New application of recombinant XVII type humanized collagen

Abstract

The invention provides an application of a recombinant XVII type humanized collagen or a composition containing the recombinant XVII type humanized collagen in preparation of a preparation for preventing and / or treating lupus erythematosus, which can overcome the defect that more adverse reactions are generated in treatment in the existing scheme.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the 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, which is more common in women aged 15 to 40. It is a spectrum disease and can be divided into subtypes such as discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), deep lupus erythematosus (LEP), neonatal lupus erythematosus (NLE), drug-induced lupus erythematosus (DIL), and systemic lupus erythematosus (SLE).

[0003] Discoid lupus erythematosus mainly invades the skin and is the mildest type of lupus erythematosus. A few may have mild visceral damage, and a few cases may turn into systemic lupus erythematosus. Skin lesions initially appear as one or several bright red spots, ranging in size from mung beans to soybeans, with adhesive scales on the surface, which gradually expand and become round or irregular in shape, with significantly darker pigmentation at the edges, slightly higher than the center. Lesions are mainly distributed in sun-exposed areas, such as the face, helix, and scalp, and a few may involve the upper chest, back of the hands, forearms, lips, and oral mucosa. Most patients have no symptoms of skin lesions, but it is difficult for them to completely disappear. New lesions may gradually increase or remain unchanged for many years. Discoid lesions worsen after sun exposure or fatigue. Lesions on the scalp can cause permanent hair loss. Old lesions can occasionally develop into squamous cell carcinoma of the skin.

[0004] Subacute cutaneous lupus erythematosus is a special intermediate type that is less common in clinical practice. There are two types of skin lesions: annular erythema and papule-squamous. In most cases, the two types of skin lesions exist alone, but in a few cases, they can exist together. Skin lesions often recur, and most patients have visceral damage, but severe cases are rare. The main symptoms are joint pain, muscle pain, and repeated low fever. A few have nephritis and changes in the blood system.

[0005] Lupus erythematosus profundus, also known as lupus panniculitis, is also an intermediate type of lupus erythematosus. Skin lesions are nodules or plaques located in the deep dermis or subcutaneous fat tissue. They are of varying sizes and numbers. The surface skin color is normal or light red, and the texture is firm and non-mobile. Lesions can occur in any part of the body, most commonly in the cheeks, buttocks, arms, and the lower legs and chest. Lupus erythematosus profundus is unstable and can exist alone, and later transform into discoid lupus erythematosus, or systemic lupus erythematosus, or coexist with them.

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

[0007] Drug-induced lupus erythematosus is mainly manifested by fever, joint pain, myalgia, facial butterfly rash, oral ulcers, and serositis. ANA, anti-histone antibodies, anti-ss-DNA antibodies, etc. may be positive. The condition gradually improves after stopping the drug. For patients with severe conditions, appropriate amounts of glucocorticoids can be given.

[0008] Systemic lupus erythematosus (SLE) is a complex autoimmune disease that affects multiple organs and tissues. It is mainly characterized by the production of multiple specific autoantibodies and abnormal activation of the immune system, often accompanied by abnormal coagulation and fibrinolysis. Clinical lesions mainly manifest in the kidneys, skin, nerves, and cardiovascular systems. Kidney involvement is one of the most serious manifestations of SLE, with clinical manifestations such as glomerulonephritis and impaired renal function. The blood system abnormalities caused by SLE lead to spleen damage, and obvious splenomegaly was observed in lupus model mice. The skin is the most commonly affected organ in SLE patients. Among SLE patients, 60% to 85% have varying degrees of skin damage, manifested as cutaneous lupus erythematosus.

[0009] The mechanism of skin damage caused by SLE is very complex. Under the influence of multiple factors such as genes, environment, and hormones, chronic interferon overexpression leads to barrier damage, inflammatory cell infiltration, autoantigen release, and autoantibody production, which leads to skin damage, clinically manifested as rash, dermatitis, butterfly spots, etc. Due to the incurable nature of SLE and its chronic nature, the skin damage caused by SLE is characterized by persistence and recurrence, and often occurs in exposed parts of the body, seriously affecting the daily life of patients.

[0010] SLE varies greatly in age, sex, race, and region of onset. The disease is more common in young and middle-aged people, mostly in the age group of 15 to 45 years old, with a male:female ratio of 1:7 to 1:9. In recent years, there has been an increasing trend in reported cases worldwide. Epidemiological surveys in many regions of the United States reported that the prevalence of SLE was 14.6 to 122 / 100,000; a one-time survey of a large sample in my country (>30,000 people) showed that the prevalence of SLE was 70 / 100,000, and as high as 113 / 100,000 in women. According to a latest epidemiological survey report, between 1995 and 2013, the survival rate of SLE patients in my country was 94% within 5 years and 89% within 10 years. The main causes of death were infection (33.2%), lupus nephritis (18.7%), lupus encephalopathy (13.8%), and cardiovascular disease (11.5%).

[0011] At present, most treatment methods are glucocorticoids combined with immunosuppressants. Although they have immediate effects, long-term medication can easily cause the body to develop drug resistance, and common complications can seriously affect the quality of life of SLE patients. Corticosteroids cannot be used for a long time because of 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, it is urgent to actively explore new treatment methods and therapeutic drugs. Summary of the invention

[0012] Collagen is the main component of the extracellular matrix. It is the most abundant and widely covered 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. So far, at least 29 different types of collagen have been found in the human body, which can be divided into two categories: fibrous and non-fibrous according to whether the structure is fibrous. Fibrous collagen mainly serves as a cell scaffold, fixing the position of cells, playing an anchoring role, and also providing tensile strength and stiffness for tissues. The main representative types are collagen types I, II, III, V, and XI. Non-fibrous collagen is further subdivided into basal collagen, short-chain collagen, transmembrane collagen, etc., each of which has different functions.

[0014] Collagen is a helical fibrous protein composed of three peptide chains, and its unique hallmark is a right-handed triple helical domain, i.e., a protein with repeated Gly-XY triplets, where proline and lysine usually appear at positions X and Y. However, concerns about the purity, disease transmission, and reproducibility of animal-derived collagen limit its application, and recombinant collagen is needed to replace animal-derived collagen.

[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, expressed in yeast, bacteria or animal cells, and can be produced on a large scale with controllable quality. 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 recombinant technology, or a combination of functional fragments of human collagen.

[0016] The Chinese patent application with application number 201911051106.3 and invention name “Human collagen type 17 polypeptide, its production method and use” records a recombinant type XVII humanized collagen (RhCOLXVII). This technical research is based on the original gene sequence of type XVII human collagen. The parts with strong water solubility and high biological activity are selected for codon optimization and splicing recombination to obtain a new recombinant human collagen sequence. Experiments have confirmed that this collagen has a large expression amount, good water solubility, and high biological activity, and its performance is better than human natural collagen. Studies have shown that Rh COL XVII has the effects of promoting wound repair, promoting proliferation, and anti-inflammatory. The repair effect of RhCOLXVII on skin damage 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 schemes 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 A's 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% and the like.

[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 of the technical solutions 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 of the technical solutions 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, and the like. As some studies have shown, 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. It is essential 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 pathophysiological mechanism of SLE by blocking Treg activity and inducing early Tfh differentiation. In addition, IL-6 is the main inducer of acute-phase response proteins produced by the liver and plays an important role in the regulation of the immune system. The study of Saiyan Jin et al. showed that IL-6 has a significant effect of promoting inflammation and is significantly increased 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, resulting in the production of interferon γ and causing immune effects. In the mouse MRL / LPR model, administration of exogenous IL-18 can aggravate the severity of the disease in mice, while administration of anti-IL-18 can alleviate lupus. It has been reported that IL-18 levels are increased in SLE patients and are positively correlated with the activity of the disease. In addition, it has been determined that serum IL-18 is associated with the severity of lupus nephritis. The study by Renge et al. shows that IL-18 may be involved in the pathogenesis of neuropsychiatric systemic lupus erythematosus (NPSLE)-related epileptic seizures and is associated with disease activity. However, no obvious correlation has been reported in 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 mainly produced by monocytes and macrophages, and is also produced by lymphocytes, neutrophils, endothelial cells, and fibroblasts. The expression (synthesis, processing, and secretion) of IL-1β is regulated by dual signals. The initial signal induces cells to express pro-IL-1β, which is inactive. The activation signal activates the inflammasome. Subsequently, mature Caspase-1 cuts pro-IL-1β into bioactive IL-1β, which is finally secreted to the extracellular space through different pathways to exert its effects. IL-1β can exert its effects in a variety of ways, such as autocrine, paracrine, and endocrine. Therefore, IL-1β can act on the cells that produce it or adjacent cells to exert its effects locally, or it can act on distant target cells through endocrine to mediate systemic reactions. However, it has no precursor state storage and is easily degraded. After the relevant cells are stimulated by inflammatory factors, they will immediately start the gene transcription of IL-1β, which is short-lived and the transcription products are easily degraded. IL-1β is considered a typical multifunctional cytokine that affects almost all types of cells, either alone or in combination with other cytokines. IL-1β is essential for cellular defense and tissue repair of almost all tissues and is associated with pain, inflammation, and autoimmunity. Studies have shown that platelet activity is increased in SLE patients, and platelet-endothelial interactions may play an important role in the pathogenesis of cardiovascular disease in SLE patients by inducing activation of systemic lupus erythematosus endothelial cells through the IL-1β pathway. If IL-1β secretion is blocked, not only can this highly reactive platelet phenotype in SLE be inhibited, but also the interaction between platelets and endothelial cells can be weakened, 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 the progression of the disease in SLE patients.

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

[0034] TNF is directly induced by immune complexes and is closely related to the activity of the disease. It is present in higher levels in SLE inflammatory organ diseases (such as lupus nephritis) and is a good inflammatory marker for assisting the diagnosis of active SLE. Saiyan et al. believe that endogenous immune complexes stimulate the activity of plasma dendritic cells, and a large number of dendritic cells promote the secretion and synthesis of TNF-α, regulate the immune system in the body, and lead to the occurrence of SLE. TNF-α acts on immune-active cells to stimulate the secretion of inflammatory factors such as interleukins. It can also induce vascular endothelial cells to express intercellular adhesion molecule-1 to complete adhesion, causing leukocyte aggregation and adhesion, thereby destroying vascular endothelial cells. Due to the use of different detection methods, the accuracy of TNF is not very reliable. Some studies believe that the level of human soluble tumor necrosis factor receptor 2 (sTNFR2) is related to the activity of TNF and SLE, and may become 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 BAFF helps maintain normal immunity levels, while insufficient levels may cause immunodeficiency and excessive levels may cause abnormally high antibody production. When a patient exhibits autoimmunity, it produces antibodies against its own tissues. Both systemic lupus erythematosus and rheumatoid arthritis are caused by higher levels of BAFF in the body. Therefore, regulating the level of BAFF is quite important for the treatment of autoimmune diseases.

[0036] (3) Interferon (IFN)

[0037] With the development of sequencing technology, SLE was found to have obvious interferon gene characteristics. Higher levels of type I (IFN-α), type II (IFN-γ), and type III (IFN-λ) interferons can be observed in SLE patients and are related to the activity of the disease. Many previous studies have shown that type I interferon is related to the susceptibility of SLE and is the main pathogenic factor. The study by Wenping et al. believes that the level of IFN-γ in SLE patients is higher than that in normal people, and there is accumulation of IFN-γ in the body before SLE is diagnosed. Its role in SLE is as important as type I interferon. In addition, studies have shown that specific organ damage in SLE patients is related to different interferon subtypes. For example, IFN-κ is related to the pathogenesis of cutaneous lupus, while IFN-γ is associated with nephritis and arthritis. Therefore, these results are of great significance for understanding the pathogenesis and targeted treatment of SLE. In addition, compared with blocking one interferon pathway, blocking several interferon pathways in cells may be a more promising treatment method.

[0038] (4) Chemokines

[0039] Some chemokines have been found to play a role in the pathological process of SLE patients. CCL2 binds to CCR2 and CCR4 receptors on the surface of target cells, causing a large number of immune cells to move in a directional manner, increasing the adhesion of leukocytes and the permeability of renal endothelial cells. Other studies have shown that the level of urinary CCL2 is associated with SLEDAI scores and proteinuria, urea nitrogen and creatinine levels, but negatively correlated with hemoglobin and creatinine clearance. For this reason, urinary CCL2 is expected to become a new indicator for the diagnosis of lupus nephritis. CXCL10 can enhance the adhesion of T cells to endothelial cells, has the highest correlation with SLE activity, and has the strongest predictive ability for disease outbreaks. In addition, in SLE patients, the concentration of CXCL10 in cerebrospinal fluid is high, which may be related to the manifestation 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. In addition, CXCL13 is also related to SLE-related autoimmune hemolytic anemia, and its level is negatively correlated with hemoglobin, which can be used as a marker to assess the severity of autoimmune hemolytic anemia. In addition to the above chemokines, CCL3, CCL4, CCL11, CCL20, CXCL9, CXCL16, etc. are also related to 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 of lupus erythematosus patients, including but not limited to TNFSF13B.

[0041] The disease was 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 death, 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, 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 body's immune system. The disease occurs due to autoantibody disorders. After the disease occurs, a large amount of antibodies and immune complexes are produced in the body, causing tissue damage, and because of the targeted relationship between autoantibodies and nuclear antigen components, multiple organs of the body are damaged, including the kidneys, joints, and nervous system. Analysis of the causes of the disease found that hormones, genetics, and the environment can all cause the disease, and the incidence of lupus nephritis (LN) in SLE patients is more than 70%. With the emergence of hormones and cyclophosphamide drugs, the survival rate of the disease has been significantly improved, and the survival rate of more than 10 years can reach more than 90%. Studies have shown that immunosuppressive drugs combined with hormones can play a certain role in the treatment of LN (Zeng Huilin, et al. Research progress in the pathogenesis or treatment of lupus nephropathy. World Latest Medical Information Digest, 2020). In a further technical solution of the present invention, recombinant type XVII humanized collagen or a composition containing recombinant type XVII humanized collagen can improve kidney damage in patients with lupus erythematosus.

[0043] The spleen is an important immune organ of the body. The enlargement of the spleen in SLE patients is often ignored. The symptoms of anemia, leukopenia and thrombocytopenia in SLE patients are speculated to be related to the changes in blood circulation and immune microenvironment caused by splenomegaly. Some studies have shown that IgG immune complexes are deposited in the spleen, causing damage to the spleen immune barrier structure. IgG may interact with FcyR of macrophages in the marginal zone of the barrier, causing migration and activation of B cells in the marginal zone, and then producing a large number 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 type XVII humanized collagen or a composition containing recombinant type XVII humanized collagen can improve spleen damage in patients with lupus erythematosus.

[0044] In actual treatment, recombinant humanized type XVII collagen 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. Chinese Drug Application and Monitoring, 2005):

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

[0047] About 70% to 80% of SLE patients use NSAIDs during the course of their disease. All NSAIDs can relieve lupus-related symptoms such as fever, arthritis, serositis and headache. However, the side effects of NSAIDs are still of concern. Studies have found that lupus nephritis is a risk factor for NSAID-related acute renal insufficiency, and SLE patients are more likely to experience drug-induced liver damage when using NSAIDs. In addition, for some patients who cannot tolerate oral NSAIDs, NSAIDs gels can be used topically 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 delaying the progression of atherosclerosis in SLE patients. In addition, a randomized placebo-controlled study by LevyRA et al. showed that hydroxychloroquine is also safe for use during pregnancy. The newborns born by 10 patients (8 SLE and 2 DLE) who took hydroxychloroquine during pregnancy had no congenital abnormalities, and no abnormalities including neurological vision and hearing were found during follow-up for 1.5 to 3 years. Hydroxychloroquine can also inhibit the activation of cardiolipin antibodies on platelet surface activation markers, prevent thrombosis, and improve the symptoms of Sjögren's syndrome.

[0050] (3) Steroid hormones

[0051] Although long-term use can lead to multiple side effects such as infection, diabetes, osteoporosis, and femoral head necrosis, steroid hormones are still the most important and indispensable drugs for the treatment of SLE and have attracted the attention of researchers. A prospective, multicenter study evaluated the role of moderate doses of prednisone in preventing severe lupus activity. 41 SLE patients with serological abnormalities (increased C3a and anti-ds-DNA) and stable clinical symptoms were randomly assigned to receive 30 mg / d of prednisone or placebo, and the dose was gradually reduced after 4 weeks. During the follow-up of 12 to 18 months, 6 patients in the placebo group had severe lupus activity, while none in the prednisone group had such an occurrence. The authors believe that moderate doses of prednisone can prevent severe lupus relapses, thereby avoiding the use of high-dose hormone therapy in the future.

[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 have 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 drug side effects has always been a research direction. The "Euro-Lupus Nephritis Trial" came to 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 then every 3 months after 6 months; the second group received low-dose CYC treatment: 500 mg, once every 2 weeks, pulse treatment for 6 times, and then 1 mg kg -1 ·d -1 The results showed that the remission rate of kidney disease was 71% in the low-dose group and 54% in the high-dose group, 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 refer to preparations that take drugs into the body through the oral route, absorb them into the blood circulation through the gastrointestinal tract, and thus exert therapeutic effects. Such preparations are usually designed to be easy to swallow and absorb, such as tablets, capsules, granules, solutions, etc. Due to the particularity of the raw materials in the present invention, if they are prepared into oral preparations, the degradation of proteases in the body, the ease of absorption, etc. are comprehensively considered, and the appropriate preparation method is selected.

[0058] Topical preparations refer to pharmaceutical preparations that are directly applied to the skin, mucous membranes or body cavity surfaces and exert therapeutic effects through local administration. Such preparations usually have specific dosage forms, such as ointments, creams, gels, patches, lotions, etc.

[0059] Injections refer to sterile or aseptic drug preparations for injection into the human body, including injection solutions, concentrated injection solutions and sterile injection powders, etc. These preparations are injected through veins, muscles, subcutaneous injections, etc., and directly enter the blood circulation or tissue spaces to exert 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 the drug except the main drug. The 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 micropowder 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 , pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium dodecyl 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 hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan oleate, poloxamer F-68, lecithin, soybean 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 starch 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 glycol, 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, pills, 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 opacifiers, and the release of the active substance in such compositions may be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active substance can also be formed into microcapsules 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, isopropanol, 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 of these substances, etc.

[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] Suspensions, in addition to the active ingredients, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0069] Compositions for parenteral injection may include 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 for injection preparations. The injection is selected from liquid injection (water injection), sterile powder for injection (powder injection) or injection tablets (referring to molded tablets or machine-pressed tablets made by aseptic operation of the drug, dissolved in injection water before use, for subcutaneous or intramuscular injection).

[0072] The powder for injection contains at least an excipient in addition to the active substance. The excipient in the present invention is a component intentionally added to the drug, which should not have pharmacological properties in the amount used, but the excipient can help the processing, dissolution or dissolution of the drug, delivery of the drug through a targeted administration route or stability.

[0073] In the medicine of the present invention, the content of recombinant humanized type XVII collagen is above 0.001% (equivalent to 0.01 mg / mL), and can be further above 0.01%, and can be further 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 an effective dose of recombinant humanized type XVII collagen or a composition containing recombinant humanized type XVII collagen is administered to the patient. 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., such as 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 recombinant humanized type XVII collagen for treating SLE skin damage. Research data show that RhCOLXVII can promote the proliferation and migration of skin fibroblasts, accelerate the growth of the epidermis, and inhibit the expression of inflammatory factors, thereby playing a role in treating SLE skin damage. The RhCOLXVII used in this study is based on the original gene sequence of human COL17. The parts with strong water solubility and high biological activity are selected for codon optimization and splicing recombination to obtain a new RhCOLXVII sequence, which can be mass-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 damage 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 corticosteroid drugs and calcineurin inhibitor drugs, 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 concentration 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 This is the repairing effect of rhCol XVII on skin damage in MRL / lpr mice.

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

[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] Embodiments of the present invention will be described below, but the present invention is not limited thereto.

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

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

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

[0087] In the present invention, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., refer to specific elements (e.g., features, structures, properties and / or characteristics) related to the embodiments described herein, which are included in at least one embodiment described herein, and may or may not exist 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 “a numerical value A to a numerical value B” or “a numerical value A-a numerical value B” means a range including the endpoints 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 suffering from a disease, a 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 must be completely suppressed.

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

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

[0093] In the present invention, the recombinant type XVII humanized 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 the sequence shown in (A)m 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 IDNo.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 IDNo.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 IDNo.3; m is an integer between 1 and 10; wherein each A is the same or different and two adjacent A's are directly connected by a peptide bond or by one or more amino acid residues.

[0095] The SEQ ID No.1, SEQ ID No.2 or SEQ ID No.3 described in the present invention is 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 3 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, 3, preferably 2 amino acids or 1 amino acid are replaced by amino acids with similar or close properties to form a peptide. These conservative variant peptides can be generated according to 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 Glu or Asn for Asp, 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 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 of an amino acid sequence, such as the sequence of SEQ ID No. 4. The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not 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 article, the insertion position of the amino acid is not between each repeating sequence.

[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 combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, a person of ordinary skill in the art can make any modification or change to the technical solution of the present invention, and such modification and change are also included in the scope of the present invention.

[0115] In the examples, if the specific conditions are not specified, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. 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 gist 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 was 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] MRL / lpr mice are spontaneous lupus erythematosus-like animal models. Lupus-like phenotypes are mainly manifested by lymphadenopathy, splenomegaly, production of autoantibodies, and immune complex-mediated glomerulonephritis. In addition, MRL / Lpr can also show 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 treating lupus erythematosus. It should be considered that amino acid sequences that replace, 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 collagen (Shanxi Jinbo Biopharmaceutical Co., Ltd.) was subcutaneously injected into the injured part of the back of the mice. RhCOLXVII was dissolved in saline, the dosage was 20 mg / kg, 100 μL was injected, once every two days, and the injection was continued for 4 weeks. In the negative control group, 100 groups of saline were subcutaneously injected into the injured part of the back of the mice, and the frequency was the same as that of the experimental group. In the positive control group, tacrolimus ointment (Astellas Pharmaceuticals (China) Co., Ltd.) was applied to the injured part of the back of the mice, once in the morning and evening every day, and continued to be applied for 4 weeks. During the experiment, photos were taken every week to observe and record the body weight. After 4 weeks, the mouse serum was collected and dissected, and the skin tissue, kidneys, and spleens of the injured part of the mouse skin were collected.

[0124] After the animal experiment, the mouse blood was collected by retro-orbital venous plexus blood collection, and the blood was allowed to stand for 2 hours and centrifuged at 3000 rpm and 4°C for 10 minutes to obtain mouse serum. The expression levels of anti-dsDNA ab and anti-ANA ab in the serum of each group of mice were quantified using ELISA kits (Wuhan Elaruite Biotechnology Co., Ltd.). The biological experiments were performed in triplicate.

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

[0126] Subcutaneous injection of RhCOLXVII can reduce the concentration of anti-dsDNA ab and anti-ANA ab in mouse serum. Compared with the negative control group, RhCOLXVII reduced the concentration of anti-dsDNA ab in serum by 18% and the concentration of anti-ANA ab by 66%. The animal models of other embodiments of the present invention are the same as those of 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, paraffin-embedded, and sliced ​​at 4 μm. Dewaxed with xylene, hydrated with ethanol (100%, 95%, 80%, 70%) in a gradient manner. Hematoxylin staining, differentiation, blueing, eosin staining, ethanol (70%, 80%, 95%, 100%) gradient dehydration, and xylene transparent. After transparentization, the slides were sealed with neutral gum, and the tissues of each test group were taken for light microscopic pathological examination on a DM6000B / DFC450 biological microscope (LEICA, DE).

[0132] (3) MASSON staining

[0133] The sections were routinely dewaxed and hydrated, stained with iron hematoxylin, differentiated, blued, Ponceau fuchsin, fast green, dehydrated with 95% ethanol and anhydrous ethanol, and transparentized with xylene. After transparency, the sections were sealed with neutral gum, and the tissues of each test group were taken for light microscopic pathological examination under a DM6000B / DFC450 biological microscope (LEICA, DE).

[0134] result:

[0135] The results of mouse skin photography are as follows Figure 2 The results of HE staining and MASSON staining are shown as follows Figure 3 Show. 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 was 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 redness area of ​​the skin of the mice in the positive control group expanded. Long-term use of positive drugs can lead to adverse reactions. Focal necrosis and scab formation occurred in the epidermis of the skin tissue of the mice in the negative control group, accompanied by inflammatory cell infiltration, epidermal squamous cell hyperplasia, etc. The positive control mice had epidermal scab formation, epidermal squamous cells proliferated in a spinous manner, inflammatory cells infiltrated in the dermis, and fibrous tissue in the dermis proliferated.

[0136] Compared with the negative control group, mice injected subcutaneously with RhCOLXVII showed no obvious pathological conditions. At the same time, compared with the negative control group, the average dermis thickness of the experimental group mice increased by 72%, which was significantly higher than that of the negative control group mice. Compared with the negative control group mice, the collagen content of the dermis of the experimental group mice was significantly higher than that of the negative control group, which was 2.26 times that of the negative control group. The increase in collagen content helps the repair of ECM and the growth of fibroblasts, promotes the thickening of the dermis, the proliferation of epidermal basal cells and squamous epithelial cells, and accelerates the recovery of injuries, 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

[0139] The paraffin-embedded skin tissue was sliced ​​into 5 μm sections and baked at 60°C for 1 hour. Dewaxed with xylene and hydrated with gradient ethanol (100% to 75%). Washed with PBS for 3 minutes / 3 times. Boiled in EDTA antigen retrieval solution (95°C, 15 minutes), high heat for 4 minutes, low heat for 10 minutes, and cooled naturally to room temperature. Washed with PBS for 5 minutes / 3 times. Incubated at room temperature for 30 minutes with 10% BSA serum blocking solution. Added primary antibodies diluted as required, which were IL-1β antibody, IL-6 antibody, and TNFSF13B antibody purchased from Beijing Biosen Biotechnology Co., Ltd. Incubated overnight at 4°C.

[0140] Wash with PBS for 3 min / 3 times, add secondary antibody (rabbit two-step kit, Zhongshan Jinqiao), and let stand at room temperature for 20 min. Wash with PBS for 3 min / 3 times, develop with DAB in the dark for 3-5 min, and stop washing with water. Perform microscopic examination to observe whether the positive color on the tissue is stained. Then perform hematoxylin counterstaining for 20s-2min, differentiate with hydrochloric acid alcohol for a few seconds, and rinse with tap water for 5-15min. Routine dehydration, transparency, sealing, microscopic examination with OLYMPUS microscope (BX51), and scanning with 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. The experimental results are as follows Figure 4 The results showed that compared with the negative control group, the positive area of ​​inflammatory factor IL-1β in the experimental group of mice 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 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 mice, the multifocal infiltration of inflammatory cells in the renal cortex, medullary interstitium and around blood vessels in the experimental group mice was reduced, the focal infiltration of inflammatory cells under the renal pelvic mucosa was significantly alleviated, and the degree of glomerular lesions was reduced. The results of renal tissue HE lesion scoring 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 control group and the positive control group. The above results suggest that subcutaneous injection of RhCOLXVII can reduce the harm of SLE to the kidneys and improve the condition of kidney enlargement.

[0147] The results of spleen tissue staining showed that compared with the negative control group mice, the experimental group mice had significantly reduced splenic lymph node volume and cell proliferation, and the extramedullary hematopoiesis was alleviated. The results of spleen tissue HE lesion scoring showed that the spleen lesion score of the experimental group mice was significantly lower than that of the negative control group mice (Ordinary one-way ANOVA test). The spleen mass results showed that although there was no statistical difference in spleen mass between the groups of mice (Ordinary one-way ANOVA test), the spleen mass of the experimental group mice was lower than that of the negative control group and the positive control group mice. Subcutaneous injection of RhCOLXVII can reduce the damage of SLE to the spleen and improve the enlargement of the spleen.

[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 preventing and / or treating lupus erythematosus.

2. The use according to claim 1, characterized in that 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 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 A's are directly connected via a peptide bond or via one or more amino acid residues.

3. The use according to claim 2, characterized in that: 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.

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

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

6. The use according to claim 1, characterized in that: The recombinant humanized type XVII collagen or the composition containing the recombinant humanized type XVII collagen has one or more of the following effects: (1) Reduce the expression of inflammatory factors in patients with lupus erythematosus; (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.

7. The use according to claim 6, characterized in that The inflammatory factors include but are not limited to one or more of IL-6, IL-1β, and TNFSF13B.

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

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

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