Use of keratin 14 or glycosylation modifications thereof in the manufacture of a product for the treatment of systemic lupus erythematosus

Abstract

The application discloses application of keratin 14 or a glycosylation modifier thereof in preparation of a product for treating systemic lupus erythematosus. The keratin 14, as a component of skin keratinocytes, has small toxic and side effects. The first research of the application shows that the keratin 14 has certain potential in treating systemic lupus erythematosus, and use of the keratin 14, a body self component, can greatly shorten research and development cost and research and development risk. The application first explores the effect of the keratin 14 in treating systemic lupus erythematosus, and provides an experimental basis for treatment of systemic lupus erythematosus.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the therapeutic use of keratin 14, particularly the use of keratin 14 or its glycosylated modifications in the preparation of products for the treatment of systemic lupus erythematosus. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a complex autoimmune disease with diverse clinical manifestations, characterized by the overproduction of type I interferon (IFN) and autoantibodies. The pathogenesis of SLE remains unclear, but genetic susceptibility, environmental triggers, and hormone levels interact to promote its development. In recent years, the incidence of SLE has been rising worldwide, with a global prevalence of 43.7 per 100,000 people (Tian J, Zhang D, Yao X, Huang Y, Lu Q. Global epidemiology of systemic lupus erythematosus: a comprehensive systematic analysis and modelling study). Ann Rheum Dis . 2023;82(3):351-356), seriously endangering human health and even threatening life.

[0003] SLE commonly affects women of childbearing age. The main treatments are glucocorticoids and immunosuppressants. Most patients achieve a stable or low-activity state, but a complete cure is not possible. Furthermore, some patients experience low or no response to medications, and long-term medication use can have significant side effects. Therefore, the treatment of SLE remains a key focus and challenge in research.

[0004] Keratin 14 (Krt14) is primarily expressed in the basal layer of epithelial cells and is a crucial component for maintaining cellular structural integrity and mechanical strength. Abnormal expression or dysfunction of Krt14 is associated with various diseases. N-acetylgalactosamine (GalNAc) modification is used to target drug delivery to the liver for the treatment of liver diseases, or to induce immune tolerance to specific antigens using the liver. GalNAc-modified myelin oligodendrocyte glycoprotein (MOG) fragments have shown good efficacy in treating multiple sclerosis (MS) models in mice, and clinical trials of "reverse vaccines" based on GalNAc modification strategies for celiac disease or MS are underway. Over 75% of SLE patients have skin involvement, and in some patients, skin involvement is the initial symptom; anti-keratin antibodies are detected in the serum of approximately 30% of SLE patients, suggesting that keratin may be one of the autoantigens that induce SLE. However, to date, no studies or clinical trials have elucidated the impact of keratin 14 or other types of keratin on SLE disease. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the problem of large side effects of traditional SLE treatment drugs, and to provide the use of keratin 14 and its glycosylation modifiers in the treatment of SLE, so as to provide an experimental basis and new treatment methods for SLE.

[0006] This invention is the first to propose the application of keratin 14 or its glycosylated modifications in the preparation of products for the treatment of systemic lupus erythematosus (SLE). Specifically, the SLE described in this invention is an autoimmune disease characterized by immune dysregulation and the production of large amounts of autoantibodies, which distinguishes it from other types of lupus.

[0007] Intraperitoneal injection of GalNAc-modified keratin 14 or its glycosylated derivatives into SLE model mice revealed that this treatment significantly reduced the levels of anti-dsDNA, ANA, and urinary protein in peripheral blood, and decreased the production of autoantibodies. The volume and weight of draining lymph nodes were significantly reduced compared to the untreated group, suggesting that the overactive autoimmune response was suppressed. These results indicate that GalNAc-modified keratin 14 can reduce autoantibody production, protect the kidneys, and exert a therapeutic effect on SLE. Simultaneously, keratin 14 or its glycosylated derivatives alleviated SLE-induced lymph node hyperplasia.

[0008] In application, keratin 14 or its glycosylated modifications are used as the active ingredient in drugs for treating systemic lupus erythematosus.

[0009] The present invention further proposes the use of a pharmaceutical composition in the preparation of a product for the treatment of systemic lupus erythematosus, said composition comprising keratin 14 or a glycosylated modification thereof.

[0010] Furthermore, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.

[0011] Preferably, the pharmaceutical composition is selected from the following dosage forms: solution, gel, ointment, paste, powder, tablet, injection, capsule or oral liquid, wherein the solution is a pharmaceutical solution applied topically to the skin lesion.

[0012] The above-mentioned pharmaceutical composition can be formulated using known methods and administered to subjects via the following routes, including but not limited to: parenteral, oral, local, intradermal, intramuscular, intraperitoneal, subcutaneous, and intravenous.

[0013] Preferably, the pharmaceutical composition is N-acetylgalactosamined keratin 14 injection.

[0014] Beneficial Effects: Currently, the main treatments for SLE are glucocorticoids and immunosuppressants. Most patients achieve a stable or low-activity state, but a complete cure is not yet possible. Furthermore, some patients experience low or no response to medications, and long-term medication use can have significant side effects. Keratin 14, as a naturally occurring component of the body, has fewer toxic side effects. This invention is the first to demonstrate that keratin 14 has potential in treating SLE, and its presence as a naturally occurring component can significantly reduce research and development costs and risks. This invention also explores the efficacy of keratin 14 in treating systemic lupus erythematosus (SLE), providing an experimental basis for SLE treatment. Attached Figure Description

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0016] Figure 1 Intraperitoneal injection of keratin 14 or GalNAcized keratin 14 was used to treat SLE in mice. Figure A shows that GalNAcized keratin 14 can alleviate the severity of skin lesions in SLE mice, and both keratin 14 and GalNAcized keratin 14 can improve the survival rate of mice. Figure B shows that keratin 14 and GalNAcized keratin 14 can reduce the levels of urinary protein, peripheral blood anti-dsDNA, and ANA in SLE mice, with GalNAcized keratin 14 showing a more significant effect. Figure C shows that keratin 14 and GalNAcized keratin 14 can alleviate lymph node hyperplasia in SLE mice. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments, but it should not be construed that the scope of the present invention is limited to the following embodiments. Various substitutions and modifications made based on common technical knowledge and methods in the art without departing from the above-described technical concept of the present invention should be included within the scope of protection of the present invention.

[0018] 1. Experimental Materials 1. Experimental drugs and reagents: Keratin 14 (OriGene), GalNAc (Bide), Acetic anhydride (Hushi), LA-C2OH (Bailinwei), TMSO-Tf (Annaiji), PBS (Sigma-Aldrich), DMSO (Sigma-Aldrich), 4-hydroxytamoxifen (Absin), Corn oil (Beyotime), Proteinuria test strips (Ulite), Urine protein quantification kit (Nanjing Jiancheng Bioengineering Institute), Antinuclear antibody and anti-double-stranded DNA antibody ELISA kit (Cusabio).

[0019] The GalNAc-modified keratin 14 used in this experiment was synthesized in-house. Specifically, GalNAc-modified keratin 14 was prepared using the following method: (1) GalNAc was protected with 5.0 eq of acetic anhydride and directly condensed with LA-C2OH via TMSO-Tf. The LA-GalNAc was obtained by column chromatography purification and deprotection with NaOMe / MeOH system.

[0020] (2) Keratin 14 was diluted to 0.2 mg / mL, and LA-GalNAc was added to a final concentration of 300 mM. The mixture was frozen at -20 ℃ for 3 h. The successful synthesis of GalNAcized keratin 14 was confirmed by SDS-PAGE.

[0021] (3) Dilute the reaction mixture to 2.5 mL and perform preliminary purification using a PD-10 desalting column (Cytiva). Collect 3.5 mL of eluent. Further purify the obtained GalNAc-modified keratin 14 using a Mono S cation exchange column (20 mM NaOAc, pH = 4.5). Concentrate the collected flow-through solution through a 10000 kDa ultrafiltration tube and replace it with PBS buffer.

[0022] 1.2 Laboratory Animals The mice used in this experiment were Krt5-CreERT2 / + PPARγ flox / flox SLE mice. The mice were approximately 6-12 weeks old, weighed 20-30g, and were provided by Shanghai Southern Model Biotechnology Co., Ltd.

[0023] Specifically, the Krt5-CreERT2 / + PPARγ flox / flox SLE mouse was constructed using the following method: (1) Preparation of Krt5-CreERT2 / + PPARγ flox / flox mice: Krt5-CreERT2 mice were mated with PPARγ flox / flox mice containing loxP flanking sequences to generate offspring mice with genotypes of Krt5-CreERT2 / + PPARγ flox / + and PPARγ flox / +. Krt5-CreERT2 / + PPARγ flox / + mice were retained, and heterozygous mice were mated to obtain mice with genotypes of Krt5-CreERT2 / + PPARγ flox / flox. Among them, PPARγ flox / flox and Krt5-CreERT2 mice were provided by Shanghai Southern Model Biotechnology Co., Ltd. Krt5-CreERT2 mice can directionally express CreERT2 fusion protein in keratinocytes through the endogenous promoter / enhancer element of the krt5 locus. The CreERT2 fusion protein consists of a mutant of the ligand-binding region (ERT) of the estrogen receptor (ER) and the Cre recombinase protein. PPARγ flox / flox mice, or PPARγ conditional knockout mice, have loxP sites inserted at both ends of specific exons of the PPARγ gene. These loxP sites can be cleaved by active Cre recombinase.

[0024] (2) Specific knockout of PPARγ in keratinocytes: 50 mg of 4-hydroxytamoxifen was mixed in 1 mL of DMSO, 9 mL of corn oil was added, the suspension was thoroughly mixed using a vortex mixer, and the mixture was sonicated in an ultrasonic bath at 37°C for 20 minutes to prepare a 4-hydroxytamoxifen solution (5 mg / mL) for use. Then, 50 μL of the 5 mg / mL 4-hydroxytamoxifen solution was applied to both ears of Krt5-CreERT2 / + PPARγ flox / flox mice. The corresponding solution was applied to both ears for 5 consecutive days, and the phenotype of the mice was observed to obtain Krt5-CreERT2 / + PPARγ flox / flox SLE mice.

[0025] Mice were observed for skin lesions weekly, and photographs were taken for documentation. Clean-catch midstream urine was collected from mice periodically, and urinary protein was detected using a Unirit proteinuria test strip. The CBB method was used to quantify the urinary protein content. The levels of peripheral blood serum antinuclear antibodies and anti-double-stranded DNA antibodies were detected using an ELISA kit (Cusabio).

[0026] On day 14, mice were euthanized by cervical dislocation after anesthesia. Draining lymph nodes were collected from the same location at the same time using a sharp scalpel and ophthalmic scissors, and the weight of the draining lymph nodes was analyzed.

[0027] The results showed that the constructed Krt5-CreERT2 / + PPARγ flox / flox SLE mice, after activating the PPARγ gene knockout in local keratinocytes, exhibited highly efficient, stable, and spontaneous SLE-like phenotypes such as immune cell disorder, inflammatory skin lesions and hair loss, proteinuria, significant increases in peripheral blood dsDNA and ANA autoantibodies, and hyperplasia of draining lymph nodes.

[0028] Husbandry conditions: room temperature 18-20℃, humidity 50-60%, alternating light and dark (12 h), moderate light intensity, and good ventilation and cleanliness. All experiments were approved and conducted in accordance with the guidelines of the Ethics Committee of the Institute of Dermatology, Chinese Academy of Medical Sciences (CASD).

[0029] 2. Test Methods

[0030] 2.1 Drug administration to mice SLE mice were divided into three groups according to requirements: control group (PBS), keratin 14 group, and GalNAcized keratin 14 group, with 6 mice in each group. Keratin 14 or GalNAcized keratin 14 was dissolved in PBS to prepare an injection solution (0.01 mg / mL). The control group was injected intraperitoneally with 100 μL of PBS, the keratin 14 group was injected intraperitoneally with 100 μL of 0.01 mg / mL keratin 14 injection solution, and the GalNAcized keratin 14 group was injected intraperitoneally with 100 μL of 0.01 mg / mL GalNAcized keratin 14 injection solution. The mice in all three groups were injected intraperitoneally once on day 4 before SLE modeling, day 1 before modeling, and day 3 after modeling.

[0031] 2.2 Monitoring of mouse skin lesions, urinary protein, and autoantibodies Mice were observed for skin lesions weekly, and photographs were taken for documentation. Clean-catch midstream urine was collected from mice periodically, and urinary protein was detected using a Unirit proteinuria test strip. The CBB method was used to quantify the urinary protein content. The levels of peripheral blood serum antinuclear antibodies and anti-double-stranded DNA antibodies were detected using an ELISA kit (Cusabio).

[0032] 2.3 Organizational Observation On day 14, mice were euthanized by cervical dislocation after anesthesia. Drainage lymph nodes from the neck and axilla of the three groups of mice were harvested at the same time and location using a sharp scalpel and ophthalmic scissors, and the weight of the drainage lymph nodes was analyzed.

[0033] 3. Experimental Results

[0034] Figure 1 GalNAc-modified keratin 14 alleviated the severity of skin damage in SLE mice, and both keratin 14 and GalNAc-modified keratin 14 improved the survival rate of mice. On day 14 of treatment, mice in the control group and the keratin 14 group showed significant hair loss and inflammatory skin lesions covered with scales. Mice in the GalNAc-modified keratin 14 group experienced less hair loss and reduced skin lesions. On day 14 of treatment, the survival rate of mice in the control group was only 50%, while the survival rate of both the keratin 14 group and the GalNAc-modified keratin 14 group was 100%. This indicates that GalNAc-modified keratin 14 can alleviate skin damage in SLE mice, and both keratin 14 and GalNAc-modified keratin 14 can improve the survival rate of SLE mice.

[0035] Figure 1B. Keratin 14 and GalNAcized keratin 14 can reduce urinary protein, peripheral blood anti-dsDNA and ANA levels in SLE mice. Compared with the control group, the keratin 14 group showed significantly lower levels of urinary protein (control group vs. keratin 14 group: 86.36±17.95 mg / dL vs. 45.93±5.175 mg / dL, *P<0.05) and peripheral blood autoantibody anti-dsDNA (control group vs. keratin 14 group: 185.3±10.20 ng / mL vs. 118.1±17.82 ng / mL, *P<0.05). Compared with the control group, the GalNAc keratin 14 group showed significantly lower levels of urinary protein (control group vs GalNAc keratin 14 group: 86.36±17.95 mg / dL vs 23.13±5.045 mg / dL, ***P<0.001), peripheral blood autoantibodies anti-dsDNA (control group vs GalNAc keratin 14 group: 185.3±10.20 ng / mL vs 37.43±8.176 ng / mL, ***P<0.001), and ANA (control group vs GalNAc keratin 14 group: 129.1±9.702 ng / mL vs 83.05±5.980 ng / mL, **P<0.01). Compared to the keratin 14 group, the GalNAc-modified keratin 14 group showed a significant decrease in peripheral blood autoantibodies (anti-dsDNA, keratin 14 vs. GalNAc-modified keratin 14: 118.1±17.82 ng / mL vs. 37.43±8.176 ng / mL, **P<0.01) and ANA (keratin 14 vs. GalNAc-modified keratin 14: 123.6±12.69 ng / mL vs. 83.05±5.980 ng / mL, **P<0.01). These results indicate that keratin 14 and GalNAc-modified keratin 14 can significantly reduce urinary protein and autoantibodies in SLE mice, with GalNAc-modified keratin 14 showing a more significant therapeutic effect.

[0036] Figure 1C-keratin 14 and GalNA-cated keratin 14 can alleviate the hyperplasia of draining lymph nodes in SLE mice. The draining lymph nodes of mice were weighed, and compared with the control group, the weight of draining lymph nodes at the same location was significantly lower in the keratin 14 and GalNA-cated keratin 14 groups (control group vs. keratin 14 group: 68.20±7.211 mg vs. 45.77±4.141 mg, *P<0.05; control group vs. GalNA-cated keratin 14 group: 68.20±7.211 mg vs. 35.02±7.350 mg, *P<0.05). These results demonstrate that both keratin 14 and GalNA-cated keratin 14 can alleviate the hyperplasia of draining lymph nodes in SLE mice.

[0037] The above results indicate that both intraperitoneal injection of keratin 14 and GalNAcized keratin 14 can treat SLE mice, with GalNAcized keratin 14 showing more significant efficacy.

[0038] This invention provides a concept and method for the application of keratin 14 in the preparation of drugs for treating systemic lupus erythematosus. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The use of keratin 14 in the preparation of drugs for the treatment of systemic lupus erythematosus.

2. The use of a pharmaceutical composition in the preparation of a medicament for treating systemic lupus erythematosus, characterized in that, The composition contains keratin 14.

3. The application according to claim 2, characterized in that, The pharmaceutical composition contains pharmaceutically acceptable excipients.

4. The application according to claim 2, characterized in that, The pharmaceutical composition contains a pharmaceutically acceptable carrier.

5. The application according to claim 2, characterized in that, The pharmaceutical composition is selected from the following dosage forms: solution, powder, tablet and capsule.

6. The application according to claim 2, characterized in that, The dosage form of the pharmaceutical composition is an oral liquid.

7. The application according to claim 2, characterized in that, The dosage form of the pharmaceutical composition is an injection.

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