ala (Alanine; A) at position 13 is replaced by Pro (Proline; P), and the recombinant stable galectin-9 protein may include an amino acid sequence, the amino acid sequence having a sequence homology with the amino acid sequence shown in SEQ ID NO: greater than or equal to 75%, preferably greater than or equal to 80%, further preferably greater than or equal to 90%, most preferably greater than or equal to 95%, and may also include an amino acid sequence made for a specific purpose (targeting sequence, tag, labeling residue, improving half-life or peptide stability).

[0038] In addition, the recombinant stabilized galectin-9 protein may include a deletion of the first amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, and specifically may consist of the amino acid sequence shown in SEQ ID NO: 4.

[0039] The term "polynucleotide" as used in the present invention is a polymer of nucleotide bonds that serves to transmit genetic information. For the purpose of the present invention, it encodes the recombinant protein of SEQ ID NO: 1, and may include a sequence having a sequence homology greater than or equal to 75%, preferably greater than or equal to 80%, further preferably greater than or equal to 90%, and most preferably greater than or equal to 95% with the sequence homology of the polynucleotide sequence encoding the recombinant protein.

[0040] The term "homology" used in the present invention is intended to indicate the degree of similarity to a wild-type amino acid sequence or polynucleotide sequence. Such homology comparisons can be performed using comparison programs known in the art, and the homology of two or more sequences can be calculated as a percentage (%).

[0041] The term "lupus" as used in the present invention is an antibody-related autoimmune disease that attacks connective tissue, and may include chronic inflammatory autoimmune diseases characterized by the presence of autoantibodies, rashes, oral ulcers, serositis, neurological diseases, low blood cell counts, joint pain, and edema. The term "lupus" in the present invention has the conventional meaning used in the technical field to which the present invention belongs, unless otherwise specified. In the present invention, lupus may include, but is not limited to, systemic lupus erythematosus (systemic lupus erythematosus; SLE), systemic lupus, discoid lupus, drug-induced lupus (Druginduced lupus) or neonatal lupus, and may further include various other forms of lupus. In addition, lupus may cause chronic nephritis such as lupus nephritis or glomerulonephritis.

[0042] The systemic lupus erythematosus (SLE) is systemic lupus, also known as systemic lupus erythematosus, and has the conventional meaning used in the technical field to which the present invention belongs. SLE is a multi-system autoimmune disease that produces anti-nuclear antibodies including anti-dsDNA antibodies, and produces antigen-antibody immune complexes that are deposited in small blood vessels, thereby causing verification and damage to various organs such as the skin and kidney basement membrane.

[0043] Discoid lupus is a disease limited to the skin. It usually causes rashes on the face, neck, limbs, etc. Although the range of skin symptoms is smaller than that of systemic lupus, the symptoms are usually more severe and may leave pigmentation or scars if not treated properly. About 10% of patients with discoid lupus will eventually develop systemic lupus, and in the case of systemic lupus, 10% to 20% of patients present with discoid skin changes.

[0044] Drug-induced lupus occurs after taking certain drugs and presents symptoms similar to those seen in systemic lupus erythematosus, such as fever, rash, and joint pain. The most relevant drugs include hydralazine, a hypertension treatment, and procainamide, a heart rhythm treatment.

[0045] In the present invention, the pharmaceutical composition can prevent or treat lupus by reducing skin lesions, lymphadenopathy or proteinuria caused by lupus and the concentration of anti-dsDNA antibodies in the blood.

[0046] Furthermore, the pharmaceutical composition inhibits glomerular mesangium and reduces the infiltration of inflammatory cells in the glomerulus and the renal infiltration of immune complexes, thereby being able to prevent or treat lupus nephritis or glomerulonephritis.

[0047] In a specific embodiment of the present invention, the inventors prepared a recombinant stable galectin-9 protein (sGal-9), in which the amino acids of the C-terminal domain (CCRD) and the connecting peptide in the two sugar chain recognition regions of the existing wild-type galectin-9 were deleted and replaced.

[0048] Furthermore, the present inventors demonstrated that no change in body weight occurred due to repeated administration of the sGal-9 in an in vivo model of systemic lupus erythematosus.

[0049] Furthermore, the present inventors have demonstrated that, in an in vivo model of systemic lupus erythematosus, sGal-9 alleviates clinical symptoms caused by lupus by reducing skin lesions, inhibiting lymphadenopathy, and reducing the amount of proteinuria.

[0050] Furthermore, the present inventors confirmed that sGal-9 inhibited mesangial proliferation and reduced renal infiltration of inflammatory cells and immune complexes in and around glomeruli in an in vivo model of systemic lupus erythematosus, thereby showing therapeutic effects on lupus nephritis and glomerulonephritis.

[0051] Furthermore, the present inventors confirmed that sGal-9 exhibited an effect of reducing the anti-dsDNA antibody concentration in plasma in an in vivo model of systemic lupus erythematosus.

[0052] Therefore, the present inventors confirmed that the recombinant stable galectin-9 protein according to the present invention showed safety in an animal model of systemic lupus erythematosus, alleviated clinical symptoms caused by lupus as well as lupus nephritis and glomerulonephritis, and reduced the concentration of anti-dsDNA antibodies in the blood. Therefore, the recombinant stable galectin-9 protein according to the present invention or the polynucleotide encoding the same can be effectively used as an active ingredient of a pharmaceutical composition for preventing or treating lupus or glomerulonephritis.

[0053] In the present invention, the recombinant stabilized galectin-9 protein of the present invention or the polynucleotide encoding the same can be contained in a pharmaceutically acceptable carrier, such as a colloidal suspension, a powder, a salt solution, a lipid, a liposome, a microsphere (microspheres) or a nano-spherical particle. It can form a complex with a delivery carrier or be associated with it, and can be delivered to the body through a delivery system known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids.

[0054] In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. In addition, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. may be further included.

[0055] In the present invention, the pharmaceutical composition of the present invention can be administered orally or parenterally (for example, suitable for intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal or local) according to the purpose, and the dosage varies according to the patient's condition and weight, the degree of illness, the type of drug, the route of administration and the time, but can be appropriately selected by a technician in the field.

[0056] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

[0057] The term "pharmaceutically effective amount" used in the present invention refers to an amount sufficient to treat at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined based on factors including the patient's disease type, severity, drug activity, sensitivity to the drug, administration time, route of administration and excretion rate, treatment time, drugs used simultaneously, and other known factors in the medical field. The pharmaceutical composition according to the present invention can be used as a separate therapeutic agent, or used in combination with preparations for surgery, hormone therapy, drug therapy, and biological response modifiers, and can be used simultaneously, separately or sequentially with the preparation, and can be used once or multiple times. It is important that the amount that can achieve the maximum effect with the minimum amount without side effects considering all the above factors can be easily determined by a technician in the field.

[0058] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, state, body weight, absorbability of the active ingredient in the body, inactivation rate, excretion rate, disease type, and drugs administered in combination, and may be increased or decreased depending on the route of administration, severity of obesity, sex, body weight, age, etc.

[0059] In the present invention, the pharmaceutical composition can be formulated into a dosage form selected from the group consisting of tablets, capsules, injections, lozenges, powders, granules, solutions, suspensions, oral solutions, emulsions, syrups, suppositories, vaginal tablets and pills, but is not limited thereto and can be formulated into an appropriate dosage form as required. In addition, when formulated into the composition, it is prepared by using a commonly used diluent or excipient such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant and a surfactant.

[0060] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, lozenges, etc., which are prepared by mixing at least one excipient (e.g., starch, calcium carbonate, sucrose, lactose or gelatin, etc.) with at least one recombinant protein of the present invention. In addition, lubricants such as magnesium stearate talc are also used in addition to simple excipients. Liquid preparations for oral administration include suspensions, internal liquids, emulsions or syrups, etc., and in addition to commonly used simple diluents (e.g., water, liquid paraffin), various excipients may be included, such as wetting agents, sweeteners, aromatics, preservatives, etc.

[0061] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories and the like.

[0062] Non-aqueous solvents and suspending solvents include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), etc. The base of the suppository may include witepsol, Macrogol, Tween 61, cocoa, laurel, cocoa butter, laurin, glycerin, gelatin, etc.

[0063] The present invention provides as follows: a health functional food composition for preventing or improving lupus or glomerulonephritis, comprising a recombinant stable galectin-9 protein or a polynucleotide encoding the same as an effective ingredient, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; a use of the recombinant stable galectin-9 protein or a polynucleotide encoding the same in preventing or improving lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1; and a use of the recombinant stable galectin-9 protein or a polynucleotide encoding the same in preparing a health functional food composition for preventing or improving lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0064] In the present invention, the contents about the recombinant stable galectin-9 protein and lupus are as described above, and the specific description is referred to the above contents.

[0065] The term "improvement" as used herein refers to all actions that at least reduce parameters (e.g., degree of illness) associated with a cured state. At this point, the health functional food composition can be used with therapeutic drugs simultaneously or alone before or after the disease occurs to prevent or improve the disease.

[0066] In addition, in the present invention, the recombinant stable galectin-9 protein according to the present invention exhibits safety in a systemic lupus erythematosus animal model, alleviates clinical symptoms caused by lupus, lupus nephritis and glomerulonephritis, and reduces the concentration of anti-dsDNA antibodies in the blood. Therefore, the recombinant stable galectin-9 protein according to the present invention or the polynucleotide encoding it can be effectively used as an active ingredient of a health functional food composition for preventing or improving lupus.

[0067] In the health functional food of the present invention, the active ingredient can be directly added to the food, or used together with other foods or food ingredients, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use (prevention or improvement). When food or beverage is usually manufactured, the health functional food of the present invention is preferably able to be added in an amount of 15% by weight or less relative to the raw material, preferably in an amount of 10% by weight or less. However, when long-term intake is for health and health care or regulating health conditions, the amount may be less than or equal to the range.

[0068] In addition to the above-mentioned active ingredients, the health functional food of the present invention can also contain other ingredients as essential ingredients without any particular restrictions. For example, various flavor enhancers or natural carbohydrates can be contained as additional ingredients as conventional beverages. The above-mentioned natural carbohydrates can exemplify monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin as conventional sugars, sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-mentioned flavorings, natural flavorings (thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) can be advantageously used. Those skilled in the art can appropriately determine the ratio of the above-mentioned natural carbohydrates.

[0069] In addition to the above, 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, coloring agents and thickeners (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents for carbonated beverages, etc. These ingredients can be used alone or in combination, and the proportion of such additives can also be appropriately selected by those skilled in the art.

[0070] Hereinafter, the present invention is described in detail by preparation examples and examples. However, the following preparation examples and examples are intended to help understand the present invention, but do not limit the scope of the present invention.

[0071] <Preparation Example 1> Preparation of recombinant stable Galectin 9 protein (sGal-9)

[0072] After the expression vector is prepared, the expression vector is introduced into Escherichia coli by heat shock, wherein the expression vector comprises a gene encoding a recombinant stable galectin-9 protein having an amino acid sequence of SEQ ID NO: 1. Escherichia coli was cultured in LB medium containing kanamycin, and arabinose was added when the absorbance at 600 nm reached 0.7 to induce the expression of the recombinant protein. Then, the cells induced to express the recombinant protein were lysed and filtered, and the target protein was captured by cation exchange and affinity column, thereby obtaining a high-purity recombinant stable galectin-9 protein with high yield.

[0073] <Example 1> Evaluation of the safety of sGal-9 in an in vivo systemic lupus erythematosus model

[0074] To confirm the efficacy of sGal-9 on 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).

[0075] <1-1> Preparation of an in vivo systemic lupus erythematosus model and administration of sGal-9

[0076] To confirm the efficacy of sGal-9 on lupus, an in vivo systemic lupus erythematosus model was prepared and sGal-9 of the present invention was administered.

[0077] Specifically, it is known that MRL / FASlpr mice usually develop the disease due to MRL-lpr or lpr mutations. It is a homozygote of spontaneous lymphoproliferation mutation (Faslpr) and exhibits systemic autoimmunity, a large number of lymphadenopathy associated with abnormal T cell proliferation, arthritis, and immune complex glomerulonephritis. In these mice, the level of circulating immune complexes increases significantly from about 3 months of age, the lymph node proliferation in MRL / Mp-Faslpr / Faslpr is about 75 times the weight of the lymph nodes in the control group, and the differences in renal pathology occur widely in 4 to 7 months, so it can be effectively used as a model for evaluating the therapeutic efficacy of systemic lupus erythematosus. Therefore, 11-week-old MRL / FASlpr female mice were purchased from Central Lab.Animal Inc. and used as an in vivo model of systemic lupus erythematosus. The feeding and experiments of MRL / FASlpr mice were carried out in the SPF (specific pathogen free) experimental animal room, and the temperature was maintained at 21°C to 23°C and the relative humidity was maintained at 40% to 45%. The experimental animals were fed with rodent chow (Envigo Co., Ltd.) ad libitum.

[0078] As shown in Table 1 below, the 11-week-old MRL / FASlpr female mice were randomly arranged into four groups, namely, a negative control group (vehicle), a positive control group (mycophenolate), a sGal-9 2 mg / kg administration group (sGal-9 2 mg / kg), and a sGal-9 4 mg / kg administration group (sGal-9 4 mg / kg). The negative control group was subcutaneously injected with vehicle once a week, the positive control group was orally administered with 60 mg / kg of mycophenolate (Roche) for the treatment of lupus nephritis once a week, and the sGal-9 2 mg / kg administration group (sGal-9 2 mg / kg) and the sGal-9 4 mg / kg administration group (sGal-9 4 mg / kg) were subcutaneously administered with 2 mg / kg and 4 mg / kg of sGal-9 of Preparation Example 1, respectively, once a week. Each experimental substance was administered for 9 weeks.

[0079] Table 1

[0080]

[0081] In addition, based on the blind evaluation data of each evaluation index, statistical analysis was performed between the negative control group and the experimental group or between the two experimental groups by SPSS. To compare the two groups, Student's t-test and Mann Whitney U test were used. When comparing the differences between the treatment groups at different time points, repeated measures ANOVA and Tukey's post-hoc test were used. A p value of less than or equal to 0.05 was set as a significant level.

[0082] <1-2> Body weight evaluation in an in vivo systemic lupus erythematosus model

[0083] In order to determine the safety of sGal-9 of the present invention in the in vivo model of systemic lupus erythematosus, the body weights of the experimental animals in the above Example 1-1 were measured.

[0084] Specifically, body weight was measured during 9 weeks of repeated administration of the test substance by the method described in Example 1-1.

[0085] Results, such as Figure 1 As shown, it was confirmed that the negative control group (vehicle), the positive control group (mycophenolic acid), the sGal-9 2 mg / kg administration group (sGal-9 2 mg / kg), and the sGal-9 4 mg / kg administration group (sGal-94 mg / kg) did not show statistically significant changes in body weight.

[0086] <Example 2> Evaluation of clinical indicators after administration of sGal-9 in an in vivo systemic lupus erythematosus model

[0087] In order to determine the efficacy of sGal-9 of the present invention in the in vivo model of systemic lupus erythematosus, the clinical indicators of the experimental animals in the above Example 1-1 were evaluated.

[0088] Specifically, the occurrence and severity of lupus were regularly observed from the grouping of the experimental animals in Example 1-1 to the end of the experiment, and evaluated according to the criteria in Table 2 below.

[0089] Table 2

[0090]

[0091]

[0092] In addition, after the experiment, the experimental animals of Example 1-1 were sacrificed, and their weights were measured after the spleen was removed. Figure 2 As shown, the skin lesions of the positive control group (mycophenolic acid) and the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) were reduced compared to the negative control group (vehicle). In particular, the skin lesion reduction effect of the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) was excellent compared to the positive control group (mycophenolic acid).

[0093] In addition, if Figure 3 As shown in the figure, the lymphadenopathy scores of the positive control group (mycophenolic acid) and the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) were decreased compared with the negative control group (vehicle). In particular, the inhibitory effect on lymphadenopathy in the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) was excellent compared with the positive control group (mycophenolic acid).

[0094] In addition, if Figure 4 As shown, proteinuria was reduced in the positive control group (mycophenolic acid) and sGal-9 administration groups (sGal-9 2 mg / kg and sGal-9 4 mg / kg) compared to the negative control group (vehicle).

[0095] In addition, if Figure 5 As shown, the spleen weight of the sGal-9 2 mg / kg administration group was reduced compared to the negative control group (vehicle).

[0096] <Example 3> Evaluation of tissue morphology after administration of sGal-9 in an in vivo systemic lupus erythematosus model

[0097] In order to determine the efficacy of sGal-9 of the present invention on the in vivo model of systemic lupus erythematosus, the histological changes of the kidney tissues of the experimental animals in the above Example 1-1 were confirmed by H&E (hematoxyline & Eosin) staining and IgG immunofluorescence staining.

[0098] Specifically, after the experiment, the experimental animals of the above-mentioned embodiment 1-1 were sacrificed and the kidney tissues were collected. The collected kidney tissues were fixed in 10% formaldehyde and dehydrated, and fixed with paraffin. The fixed kidney tissues were cut into 2 μm thickness and stained with H&E. The cut tissues were covered with a coverslip and observed after magnification 400 times with an optical microscope. Four parts of each tissue were photographed and scored by two or more researchers according to the tissue morphology evaluation items and indicator standards in Table 3 below. The average of the researchers' scores was used as the score value, and the score values ​​of the four parts were averaged as the score of each tissue.

[0099] Table 3

[0100]

[0101] In addition, the collected kidney tissue was frozen, cut into a thickness of 4 μm, and fixed in cold acetone for 5 minutes, and then washed twice with PBS (phosphate buffered saline) for 5 minutes each time. To eliminate nonspecific reactions, it was blocked with a PBS solution containing 1% bovine serum albumin and 0.05% tween-20 for 30 minutes, and then reacted with FITC-labeled goat anti-mouse IgG antibody (1:200, AP308F, Merck Millipore) at room temperature for 1 hour. Then, it was washed with PBS 3 times for 5 minutes each time, stained with DAPI and covered with a coverslip with a sealing agent, and observed with a confocal laser scanning microscope (C2 Plus, Nikon). The results, as shown in FIG. Figure 6 As shown in the figure, glomerulonephritis was clearly observed in the negative control group (vehicle), and inflammatory cells in and around the glomeruli and renal tubules were destroyed while mesangial proliferation occurred. In contrast, in the positive control group (mycophenolic acid) and the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg), inflammatory cell infiltration in the glomeruli was reduced. In addition, the glomerular lesion scores of the positive control group (mycophenolic acid) and the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) were reduced compared to the negative control group (vehicle).

[0102] In addition, if Figure 7As shown, IgG deposition was decreased in the positive control group (mycophenolic acid) and sGal-9 administration groups (sGal-9 2 mg / kg and sGal-9 4 mg / kg) compared to the negative control group (vehicle).

[0103] From the above results, it can be seen that sGal-9 of the present invention exhibits therapeutic effects on lupus nephritis and glomerulonephritis by reducing the infiltration of inflammatory cells in and around the glomeruli and the infiltration of immune complexes into the kidneys.

[0104] <Example 4> Analysis of anti-dsDNA antibody concentration in plasma after administration of sGal-9 in an in vivo systemic lupus erythematosus model

[0105] Anti-dsDNA antibodies are anti-nuclear antibodies (antibodies to substances in the cell nucleus), which are present in high titers in systemic lupus erythematosus and are associated with the continued progression of kidney inflammation and damage. In order to determine the efficacy of sGal-9 of the present invention in an in vivo model of systemic lupus erythematosus, the concentration of anti-dsDNA antibodies in the blood of the experimental animals of Example 1-1 above was detected.

[0106] Specifically, after the experiment, blood was collected from the kidneys of the experimental animals of Example 1-1. The blood was coagulated for 30 minutes using a capillary blood collection tube (Cat. 365967, BD), and then centrifuged at 6000×g for 10 minutes to obtain plasma.

[0107] The results are as follows Figure 8 As shown in the figure, compared with the negative control group (vehicle), the anti-dsDNA antibody concentrations of the positive control group (mycophenolic acid) and the sGal-9 administration group (sGal-9 2 mg / kg and sGal-9 4 mg / kg) decreased. In particular, compared with the positive control group (mycophenolic acid), the anti-dsDNA antibody concentration decrease effect of the sGal-9 4 mg / kg administration group (sGal-9 4 mg / kg) was more excellent.

[0108] Industrial Applicability

[0109] The recombinant stable galectin-9 protein of the present invention is safe in a systemic lupus erythematosus (SLE) animal model, reduces skin lesions, lymphadenopathy and proteinuria caused by lupus, alleviates lupus nephritis and glomerulonephritis, and reduces the concentration of anti-dsDNA antibodies in plasma, and can therefore be effectively used as an active ingredient of a composition for preventing or treating lupus or glomerulonephritis.

Claims

1. A pharmaceutical composition for preventing or treating lupus or glomerulonephritis, comprising: The recombinant stable galectin-9 protein or a polynucleotide encoding the same is used as an effective ingredient, and the recombinant stable galectin-9 protein has the amino acid sequence shown in SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, wherein The recombinant stable galectin-9 protein also lacks the first amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO:

1.

3. The pharmaceutical composition according to 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. The pharmaceutical composition according to 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 reduces the concentration of anti-dsDNA antibodies in the blood.

6. The pharmaceutical composition according to claim 1, further comprising: A pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is formulated as a composition for oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, or topical administration.

8. 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, solutions, suspensions, oral solutions, emulsions, syrups, suppositories, vaginal tablets and pills.

9. A health functional food composition for preventing or improving lupus or glomerulonephritis, comprising: The recombinant stable galectin-9 protein or a polynucleotide encoding the same is used as an effective ingredient, and the recombinant stable galectin-9 protein has the amino acid sequence shown in SEQ ID NO:

1.

10. A method for preventing or treating lupus or glomerulonephritis, comprising: A pharmaceutical composition is administered to an individual, wherein the pharmaceutical composition comprises a recombinant stable galectin-9 protein or a polynucleotide encoding the same, wherein the recombinant stable galectin-9 protein has an amino acid sequence shown in SEQ ID NO:

1.

11. Use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in the preparation of a pharmaceutical composition for preventing or treating lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has the amino acid sequence shown in SEQ ID NO:

1.

12. Use of a recombinant stable galectin-9 protein or a polynucleotide encoding the same in preparing a health functional food composition for preventing or improving lupus or glomerulonephritis, wherein the recombinant stable galectin-9 protein has the amino acid sequence shown in SEQ ID NO: 1.

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