New applications of recombinant humanized type XVII collagen

Abstract

The present invention provides use of recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen in the preparation of a preparation for preventing and / or treating lupus erythematosus, which can overcome the defect of existing solutions in causing many adverse reactions during treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to use of recombinant humanized type XVII collagen in the treatment of lupus erythematosus. Background Art

[0002] Systemic lupus erythematosus (LE) is a typical autoimmune connective tissue disease, most commonly affecting women aged 15 to 40. LE encompasses a spectrum of diseases, including discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), profundus lupus erythematosus (LEP), neonatal lupus erythematosus (NLE), drug-induced lupus erythematosus (DIL), and systemic lupus erythematosus (SLE).

[0003] Discoid lupus erythematosus primarily affects the skin and is the mildest form of lupus erythematosus. A minority may experience mild visceral damage, and a few cases may develop into systemic lupus erythematosus. Skin lesions initially appear as one or several bright red patches, ranging in size from mung beans to soybeans, with adherent scales on the surface. These lesions gradually expand, becoming round or irregular in shape, with noticeably darker edges and slightly higher pigmentation than the center. Lesions are primarily located in sun-exposed areas, such as the face, helices, and scalp. A few may also affect the upper chest, backs of the hands, forearms, lips, and oral mucosa. Most patients experience no symptoms, but complete resolution of these lesions is difficult. New lesions may gradually increase or remain unchanged for years. Discoid lesions worsen after sun exposure or exertion. Lesions on the scalp can cause permanent hair loss. Older lesions can occasionally develop into squamous cell carcinoma of the skin.

[0004] Subacute cutaneous lupus erythematosus (SULE) is clinically less common and represents a distinct intermediate type. Two types of skin lesions occur: annular erythema and papulesquamous. In most cases, either type is present alone, though a few may present simultaneously. Lesions often recur, and the vast majority of patients experience visceral damage, though severe cases are rare. Key symptoms include arthralgia, myalgia, and recurrent low-grade fever. A few may also experience nephritis and hematologic changes.

[0005] Lupus erythematosus profundus, also known as lupus panniculitis, is also an intermediate form of lupus erythematosus. Skin lesions are nodules or plaques located deep in the dermis or subcutaneous adipose tissue. They vary in size and number, have a normal or reddish skin color, are firm, and non-mobile. Lesions can occur anywhere, but are most common on the cheeks, buttocks, and arms, followed by the calves and chest. Lupus erythematosus profundus is unstable and can exist in isolation, later transforming into discoid lupus erythematosus, systemic lupus erythematosus, or both.

[0006] Neonatal lupus erythematosus presents as annular erythema on the skin and congenital heart block, which is self-involved and usually disappears on its own within 4 to 6 months after birth. Heart disease often persists.

[0007] Drug-induced lupus erythematosus (DISE) presents primarily with fever, arthralgia, myalgia, facial butterfly rash, oral ulcers, and possibly serositis. ANA, anti-histone antibodies, and anti-ss-DNA antibodies may be positive. Improvement gradually occurs after discontinuation of medication. Severe cases may require appropriate glucocorticoid administration.

[0008] Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects multiple organs and tissues. Its main characteristics are the production of multiple specific autoantibodies and abnormal activation of the immune system, often accompanied by abnormal coagulation and fibrinolysis. Clinical lesions primarily manifest in the kidneys, skin, nerves, and cardiovascular system. Renal involvement is one of the most serious manifestations of SLE, with clinical manifestations such as glomerulonephritis and impaired renal function. SLE-induced hematologic abnormalities lead to spleen damage, with significant splenomegaly observed in lupus model mice. The skin is the most commonly affected organ in SLE patients. Among SLE patients, 60% to 85% experience varying degrees of skin lesions, manifesting as cutaneous lupus erythematosus.

[0009] The mechanism of skin damage caused by SLE is complex. Under the influence of multiple factors, including genes, the environment, and hormones, chronic interferon overexpression leads to barrier damage, inflammatory cell infiltration, autoantigen release, and autoantibody production, resulting in skin damage with clinical manifestations such as rash, dermatitis, and butterfly spots. Due to the incurable nature and chronic nature of SLE, the skin damage caused by SLE is characterized by persistence and recurrence, often occurring on exposed areas of the body, severely impacting patients' daily lives.

[0010] SLE varies significantly in age, gender, race, and region of origin. It is most common in young adults, typically between the ages of 15 and 45, with a male:female ratio of 1:7 to 1:9. Reports of cases worldwide have been increasing in recent years.

[0011] Current treatment options primarily involve glucocorticoids combined with immunosuppressants. While these drugs offer immediate benefits, long-term use can easily lead to drug resistance, and common complications can severely impact the quality of life of SLE patients. Corticosteroids should not be used long-term due to the risk of side effects such as striae and vasodilation on fibroblasts and blood vessels. Topical calcineurin inhibitors also have mild side effects such as burning, itching, and erythema. Therefore, the search for new treatments and medications is urgent. Summary of the Invention

[0012] Collagen is the main component of the extracellular matrix. It is the most abundant and widely distributed macromolecular functional protein in various tissues and organs of mammals, accounting for about 30-40% of the body's protein. It is widely distributed in the tissues and organs of mammals in various forms, mainly including skin, bones and muscle tissues.

[0013] Due to its unique biological structure, collagen has good biocompatibility, degradability, and low antigenicity. In recent years, it has been widely used in many fields of production and life, such as biomedicine, cosmetics, health products, and food. To date, at least 29 different types of collagen have been discovered in the human body. They can be divided into two categories based on whether their structure is fibrous or not: fibrous and non-fibrous. Fibrous collagen mainly serves as a cell scaffold, fixing the position of cells and playing an anchoring role. It also provides tensile strength and stiffness to tissues. The main representative types are collagen types I, II, III, V, and XI. Non-fibrillar collagen is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each with different functions.

[0014] Collagen is a helical, fibrous protein composed of three peptide chains. Its unique hallmark is a right-handed triple-helical domain, characterized by repeated Gly-XY triplets, with proline and lysine typically occurring at positions X and Y. However, concerns about the purity, disease transmission, and reproducibility of animal-derived collagen limit its application, creating a need for recombinant collagen alternatives.

[0015] With the advancement of genetic engineering and synthetic biology, recombinant collagen with specific structures and functions has been developed and shows broad prospects for medical applications. Generally speaking, recombinant collagen is prepared by transcribing specific gene fragments and expressed in yeast, bacteria or animal cells. It can be produced on a large scale and the quality is controllable. In order to have good biocompatibility when used in the human body, a certain type of human collagen gene is currently selected to produce predictable and reliable recombinant collagen. In March 2021, the China National Medical Products Administration (NMPA) issued the "Guidelines for the Naming of Recombinant Collagen Biomaterials". Among them, recombinant humanized collagen is defined as a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination of functional fragments of human collagen.

[0016] Chinese patent application number 201911051106.3, entitled "Human Collagen Type 17 Polypeptide, Production Method and Use Thereof," describes a recombinant type XVII humanized collagen (RhCOLXVII). This technological research is based on the original gene sequence of human type XVII collagen. Codon optimization and splicing of highly water-soluble and bioactive portions were selected to produce a new recombinant human collagen sequence. Experimental studies have confirmed that this collagen has high expression levels, good water solubility, and high bioactivity, surpassing natural human collagen. Studies have shown that Rh COL XVII has the effects of promoting wound repair, promoting proliferation, and anti-inflammatory effects. However, the repair effect of RhCOLXVII on skin lesions in systemic lupus erythematosus has not been reported.

[0017] The present invention intends to use the recombinant humanized type XVII collagen in the Chinese patent application with application number 201911051106.3 as raw material to evaluate its effectiveness in the treatment of lupus erythematosus.

[0018] Specifically, the present invention provides use of recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen in the preparation of a preparation for preventing and / or treating lupus erythematosus.

[0019] In one of the technical solutions of the present invention, the recombinant type XVII humanized collagen comprises or consists of the sequence shown in (A)m, wherein each A is an amino acid sequence selected from any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, or an amino acid sequence in which one or more amino acid residues are substituted, added, inserted or deleted from any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, or a sequence having at least 83% sequence identity with the amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3; m is an integer between 1 and 10; wherein each A is the same or different and two adjacent As are directly connected by a peptide bond or by one or more amino acid residues.

[0020] Furthermore, the recombinant humanized type XVII collagen comprises or consists of the amino acid sequence shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0021] The "at least 83% sequence identity" mentioned in the present invention includes but is not limited to 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0022] In one technical solution of the present invention, the lupus erythematosus is selected from discoid lupus erythematosus.

[0023] In one technical solution of the present invention, the lupus erythematosus is selected from subacute cutaneous lupus erythematosus.

[0024] In one technical solution of the present invention, the lupus erythematosus is selected from lupus erythematosus profundus.

[0025] In one technical solution of the present invention, the lupus erythematosus is selected from neonatal lupus erythematosus.

[0026] In one technical solution of the present invention, the lupus erythematosus is selected from drug-induced lupus erythematosus.

[0027] In one technical solution of the present invention, the lupus erythematosus is selected from systemic lupus erythematosus.

[0028] In a further technical solution of the present invention, recombinant humanized collagen type XVII or a composition containing recombinant humanized collagen type XVII can reduce the expression of inflammatory factors in patients with lupus erythematosus; further, recombinant humanized collagen type XVII or a composition containing recombinant humanized collagen type XVII can reduce the expression of inflammatory factors in skin tissue. Furthermore, the inflammatory factors include but are not limited to interleukins, tumor necrosis factors, interferons, chemokines, etc. As some studies believe, the inflammatory factors associated with lupus erythematosus mainly include (Ren Hui, et al. Research progress on inflammatory indicators related to systemic lupus erythematosus. Progress in Clinical Medicine, 2023, 13(10), 15354-15360):

[0029] (1) Interleukin (IL)

[0030] IL-6 is produced by plasmacytoid dendritic cells and is crucial for the differentiation of B cells into plasma cells and the production of antibodies. It plays a key role in the host's resistance to pathogens and acute stress responses. IL-6 affects the pathophysiology of SLE by blocking Treg activity and inducing early Tfh differentiation. In addition, IL-6 is the main inducer of acute phase response proteins in the liver and plays an important role in immune system regulation. Studies by Saiyan Jin et al. have shown that IL-6 has a significant pro-inflammatory effect and is significantly elevated during the active phase of SLE. However, some people believe that the circadian rhythm of IL-6 may lead to a weak correlation with SLE activity. Therefore, for SLE, the key to solving the problem lies in overcoming the circadian rhythm problem of IL-6.

[0031] IL-18 binds to its receptor and activates the nuclear factor κB signaling pathway, leading to the production of interferon γ and immune responses. In the mouse MRL / LPR model, exogenous IL-18 administration exacerbates disease severity, whereas anti-IL-18 administration alleviates lupus. IL-18 levels have been reported to be elevated in patients with systemic lupus erythematosus (SLE) and positively correlate with disease activity. Furthermore, serum IL-18 levels have been associated with the severity of lupus nephritis. Studies by Renge et al. suggest that IL-18 may be involved in the pathogenesis of seizures associated with neuropsychiatric systemic lupus erythematosus (NPSLE) and correlate with disease activity. However, no clear association has been reported for other clinical manifestations. Therefore, IL-18 has the potential to become a promising biomarker for SLE.

[0032] IL-1β is the most studied member of the IL-1 family. It is primarily produced by monocytes and macrophages, with lymphocytes, neutrophils, endothelial cells, and fibroblasts also producing it. The expression (synthesis, processing, and secretion) of IL-1β is regulated by two signals: an initial signal induces the expression of inactive pro-IL-1β. Activation signals activate the inflammasome, followed by mature caspase-1, which cleaves pro-IL-1β into bioactive IL-1β, which is ultimately secreted and exerts its effects extracellularly through various pathways. IL-1β can exert its effects through various pathways, including autocrine, paracrine, and endocrine pathways. Therefore, IL-1β can act locally on the cells that produce it or adjacent cells, or it can act endocrinely on distant target cells to mediate systemic responses. However, IL-1β lacks a precursor state for storage and is highly susceptible to degradation. Upon stimulation by inflammatory cytokines, cells immediately initiate IL-1β gene transcription, but this is short-lived, and the transcript is highly susceptible to degradation. IL-1β is considered a typical multifunctional cytokine that affects almost all cell types, either alone or in combination with other cytokines. IL-1β is crucial for cellular defense and tissue repair in almost all tissues and is associated with pain, inflammation, and autoimmunity. Studies have shown that platelet activity is elevated in patients with SLE, and that platelet-endothelial interactions may play an important role in the pathogenesis of cardiovascular disease in patients with SLE by inducing activation of systemic lupus erythematosus endothelial cells through the IL-1β pathway. Blocking IL-1β secretion can not only inhibit this hyperreactive platelet phenotype in SLE, but also weaken the interaction between platelets and endothelial cells, thereby preventing the occurrence of cardiovascular disease to a certain extent. Therefore, IL-1β can be used as a potential therapeutic target for critical cases of SLE to slow down disease progression in SLE patients.

[0033] (2) Tumor necrosis factor (TNF)

[0034] TNF is directly induced by immune complexes and is closely associated with disease activity. Its levels are high in inflammatory organ diseases of SLE (such as lupus nephritis), making it a useful inflammatory marker for assisting in the diagnosis of active SLE. Saiyan et al. believe that endogenous immune complexes stimulate the activity of plasma dendritic cells, which in turn promote the secretion and synthesis of TNF-α, modulating the immune system and leading to the development of SLE. TNF-α acts on immune-competent cells, stimulating the secretion of inflammatory factors such as interleukins. It also induces vascular endothelial cells to express intercellular adhesion molecule-1, promoting adhesion, leading to leukocyte aggregation and adhesion, and ultimately damaging endothelial cells. However, the accuracy of TNF is uncertain due to the use of different detection methods. Some studies suggest that human soluble tumor necrosis factor receptor 2 (sTNFR2) levels are correlated with TNF and SLE activity, potentially becoming a more feasible parameter for diagnosing SLE.

[0035] B cell activating factor (BAFF, also known as TNFSF13B) is a cytokine that belongs to the tumor necrosis factor (TNF) ligand superfamily. Appropriate levels of BAFF help maintain normal immunity, while insufficient levels may cause immunodeficiency, and excessive levels may lead to abnormally high antibody production. When a patient exhibits autoimmunity, it produces antibodies against their own tissues. Both systemic lupus erythematosus and rheumatoid arthritis are caused by elevated levels of BAFF in the body. Therefore, regulating BAFF levels is crucial for the treatment of autoimmune diseases.

[0036] (3) Interferon (IFN)

[0037] With the development of sequencing technology, SLE has been found to have a distinct interferon gene signature. Elevated levels of type I (IFN-α), type II (IFN-γ), and type III (IFN-λ) interferons are observed in SLE patients and correlate with disease activity. Previous studies have shown that type I interferons are associated with susceptibility to SLE and are a major pathogenic factor. However, a study by Wenping et al. suggests that IFN-γ levels are higher in SLE patients than in healthy controls, and that IFN-γ accumulates in the body before SLE is diagnosed, suggesting that its role in SLE is as important as that of type I interferons. Furthermore, studies have shown that specific organ damage in SLE patients is associated with different interferon subtypes. For example, IFN-κ is involved in the pathogenesis of cutaneous lupus, while IFN-γ is associated with nephritis and arthritis. These findings have important implications for understanding the pathogenesis of SLE and for targeted therapy. Furthermore, blocking multiple intracellular interferon pathways may be a more promising therapeutic approach than blocking a single interferon pathway.

[0038] (4) Chemokines

[0039] Some chemokines have been found to play a role in the pathological progression of SLE patients. CCL2, by binding to CCR2 and CCR4 receptors on the surface of target cells, induces the directional movement of large numbers of immune cells, increases leukocyte adhesion, and enhances the permeability of renal endothelial cells. Other studies have shown that urinary CCL2 levels are correlated with the SLEDAI score, as well as proteinuria, urea nitrogen, and creatinine levels, and negatively correlated with hemoglobin and creatinine clearance. Therefore, urinary CCL2 is expected to become a new diagnostic indicator for lupus nephritis. CXCL10 enhances T cell adhesion to endothelial cells, has the highest correlation with SLE activity, and has the strongest predictive power for disease flare-ups. Furthermore, CXCL10 concentrations in the cerebrospinal fluid of SLE patients are high, potentially related to the manifestations of SLE in the central nervous system. CXCL13 has a selective chemotactic effect on B cells, and its level is positively correlated with the SLEDAI score and negatively correlated with C3. Furthermore, CXCL13 is associated with SLE-related autoimmune hemolytic anemia, with its level negatively correlated with hemoglobin, and can be used as a marker for assessing the severity of autoimmune hemolytic anemia. In addition to the above chemokines, CCL3, CCL4, CCL11, CCL20, CXCL9, and CXCL16 are also associated with SLE disease activity.

[0040] In a further technical solution of the present invention, recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen can reduce key markers in lupus erythematosus patients, including but not limited to TNFSF13B.

[0041] The disease is diagnosed according to the SLE diagnostic criteria revised by the American College of Rheumatology (ACR) in 1997. Skin lesions and lupus nephritis are the main risk factors for SLE progression and mortality, affecting about two-thirds of SLE patients. Patients with SLE skin lesions meet the typical cheek erythema and / or vasculitis (Xia Yuanrui, et al. Identification of biomarkers of skin lesions and lupus nephritis in patients with systemic lupus erythematosus. Chinese Journal of Disease Control and Prevention, 2021). In a further technical solution of the present invention, recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen can improve skin lesions in patients with lupus erythematosus.

[0042] Systemic lupus erythematosus (SLE) is a disease of the immune system caused by autoantibody imbalance. Following onset, large amounts of antibodies and immune complexes are produced in the body, causing tissue damage. Due to the targeted interaction between autoantibodies and nuclear antigen components, multiple organ damage occurs, including the kidneys, joints, and nervous system. Analysis of the underlying causes of the disease has revealed that hormones, genetics, and the environment all contribute to the disease, and the incidence of lupus nephritis (LN) in SLE patients exceeds 70%. With the advent of steroids and cyclophosphamide, survival rates have significantly improved, reaching over 90% over 10 years. Studies have also shown that immunosuppressive drugs combined with steroids can play a role in the treatment of LN (Zeng Huilin et al. Research Progress on the Pathogenesis and Treatment of Lupus Nephropathy. World Latest Medical Information Digest, 2020). In a further embodiment of the present invention, recombinant humanized type XVII collagen or compositions containing recombinant humanized type XVII collagen can improve kidney damage in patients with SLE.

[0043] The spleen, a crucial immune organ, is often overlooked for its enlargement in SLE patients. Symptoms such as anemia, leukopenia, and thrombocytopenia are hypothesized to be related to alterations in the blood circulation and immune microenvironment caused by splenomegaly. Studies suggest that IgG immune complexes deposit in the spleen, damaging the splenic immune barrier structure. IgG may interact with FcγR on macrophages in the marginal zone of the barrier, triggering migration and activation of marginal zone B cells, leading to the production of large amounts of autoantibodies (Zhang Qian et al. The Role of IgG in the Pathogenesis of Splenomegaly in Systemic Lupus Erythematosus. The 12th National Immunology Academic Conference. 2024). In a further technical solution of the present invention, recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen can improve splenic damage in patients with lupus erythematosus.

[0044] In actual treatment, recombinant humanized collagen type XVII can also be used in combination with other drugs for treating lupus erythematosus or prepared into a pharmaceutical composition, such as one or more of the non-steroidal anti-inflammatory drugs, antimalarial drugs, immunosuppressive drugs, and corticosteroid drugs disclosed in the prior art.

[0045] Commonly used therapeutic drugs include but are not limited to (Deng Xiaohu, et al. New progress in drug treatment of systemic lupus erythematosus. China Drug Application and Monitoring, 2005):

[0046] (1) Nonsteroidal anti-inflammatory drugs (NSAIDs)

[0047] Approximately 70% to 80% of SLE patients use NSAIDs during their disease course. All NSAIDs can relieve lupus-related symptoms such as fever, arthritis, serositis, and headache. However, the side effects of NSAIDs remain a concern. Studies have found that lupus nephritis is a risk factor for NSAID-related acute renal failure, and SLE patients are more likely to develop drug-induced liver injury using NSAIDs. In addition, for some patients who cannot tolerate oral NSAIDs, topical NSAID gels can be used to relieve joint symptoms.

[0048] (2) Antimalarial drugs

[0049] Commonly used preparations include hydroxychloroquine sulfate and chloroquine phosphate. Antimalarial drugs are very effective in treating lupus skin lesions and can also improve joint symptoms and mild systemic symptoms such as fever and fatigue. Recent evidence shows that hydroxychloroquine sulfate can also lower blood lipid levels, including cholesterol, triglycerides, and low-density lipoprotein, thereby slowing the progression of atherosclerosis in patients with SLE. Furthermore, a randomized, placebo-controlled study by Levy et al. demonstrated that hydroxychloroquine is also safe for use during pregnancy. Ten newborns born to patients (8 with SLE and 2 with DLE) who took hydroxychloroquine during pregnancy had no congenital anomalies, and no neurological abnormalities, including visual and auditory abnormalities, were found during follow-up for 1.5 to 3 years. Hydroxychloroquine can also inhibit the activation of platelet surface activation markers by cardiolipin antibodies, preventing thrombosis and improving Sjögren's syndrome symptoms.

[0050] (3) Steroid hormones

[0051] Although long-term use can lead to multiple side effects, including infection, diabetes, osteoporosis, and femoral head necrosis, steroids remain an indispensable mainstay of treatment for SLE and have garnered considerable attention from researchers. A prospective, multicenter study evaluated the role of moderate-dose prednisone in preventing severe lupus flare-ups. Forty-one SLE patients with serological abnormalities (elevated C3a and anti-ds-DNA) but stable clinical symptoms were randomized to receive either 30 mg / day of prednisone or placebo, with the dose gradually tapered after 4 weeks. During 12-18 months of follow-up, six patients in the placebo group experienced severe lupus flare-ups, while none in the prednisone group did so. The authors suggest that moderate-dose prednisone can prevent severe lupus flare-ups, thereby avoiding the need for subsequent high-dose steroid therapy.

[0052] (4) Immunosuppressants

[0053] The folic acid antagonist methotrexate (MTX) is widely used to treat various autoimmune diseases. SatoEI summarized 12 uncontrolled studies and 2 controlled studies on the treatment of SLE with MTX. Most studies confirmed the efficacy of MTX on the joint and skin lesions of SLE, but there is controversy about its therapeutic effect on lupus nephritis. Cyclophosphamide (CYC) is generally considered to be a conventional drug for the treatment of severe lupus nephritis and other important organ involvement, but it has obvious side effects, such as infection, gonadal suppression, and increased chance of tumors. Therefore, how to reduce the side effects of drugs has always been the research direction. The "Euro-Lupus Nephritis Trial" came up with a meaningful result. 90 patients with type I and IV lupus nephritis were divided into two groups.

[0054] One group received high-dose CYC treatment: 1 g, once a month pulse treatment, and after 6 months, pulse treatment every 3 months; the second group received low-dose CYC treatment: 500 mg, once every 2 weeks pulse treatment, for a total of 6 times, and then 1 mg·kg -1 ·d -1 The results showed that the remission rate of nephropathy in the low-dose group was 71%, and that in the high-dose group was 54%, while the subsequent relapse rates were 27% and 29%, respectively, with no significant difference between the two groups. The incidence of severe infection in the high-dose group was twice that of the low-dose group. This suggests that low-dose CYC treatment followed by AZA maintenance can achieve the same effect as high-dose CYC shock treatment, but the side effects can be greatly reduced. Azathioprine has no significant long-term protective effect on renal function in patients with lupus nephritis compared with CYC.

[0055] In addition, it also includes immunoglobulin, sex hormone intervention, mycophenolate mofetil (MMF), leflunomide, rituximab, LJP394, LymphoStat-B, anti-IL-10 monoclonal antibody, anti-CD154 (CD40L) monoclonal antibody, etc.

[0056] In the present invention, the drug is selected from oral preparations, external preparations or injections.

[0057] Oral preparations are drugs that are taken orally and absorbed through the gastrointestinal tract into the bloodstream to exert their therapeutic effects. These preparations are typically designed to be easy to swallow and absorb, such as tablets, capsules, granules, and solutions. Due to the specific nature of the raw materials used in this invention, when preparing oral preparations, the appropriate formulation method should be selected based on comprehensive considerations such as protease degradation in the body and ease of absorption.

[0058] Topical preparations are pharmaceutical preparations that are applied directly to the skin, mucous membranes, or body cavities to exert their therapeutic effects through local administration. These preparations typically have specific dosage forms, such as ointments, creams, gels, patches, lotions, etc.

[0059] Injections refer to sterile or aseptic pharmaceutical preparations intended for injection into the human body, including injection solutions, concentrated solutions for injection, and sterile powders for injection. These preparations are administered by injection through intravenous, intramuscular, or subcutaneous routes, directly entering the bloodstream or interstitial spaces to exert their therapeutic effects.

[0060] In the present invention, the medicine further comprises pharmaceutically acceptable excipients.

[0061] The pharmaceutically acceptable excipients described in the present invention are a general term for all additional materials in a drug other than the main drug. Excipients should have the following properties: (1) no toxic effects on the human body and almost no side effects; (2) stable chemical properties and not easily affected by temperature, pH, storage time, etc.; (3) no incompatibility with the main drug and no impact on the efficacy and quality inspection of the main drug; (4) no interaction with the packaging material.

[0062] The excipients in the present invention include but are not limited to fillers (diluents), lubricants (glidants or anti-adhesive agents), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc.

[0063] Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowdered silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, etc. , pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium lauryl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate 80, sorbitan oleate, poloxamer F-68, lecithin, soy lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0064] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active substance is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0065] Solid dosage forms such as tablets, drupes, capsules, pills, and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the release of the active substance in such compositions can be delayed in a certain part of the digestive tract. Examples of possible encapsulating components are polymeric substances and waxes. If desired, the active substance can also be microencapsulated with one or more of the above-mentioned excipients.

[0066] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active substance, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0067] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0068] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0069] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0070] Dosage forms for topical administration of the subject invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0071] The drug of the present invention can also be used in an injectable preparation. The injectable preparation is selected from a liquid injection (water injection), sterile powder for injection (powder injection), or injectable tablets (referring to molded tablets or machine-pressed tablets made using aseptic techniques, dissolved in water for injection upon use, and intended for subcutaneous or intramuscular injection).

[0072] The powder for injection contains, in addition to the active substance, at least an excipient. The excipient herein is an ingredient intentionally added to a drug and should not have pharmacological properties in the amount used. However, the excipient may aid in drug processing, solubility or dissolution, drug delivery via targeted routes of administration, or contribute to stability.

[0073] In the drug of the present invention, the content of recombinant humanized type XVII collagen is above 0.001% (equivalent to 0.01 mg / mL), and can further be above 0.01%, and further can be above 0.1%, including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% and the like.

[0074] The present invention also provides a method for preventing and / or treating lupus erythematosus, wherein the method comprises administering to the patient an effective dose of recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen. The effective dose includes, but is not limited to, 0.1 mg / Kg-500 mg / Kg, or 1-100 mg / Kg, or 1-50 mg / Kg, etc., for example, 0.1, 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / Kg, etc.

[0075] The present invention has the following beneficial effects:

[0076] (1) The present invention provides a method for treating SLE skin lesions by recombinant humanized type XVII collagen. Research data show that RhCOLXVII can promote the proliferation and migration of skin fibroblasts, accelerate epidermal growth, and inhibit the expression of inflammatory factors, thereby playing a role in treating SLE skin lesions. The RhCOLXVII used in this study is based on the original human COL17 gene sequence. The water-soluble and biologically active parts are selected for codon optimization and splicing recombination to obtain a new RhCOLXVII sequence. The RhCOLXVII sequence can be scalably produced using biofermentation technology. On the basis of wound repair, RhCOLXVII downregulates the SLE target gene TNFSF13B and reduces the expression of inflammatory factors. It can repair skin lesions caused by SLE, weaken the inflammatory response, cut off the inflammatory cycle unique to SLE, improve immune disorders, and accelerate skin repair. Compared with other corticosteroids and calcineurin inhibitors, RhCOLXVII, as a humanized collagen, has better biocompatibility and less toxic side effects, and has a good application prospect.

[0077] (2) If recombinant humanized type XVII collagen is used in combination with other drugs for treating lupus erythematosus, the dosage and frequency of administration of other drugs may be reduced, which is beneficial for reducing the adverse reactions caused by other drugs while ensuring a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The effect of rhCol XVII on the concentrations of anti-dsDNA ab and anti-ANA ab in the serum of MRL / lpr mice was compared with that of the control group.

[0079] Figure 2 The repair effect of rhCol XVII on skin damage in MRL / lpr mice.

[0080] Figure 3 Skin tissue staining results of MRL / lpr mice. A: HE and MASSON staining results of MRL / lpr mouse skin tissue. B: Area of positive area in MASSON staining. C: Dermis thickness in HE staining. **p<0.01, ***p<0.001 compared with the control group.

[0081] Figure 4 The effect of RhCOLXVII on inflammatory factors and TNFSF13B in the skin tissue of MRL / lpr mice. Compared with the control group, **p<0.01, ***p<0.001.

[0082] Figure 5 The effect of RhCOLXVII on the kidney and spleen of MRL / lpr mice. Compared with the control group, *p<0.05, ***p<0.001. DETAILED DESCRIPTION

[0083] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

[0084] In the present invention, the use of "may" includes both the meanings of performing a certain process and not performing a certain process.

[0085] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0086] In the present invention, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited elements or method steps.

[0087] In the present invention, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0088] In the present invention, a numerical range expressed using "numerical value A to numerical value B" or "numerical value A - numerical value B" means a range including the endpoints numerical values A and B.

[0089] In the present invention, the term "individual," "patient," or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0090] In the present invention, "treatment" means that after a subject suffers from a disease, the subject is exposed to (e.g., administered) the recombinant humanized type XVII collagen described in the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed to the disease, and does not necessarily mean that the symptoms of the disease are completely suppressed.

[0091] Being sick means that the body has symptoms of illness.

[0092] In the present invention, "prevention" means that before a subject develops a disease, the probability of developing the disease and / or the symptoms after developing the disease are reduced compared to when the subject does not develop the disease by exposing the subject to the recombinant humanized type XVII collagen described in the present invention, and it does not mean that the disease must be completely suppressed.

[0093] In the present invention, the recombinant humanized type XVII collagen is recorded in the Chinese patent application with application number 201911051106.3 and invention name “Human collagen type 17 polypeptide, its production method and use”.

[0094] In some embodiments, the recombinant type XVII humanized collagen described in the present invention comprises or consists of the sequence shown in (A)m, wherein each A is an amino acid sequence selected from any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, or an amino acid sequence in which one or more amino acid residues are substituted, added, or deleted from any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, or a sequence having at least 83% sequence identity with the amino acid sequence shown in any one of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3; m is an integer between 1 and 10; wherein each A is the same or different and two adjacent As are directly connected by a peptide bond or by one or more amino acid residues.

[0095] SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 described in the present invention are derived from the human type 17 collagen COL17A1 sequence. The human type 17 collagen COL17A1 sequence can be found at https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3.

[0096] SEQ ID No. 1:

[0097] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQG PKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA.

[0098] SEQ ID No. 2:

[0099] GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLP GAVGEPGAKGAMGPA.

[0100] SEQ ID No.3:

[0101] GADFAGDLDYNELAVRVSESMQRQGLLQGMAYTVQ GPPGQPGPQGPP GISKVFSAYSNVTADLMDFFQTYGAIQGPPGQKGEMGT PGPKGDRGPAGPPG HPGPPGPRGHKGEKGDKGDQ.

[0102] In some preferred embodiments, the recombinant humanized type XVII collagen of the present invention comprises or consists of the sequence shown in SEQ ID No. 4, which uses SEQ ID No. 1 as a repeating unit and is repeated three times.

[0103] SEQ ID No.4:

[0104] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQG PKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEA.

[0105] In other preferred embodiments, the recombinant humanized type XVII collagen of the present invention comprises or consists of the sequence shown in SEQ ID No. 5, which uses SEQ ID No. 2 as a repeating unit and is repeated three times.

[0106] SEQ ID No.5:

[0107] GLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPA.

[0108] In the present invention, amino acid addition refers to adding amino acids to the C-terminus or N-terminus of an amino acid sequence, such as SEQ ID No. 4, as long as the recombinant humanized type XVII collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 4.

[0109] In the present invention, amino acid substitution refers to the replacement of an amino acid residue at a certain position in an amino acid sequence, such as SEQ ID No. 4, by another amino acid residue, as long as the recombinant humanized type XVII collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 4.

[0110] In the present invention, the amino acid substitution may be a conservative amino acid substitution, which means that compared with the amino acid sequence of SEQ ID No. 4, three, preferably two, or one amino acid is replaced by an amino acid with similar or similar properties to form a peptide. These conservative variant peptides can be generated based on the following amino acid substitutions: substitution of Ala by Val, Leu, or Ile, substitution of Arg by Lys, Gln, Asn, or His, substitution of Asn by Gln, His, Lys, or Arg, substitution of Asn by Glu or Asn, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln, or Arg, substitution of Il by Leu, Met, Ala, Val, Phe, or norleucine, substitution of Cys by Ser or Ala, substitution of Gln by Asn or Glu, substitution of Glu by Asp or Gln, substitution of Gly by Ala, substitution of His by Asn, Lys, Gln, or Arg, substitution of Il by Leu, Met, Ala, Val, Phe, or norleucine, substitution of Il by The amino acid substitutions may also be non-conservative amino acid substitutions.

[0111] In the present invention, amino acid insertion refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, such as SEQ ID No. 4. The inserted amino acid residues may be completely or partially adjacent to each other, or none of the inserted amino acids may be adjacent to each other, as long as the recombinant humanized type XVII collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 4. In this context, the amino acid insertion position is not between the repeating sequences.

[0112] In the present invention, amino acid deletion means that 1, 2 or 3 or more amino acids can be deleted from an amino acid sequence, such as the sequence of SEQ ID No. 4, as long as the recombinant humanized type XVII collagen of the present invention retains the activity of the amino acid sequence of SEQ ID No. 4.

[0113] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0114] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In conjunction with this specification and common knowledge in the art, a person of ordinary skill in the art will clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0115] In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer's recommendations. All reagents or instruments that do not specify the manufacturer are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the main purpose of the present invention.

[0116] In the experiment of the present invention:

[0117] 0.1% NaCl was used as the negative control group, using normal saline;

[0118] Tacrolimus served as the positive control group, using 0.1% tacrolimus ointment;

[0119] RhCOLXVII is the treatment group of the present invention, using recombinant humanized type XVII collagen.

[0120] The MRL / lpr mouse is a spontaneous lupus erythematosus-like animal model. Lupus-like phenotypes primarily manifest as lymphadenopathy, splenomegaly, autoantibody production, and immune complex-mediated glomerulonephritis. Furthermore, MRL / Lpr mice can also display dermatitis and arthritis phenotypes commonly seen in human patients.

[0121] In the examples of the present invention, SEQ ID No. 2 is used as an example to demonstrate the effect of recombinant humanized type XVII collagen in the treatment of lupus erythematosus. It is believed that amino acid sequences that substitute, add, insert, or delete one or more amino acid residues based on SEQ ID No. 2, or sequences that have at least 83% sequence identity with the amino acid sequence shown in SEQ ID No. 2, all have similar activities.

[0122] Example 1 Recombinant humanized type XVII collagen reduces SLE serum markers in MRL / lpr mice

[0123] In the experimental group, RhCOLXVII type collagen (Shanxi Jinbo Biopharmaceutical Co., Ltd.) was subcutaneously injected into the injured area on the back of the mice. RhCOLXVII was dissolved in normal saline, and the dosage was 20 mg / kg, with 100 μL injected once every two days for 4 weeks. In the negative control group, 100 groups of normal saline were subcutaneously injected into the injured area on the back of the mice, with the same frequency as the experimental group. In the positive control group, tacrolimus ointment (Astellas Pharmaceuticals (China) Co., Ltd.) was applied to the injured area on the back of the mice, once in the morning and once in the evening every day, for 4 weeks. During the experiment, photos were taken and body weights were recorded every week. After 4 weeks, serum was collected from the mice and the mice were dissected to collect skin tissue, kidneys, and spleens from the injured area of the mice.

[0124] After the animal experiment, blood was collected from the mouse retro-orbital venous plexus, allowed to rest for 2 hours, and then centrifuged at 3000 rpm at 4°C for 10 minutes to obtain mouse serum. ELISA kits (Wuhan Elaruite Biotechnology Co., Ltd.) were used to quantify the expression levels of anti-dsDNA and anti-ANA antibodies in the serum of each group of mice. Biological experiments were performed in triplicate.

[0125] The experimental results are as follows Figure 1 The results show that:

[0126] Subcutaneous injection of RhCOLXVII reduced serum anti-dsDNA and anti-ANA concentrations in mice. Compared to the negative control group, RhCOLXVII reduced serum anti-dsDNA concentrations by 18% and anti-ANA concentrations by 66%. The animal models used in other embodiments of the present invention are similar to those used in this embodiment.

[0127] Example 2: rhCol XVII repairs skin damage in MRL / lpr mice

[0128] (1) Take photos and observe

[0129] Before and after subcutaneous injection of RhCOLXVII, the skin lesions of MRL / lpr mice were photographed and observed.

[0130] (2) HE staining

[0131] The fixed tissues were routinely dehydrated, embedded in paraffin, and sectioned at 4 μm. Dewaxed in xylene, the sections were then rehydrated with a gradient of ethanol (100%, 95%, 80%, and 70%). Hematoxylin staining, differentiation, bluing, and eosin staining were performed, followed by dehydration with a gradient of ethanol (70%, 80%, 95%, and 100%), and xylene clearing. After clearing, the sections were mounted with neutral gum. Tissues from each experimental group were examined under a DM6000B / DFC450 biological microscope (LEICA, DE) for light microscopic pathological examination.

[0132] (3) MASSON staining

[0133] The sections were routinely dewaxed and hydrated, stained with iron hematoxylin, differentiated, bluing, Ponceau fuchsin, and Fast Green, and then dehydrated using a gradient of 95% ethanol and anhydrous ethanol. Transparentation was followed by mounting with neutral gum. Tissues from each experimental group were examined under a DM6000B / DFC450 biological microscope (LEICA, DE) for light microscopic pathological examination.

[0134] result:

[0135] The results of mouse skin observation are as follows Figure 2 The results of HE staining and MASSON staining are shown as follows Figure 3 The results showed that the area of skin damage on the neck of mice injected with RhCOLXVII was significantly reduced, and the redness and inflammation of the epidermis were weakened. The skin damage was repaired and new hair had grown. The skin damage area of the mice in the negative control group expanded, and the redness and inflammation still existed. The area of skin redness of the mice in the positive control group expanded. Long-term use of positive drugs can lead to adverse reactions. The skin tissue of the mice in the negative control group showed focal necrosis and scab formation in the epidermis, accompanied by inflammatory cell infiltration and hyperplasia of epidermal squamous cells. The positive control mice showed epidermal scab formation, spinous proliferation of epidermal squamous cells, inflammatory cell infiltration in the dermis, and hyperplasia of fibrous tissue in the dermis.

[0136] Compared to the negative control group, mice injected subcutaneously with RhCOLXVII showed no significant pathological changes. Furthermore, the average dermal thickness of the experimental group increased by 72%, significantly higher than that of the negative control group. The collagen content in the dermis of the experimental group was significantly higher than that of the negative control group, reaching 2.26 times that of the negative control group. This increase in collagen content aids in ECM repair and fibroblast growth, promoting dermal thickening, proliferation of epidermal basal cells and squamous epithelial cells, and accelerated injury recovery, without any signs of adverse reactions.

[0137] Example 3 rhCol XVII reduces the expression of inflammatory factors in the skin of MRL / lpr mice

[0138] Immunohistochemistry experiments

[0139] Paraffin-embedded skin tissue was sliced into 5 μm sections and baked at 60°C for 1 hour. Dewaxed with xylene and hydrated with a gradient of ethanol (100% to 75%). Washed with PBS for 3 minutes three times. Boiled in EDTA antigen retrieval solution (95°C, 15 minutes), heated on high for 4 minutes, and then on low for 10 minutes, and cooled naturally to room temperature. Washed with PBS for 5 minutes three times. Incubated with 10% BSA serum blocking solution at room temperature for 30 minutes. Primary antibodies diluted as required were added dropwise, including IL-1β, IL-6, and TNFSF13B antibodies purchased from Beijing Biosen Biotechnology Co., Ltd. Incubated overnight at 4°C.

[0140] Wash with PBS for 3 minutes three times, then add secondary antibody (rabbit two-step kit, Zhongshan Jinqiao) and incubate at room temperature for 20 minutes. Wash with PBS for 3 minutes three times, develop with DAB in the dark for 3-5 minutes, and then stop washing with water. Microscopic examination was performed to observe whether the tissue was positively stained. Counterstain with hematoxylin for 20 seconds to 2 minutes, differentiate with hydrochloric acid and alcohol for several seconds, and rinse with tap water for 5-15 minutes. Dehydrate routinely, clear, mount, and examine with an OLYMPUS microscope (BX51) and scan with an OLYMPUS scanner (VS200).

[0141] The present invention conducted immunohistochemical analysis of IL-1β, IL-6, and TNFSF13B on the skin tissue of MRL / lpr mice. Figure 4 Results showed that compared with the negative control group, the area of IL-1β-positive cells in the experimental group decreased by 51.96%, and IL-6 decreased by 76.78%. TNFSF13B, a key marker for SLE treatment evaluation, decreased by 64.53%.

[0142] The above results suggest that subcutaneous injection of RhCOLXVII significantly reduces the expression of inflammatory factors in skin tissue and has the ability to treat SLE skin lesions.

[0143] Example 4 rhColXVII improves pathological changes in the kidney and spleen of MRL / lpr mice

[0144] The effects on the kidney and spleen of lupus erythematosus were observed by HE and MASSON staining.

[0145] The experimental results are as follows Figure 5 show.

[0146] The results showed that compared with the negative control group, the experimental group had reduced multifocal infiltration of inflammatory cells in the renal cortex, medulla, and around blood vessels, the focal infiltration of inflammatory cells in the renal pelvic mucosa was significantly alleviated, and the degree of glomerular lesions was reduced. The results of the HE lesion scoring of renal tissue showed that the renal lesion score of the experimental group mice was significantly lower than that of the negative control group mice (Ordinary one-way ANOVA test). The kidney mass results showed that although there was no statistical difference in kidney mass between the groups of mice (Ordinary one-way ANOVA test), the kidney mass of the experimental group mice was lower than that of the negative and positive control groups. The above results suggest that subcutaneous injection of RhCOLXVII can reduce the damage of SLE to the kidneys and improve kidney enlargement.

[0147] Spleen tissue staining results showed that compared with the negative control group, the experimental group had significantly increased splenic lymph node volume and reduced cellular proliferation, as well as decreased extramedullary hematopoiesis. Spleen tissue HE lesion scoring results showed that the experimental group had significantly lower spleen lesion scores than the negative control group (Ordinary one-way ANOVA test). Spleen mass results showed that while there was no statistical difference in spleen mass between the groups (Ordinary one-way ANOVA test), the experimental group had lower spleen mass than the negative and positive control groups. Subcutaneous injection of RhCOLXVII can reduce the damage of SLE to the spleen and improve splenomegaly.

[0148] The results showed that subcutaneous injection of RhCOLXVII can reduce the damage of SLE to the kidneys and spleen and improve the enlargement of the kidneys and spleen.

Claims

1. Use of recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen in the preparation of a preparation for treating lupus erythematosus; the recombinant humanized type XVII collagen is selected from SEQ ID No.

2.

2. The use according to claim 1, characterized in that The lupus erythematosus is selected from discoid lupus erythematosus, subacute cutaneous lupus erythematosus, profundal lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and systemic lupus erythematosus.

3. The use according to claim 1, characterized in that The lupus erythematosus is selected from systemic lupus erythematosus.

4. The use according to claim 1, characterized in that Recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen has one or more of the following effects: (1) reducing the expression of inflammatory factors in lupus erythematosus patients, wherein the inflammatory factors include one or more of IL-6, IL-1β, and TNFSF13B; (2) Improve skin damage in patients with lupus erythematosus; (3) Improve kidney damage in patients with lupus erythematosus; (4) Improve spleen damage in patients with lupus erythematosus.

5. The use according to claim 1, characterized in that The composition containing recombinant humanized type XVII collagen also contains one or more of non-steroidal anti-inflammatory drugs, antimalarial drugs, immunosuppressive drugs, and corticosteroid drugs that can treat lupus erythematosus.

6. The use according to claim 1, characterized in that The preparation is selected from oral preparations, external preparations or injections.

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