A polypeptide AL-8(0) targeting IL17RA molecule and its application

Abstract

The present invention relates to the field of biopharmaceuticals, and in particular to a polypeptide AL-8(0) targeting IL17RA molecules and its application. The specific sequence of the polypeptide described in the present invention is: ASPVIYHL-NH2. This polypeptide can specifically recognize IL17RA on the surface of immune cells, and further has the ability to block the mutual binding between IL17A-IL17RA molecules, thereby blocking the IL17A-IL17RA immune response pathway, thereby blocking certain excessive inflammatory reactions and achieving anti-inflammatory effects. The polypeptide AL-8(0) can be used for the prevention and treatment of a variety of autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological medicines, and in particular to a polypeptide AL-8(0) targeting an IL17RA molecule and an application thereof. Background Art

[0002] Interleukin 17 (IL17) refers to a family of pro-inflammatory cytokines, which includes a series of pro-inflammatory cytokines, which are mainly produced by T helper cells 17 (Th17). In the initial process of autoimmune diseases, a large number of initial T cells stimulated by APCs will differentiate into Th17 cells, and these differentiated Th17 cells will produce a large number of IL17 family cytokines. The first cytokine discovered in the IL17 family was IL17A, which was first cloned in the activated T cell hybridoma of rodents in 1993. When it was first discovered, it was named cytotoxic T lymphocyte-associated protein 8 (CTLA-8). Later studies found that it had effects similar to cytokines, so it was renamed IL17A, and the name CTLA-8 continued to be used on the same protein on the surface of rodent cells.

[0003] As free cytokines secreted by cells into the microenvironment, the IL17 family members cannot function without binding to the target receptors. When IL17A interacts with the receptor IL17RA, IL17A first binds to the cell surface receptor IL17RA, and then forms a heterodimer with IL17RC / RD to mediate downstream signals. In this reaction process, cytokines (TGFβ, IL6, IL23, etc.) secreted by antigen presenting cells (APCs) induce initial T cells to differentiate into Th17 cells. The differentiated Th17 will secrete a large amount of IL17A. Then the combination of IL17A and IL17RA can induce a variety of target cells (such as fibroblasts, endothelial cells, macrophages, etc.) to express a variety of inflammatory factors and chemokines to promote inflammatory responses, such as IL-6, G-CSF, GM-CSF, IL-1β, TGF-β, TNF-α, etc., and can also cause the activation of a variety of inflammatory response pathways such as NF-κB pathway, MAPKs pathway, etc., thereby activating more inflammatory responses, recruiting more immune cells, and secreting more inflammatory factors. The excessive accumulation of inflammatory cells in the inflammatory area and the retention of too many inflammatory factors in the inflammatory area often become the main cause of autoimmune diseases. As for autoimmune diseases, the defect of the immune system's ability to distinguish causes APCs to react to immunogenic substances, which triggers increased differentiation of Th17 cells and triggers a series of downstream excessive inflammatory reactions and autoimmune disease reactions.

[0004] In the treatment of autoimmune diseases, it is very important to alleviate the degree of inflammatory response at the lesion site of the patient. This is not only for the effective treatment of the patient's disease, but also a necessary process to greatly alleviate the patient's physical and mental pain during the onset of the disease. In the onset of autoimmune diseases, the interaction between IL17A and IL17RA is one of the key factors affecting the degree of inflammatory response. By inhibiting the expression of IL17A or IL17RA in immune cells in the inflammatory response area, or blocking the interaction between IL17A and IL17RA, excessive inflammatory response can be reduced, thereby achieving the purpose of alleviating the symptoms of autoimmune diseases, and cooperating with other drugs to achieve effective treatment of the disease. Therefore, from the perspective of drug development, blocking the interaction between IL17A and IL17RA is a good direction for treating the symptoms of autoimmune diseases.

[0005] Regarding this treatment direction, there are currently many autoimmune disease treatment drugs on the market that use IL17A as a target, which relieve disease symptoms by blocking the IL17A-IL17RA interaction. However, there are few targeted drug studies on IL17RA, the cell surface receptor of IL17A, and there are also few drugs on the market. Therefore, there is a large research gap in this field, but it also has broad research prospects. It should also be noted that most molecules in the IL17 family need to bind to IL17RA to exert their functions. In addition to IL17A, we cannot ignore the existence of other IL17 family molecules. IL17RA targeted drugs have the potential to block the function of multiple IL17 molecules. The reason why multiple IL17A targeted drugs have poor therapeutic effects on autoimmune diseases all points to the direction of not blocking other IL17 family molecules. In addition, the ultimate goal of inhibiting IL17A-IL17RA interaction is to inhibit the immune response of target cells. Compared with drugs that act on cytokines, drugs that act directly on target cells can directly intervene in the cell's signal transduction pathways and reduce the impact on non-target physiological processes, thereby showing higher specificity and obtaining more obvious and lasting therapeutic effects, which is expected to achieve better therapeutic effects in the treatment of autoimmune diseases.

[0006] Therefore, conducting research on the treatment of autoimmune diseases targeting IL17RA and developing related targeted drugs for IL17RA is an urgent topic with certain innovative and scientific research significance, and has a high degree of economic and social demand. Summary of the invention

[0007] Problems to be solved by the present invention

[0008] At present, there is a great shortage of drugs for treating autoimmune diseases that target IL17RA for treatment. The polypeptide of the present invention acts on IL17RA to inhibit the IL17A-IL17RA pathway, thereby achieving anti-inflammatory effects, which is highly innovative. It has been verified by multiple experimental models that it has good anti-inflammatory effects, and therefore has great development prospects.

[0009] Technical Solution

[0010] In order to solve the above problems, the technical solution adopted by the present invention is as follows:

[0011] A polypeptide AL-8(0), characterized in that its amino acid sequence is: ASPVIYHL-NH2, and an amino acid sequence that is more than 80% homologous to the polypeptide.

[0012] A pharmaceutical composition, characterized in that it contains the polypeptide AL-8(0) and pharmaceutically acceptable excipients, wherein the excipients include conventional diluents, fillers, adhesives, wetting agents, absorption enhancers, surfactants, lubricants and stabilizers in the pharmaceutical field.

[0013] The pharmaceutical composition is characterized in that the drug can prevent or treat autoimmune diseases through a variety of administration methods, including subcutaneous or intramuscular injection, intravenous injection or intravenous drip, oral administration such as pills, capsules, etc., and nasal spray.

[0014] The use of the polypeptide AL-8(0) or the pharmaceutical composition in the preparation of a drug for treating autoimmune diseases.

[0015] The application is characterized in that the autoimmune diseases include: rheumatoid arthritis, osteoarthritis, gouty arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, aplastic anemia, Sjögren's syndrome, urticaria, allergic rhinitis or asthma.

[0016] In the described polypeptide AL-8(0) sequence, "-NH2" indicates that the C-terminus of the peptide chain has been amidated.

[0017] definition

[0018] As used herein, the terms "a" or "an" may mean one.

[0019] As used herein, the term "or" means "and / or" unless explicitly indicated as only alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions referring to only alternatives as well as "and / or."

[0020] As used herein, the term "another" means at least a second or more.

[0021] As used herein, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method being employed to determine the value, or the variation that exists between study subjects.

[0022] Autoimmune diseases for which the present treatment method can be used include any type, such as those caused by exogenous or endogenous antigens. Generally, the abnormal immune response of the human body attacks normal cells. At least 80 such diseases have been found so far, which can occur in almost any part of the human body, and the symptoms are often intermittent. Exemplary disease types may include, but are not limited to, autoimmune diseases from the following organs, selected from: skin, respiratory tract, thyroid, blood, gastrointestinal tract, colon, cecum, stomach, brain, head, neck, spine, ovary, testicle, kidney, larynx, sarcoma, lung, bladder, prostate and breast. Other examples of autoimmune diseases that can be treated using the methods provided herein include, but are not limited to, atopic dermatitis, urticaria, allergic rhinitis, asthma, severe allergic reaction, anaphylactic shock, food allergy, neonatal hemolytic disease, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, bullous pemphigoid, pemphigus vulgaris, rheumatic fever, Cuban syndrome, Guillain-Barre syndrome, diffuse toxic goiter, myasthenia gravis, pernicious anemia, Henoch-Schonlein purpura, allergic vasculitis, reactive arthritis, farmer's lung, poststreptococcal glomerulonephritis, serum sickness, Arthur's reaction, systemic lupus erythematosus, subacute bacterial endocarditis, rheumatoid arthritis, allergic contact dermatitis, type I diabetes, Hashimoto's thyroiditis, multiple sclerosis, celiac disease, giant cell arteritis, reactive arthritis, hypersensitivity pneumonitis, transplant rejection, Sjögren's syndrome, autoimmune hepatitis, autoimmune polyglandular syndrome, autoimmune adrenalitis, systemic autoimmune disease.

[0023] The term "pharmaceutical composition" may include excipients. Specific excipients include buffers, surfactants, preservatives, fillers, polymers and stabilizers, which can be used with the antibody. Buffers are used to control the pH of the composition. Surfactants are used to stabilize proteins, inhibit protein aggregation, inhibit protein adsorption to surfaces, and assist protein refolding. Exemplary surfactants include Tween 80, Tween 20, Brij 35, Triton X-10, Pluronic F127 and sodium dodecyl sulfate. Preservatives are used to inhibit microbial growth. Examples of preservatives include benzyl alcohol, metacresol and phenol. Fillers are used during the freeze-drying process to increase the volume. Hydrophilic polymers (such as dextran, hydroxyethyl starch, polyethylene glycol, gelatin) can be used to stabilize proteins. Polymers with non-polar parts (such as polyethylene glycol polymers) can also be used as surfactants. Protein stabilizers can include polyols, sugars, amino acids, amines and salts. Suitable sugars include sucrose and trehalose. Amino acids include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. Proteins such as human serum albumin can also competitively adsorb to the surface and reduce aggregation of antibodies. It should be noted that specific molecules can be used for multiple purposes. For example, histidine can be used as a buffer and antioxidant. Glycine can be used as a buffer and a filler.

[0024] The area where the autoimmune disease to be treated occurs is preferably positive for IL17RA.

[0025] Beneficial results

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The anti-inflammatory effect of the polypeptide involved in the present invention is good in both in vivo and in vitro models. After a large number of experiments, the inventors have learned that the polypeptide AL-8(0) can inhibit the development of adjuvant rat rheumatoid arthritis, collagen rat rheumatoid arthritis, and monosodium urate (MSU) crystal-induced rat gouty arthritis animal models. In vivo experiments have shown that it has significant effects on the treatment of the above diseases, with fewer side effects, less dosage and lower costs. Mechanism of action: The polypeptide AL-8(0) of the present invention binds to IL17RA, and then competitively blocks the binding of IL17A to IL17RA, thereby producing an anti-inflammatory effect. Specific evidence is: Fluorescence microscopy has confirmed that the polypeptide AL-8(0) can bind to cells with high expression of IL17RA and bind to the cell membrane surface, but cannot bind to cells with defective IL17RA expression.

[0028] (2) Currently, there are very few immunotherapy drugs developed for the inflammatory response caused by IL17RA, and there are very few drugs on the market, so the invention is highly innovative.

[0029] (3) The polypeptide of the present invention has a simple structure and is easy to synthesize, separate and purify. It effectively inhibits the IL17A-IL17RA pathway and exhibits good anti-inflammatory activity.

[0030] (4) The polypeptides of the present invention have weak adverse reactions and toxic side effects;

[0031] In summary, the polypeptide designed by the present invention is scientific, reasonable, feasible and effective, and can be used as a drug for treating or preventing autoimmune inflammatory diseases. It greatly expands the disease treatment range of the polypeptide, provides new ideas and prospects for future drug development, and has significant social value and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 , the effect of polypeptide AL-8(0) on the swelling of the left paw of rats with collagenous arthritis model;

[0033] Figure 2 , the effect of polypeptide AL-8(0) on the swelling of the right paw of rats with collagenous arthritis model;

[0034] Figure 3 , the effect of polypeptide AL-8(0) on the joint scores of the limb joints in the collagen-type arthritis model rats;

[0035] Figure 4 , the effect of polypeptide AL-8(0) on the swelling of the left paw of rats with adjuvant arthritis model;

[0036] Figure 5 , the effect of polypeptide AL-8(0) on the swelling of the right paw of rats with adjuvant arthritis model;

[0037] Figure 6 , the effect of polypeptide AL-8(0) on the joint scores of the limb joints in rats with adjuvant arthritis model;

[0038] Figure 7 , the effect of polypeptide AL-8(0) on the swelling of the left paw of rats with gouty arthritis induced by MSU crystals;

[0039] Figure 8 , the effect of polypeptide AL-8(0) on the swelling of the right paw of rats with gouty arthritis induced by MSU crystals;

[0040] Fig. 9 , the effect of polypeptide AL-8(0) on the joint scores of the limb joints in rats with MSU crystal-induced gouty arthritis animal model; Fig.10, the specific binding fluorescence imaging results of polypeptide AL-8(0) with the target at the cellular level, where A is the immunofluorescence result of normal HaCaT cells; B is the immunofluorescence result of IL17RA-deficient HaCaT cells. DETAILED DESCRIPTION

[0041] Example 1 Synthesis of polypeptide AL-8(0)

[0042] The polypeptide AL-8(0) was synthesized by solid phase synthesis, purified by high performance liquid chromatography, and the purity of the polypeptide was determined by RP-HPLC. The synthesis method was based on patents 201110194918.0 and 201110370529.9.

[0043] Results: The purity of the synthesized peptide was identified by reversed liquid chromatography analysis. The results are as follows: the purity of peptide AL-8(0) is 97.21%, which is greater than 95%, meeting the design requirements. Alternatively, you can entrust Shanghai Jier to synthesize it.

[0044] Example 2 Therapeutic effect of polypeptide AL-8(0) in collagen-induced arthritis model in rats

[0045] A collagen rat arthritis model was constructed to study the therapeutic effect of peptides on collagen-induced arthritis (CIA) in rats. SD rats were used as test animals. Thirty SPF SD rats (provided by SPF (Beijing) biotechnology co., Ltd., animal production license number: SCXK (Beijing) 2024-0001) were female, 7-8 weeks old, and weighed 180-220 g. The rats were randomly divided into 6 groups, namely normal control group, model control group, positive drug control group (methotrexate 10 mg / kg), and three low, medium, and high dose groups of peptide AL-8 (0) (5, 10, and 15 mg / kg). Except for the normal control group, rat CIA models were established in each experimental group on day 0. The method was to dissolve chicken cartilage type II collagen (cⅡ) with 0.1 mol / l acetic acid into a 4 mg / ml solution and keep it in a refrigerator at 4 ℃ overnight. On the day of the experiment, type II collagen was fully emulsified with complete Freund's adjuvant (CFA) containing 4 mg / ml Myeobaeterium tuberculosis strain H37Rv. After SD rats were anesthetized with isoflurane, 50 μl of the emulsifier was injected intradermally into the tail of each rat for sensitization. 21 days later, 4 mg / ml type II collagen (cⅡ) was fully emulsified with incomplete Freund's adjuvant (IFA) and the same dose of emulsifier was injected into the tail for re-immunization. Subcutaneous injection was performed from the 30th day of modeling: three dose groups of peptide AL-8(0) (5, 10, 15 mg / kg): twice a day for 15 consecutive days; positive drug control group (methotrexate MTX 10 mg / kg): once every 5 days, for 3 consecutive times for 15 days; normal control group and model control group: saline twice a day for 15 consecutive days. The rat foot volume and joint score were measured before modeling (0 day) and every 3 days from modeling to the 45th day. The ankle diameters of the left and right hind feet were measured and the swelling was calculated to observe the effect of the drug on collagen arthritis in rats. On the 45th day after modeling, the rats were killed by dislocation.

[0046] The evaluation indicators of arthritis are as follows:

[0047] 1) Joint scoring

[0048] Limbs: Scored on a five-point scale of 0-4: 0 = no erythema or swelling; 1 = mild erythema or swelling, with erythema or swelling in one of the front / hind toe joints; 2 = erythema or swelling in more than one toe; 3 = swelling of the paw below the ankle or wrist joint; 4 = swelling of all paws including the ankle joint. All four paws of the rat were scored separately, with a maximum score of 16 points.

[0049] Joint scores were performed before modeling (0 day) and every 3 days from modeling to day 45, and the results were recorded.

[0050] 2) Measure ankle diameter and calculate swelling

[0051] The diameter from the inside to the outside of the left and right ankles and the ankle thickness were measured with a vernier caliper before modeling (0 day) and every 3 days from modeling to the 45th day to calculate the foot volume. The foot swelling was calculated and the results were recorded. The swelling calculation formula is (Vt-V0) / V0, where V0 is the foot volume before modeling (mm 3 ), Vt is the foot volume after modeling (mm 3 The foot volume was measured once before modeling and every 3 days thereafter.

[0052] The quantitative data of the test results were expressed as the mean plus or minus the standard deviation (mean ± SD). SPSS11.0 software was used to perform t-tests between the treatment groups and the control group. * express p < 0.05, ** express p < 0.01.

[0053] result:

[0054] Compared with normal rats, the rats were intradermally injected with an emulsion containing inactivated Mycobacterium tuberculosis complete Freund's adjuvant and collagen in equal volumes into the tail of the model rats after modeling. After 21 days, the rats were intradermally injected with an emulsion containing incomplete Freund's adjuvant and collagen in equal volumes into the tail for booster immunization. On the 10th day after the first immunization, the paws of CIA rats became red and swollen, and the arthritis index score increased. The swelling peaked on the 30th to 45th day in the model group, and the body weight of the model group began to decline from the 3rd day. The in vivo immune protection effect of the peptide in the collagen-induced rat arthritis animal model is shown in Table 1. The data in the table are the results of the left and right hind feet and joint scores of rats in each group at 45 days. The peptide AL-8(0) can play an in vivo immune protection role in the collagen-induced rat arthritis animal model. The specific results are as follows:

[0055] Effect of polypeptide AL-8(0) on swelling of left hind foot in rats with collagenous arthritis Figure 1 The swelling degree of the left hind foot in the positive control group, high-dose, medium-dose and low-dose groups of peptide AL-8(0) were significantly different from those in the model group ( p <0.01), the experimental results were statistically significant. The effect of polypeptide AL-8(0) on the swelling of the right hind foot of rats with collagenous arthritis is shown in Figure 2 The joint swelling degree of the positive control group, high-dose, medium-dose and low-dose groups of peptide AL-8(0) were significantly different from those of the model group ( p<0.01), the experimental results were statistically significant. The effect of polypeptide AL-8(0) on the joint score of collagen arthritis rats is shown in Figure 3 The limb scores of the positive control group, low-, medium-, and high-dose groups of peptide AL-8(0) were significantly lower than those of the model control group ( p <0.01), which was significantly different from the model control group, and the experimental results were statistically significant.

[0056] Table 1 The immune protective effect of polypeptide AL-8(0) on collagen-based rat arthritis model in vivo

[0057]

[0058] * express p < 0.05, ** express p < 0.01.

[0059] Conclusion: Peptide AL-8(0) has therapeutic effect on collagen arthritis in rats.

[0060] Example 3 The therapeutic effect of polypeptide AL-8(0) on adjuvant-induced arthritis in rats

[0061] An adjuvant-induced arthritis model was established to study the therapeutic effect of peptides on adjuvant-induced arthritis (AIA) rats. SD rats were used as test animals. Thirty SPF-grade SD rats (provided by SPF (Beijing) biotechnology co., Ltd., animal production license number: SCXK (Beijing) 2024-0001), female, 7-8 weeks old, weighing 180-220 g, were used as test animals. They were randomly divided into 6 groups, namely normal control group, model control group, positive drug control group (methotrexate 10 mg / kg), and 3 low, medium and high dose groups of peptide AL-8(0) (5, 10, 15 mg / kg). Except for the normal group, rat AIA models were established in all experimental groups on day 0 by injecting 0.08 ml of complete Freund's adjuvant containing inactivated Mycobacterium tuberculosis (H37RA, 10 mg / ml) into the left hind foot of the rats to establish the rat adjuvant arthritis model. Subcutaneous injection of drugs was performed from day 10 after modeling: three dose groups of peptide AL-8(0) (5, 10, 15 mg / kg): twice a day for 15 consecutive days; positive drug control group (methotrexate MTX 10 mg / kg): once every 5 days for 3 consecutive times for 15 consecutive days; normal control group and model control group: saline twice a day for 15 consecutive days. Joint scores of all rats were performed on days 4, 7, 10, 14, 17, 20, 23, and 25 after modeling, and the swelling of the left and right hind feet was measured to observe the effects of drugs on rat adjuvant arthritis. On day 25 after modeling, the rats were killed by dislocation.

[0062] The evaluation indicators of arthritis are as follows:

[0063] 1) Joint scoring

[0064] Limbs: Scored on a five-point scale of 0-4: 0 = no erythema or swelling; 1 = mild erythema or swelling, with erythema or swelling in one of the anterior / posterior toe joints; 2 = erythema or swelling in more than one toe; 3 = swelling of the paw under the ankle or wrist joint; 4 = swelling of all paws including the ankle joint. The four paws of the rats were scored separately, with the highest score being 16 points. Joint scores were performed on all rats before modeling (0 day) and on days 4, 7, 10, 14, 17, 20, 23, and 25 after modeling, and the results were recorded.

[0065] 2) Measure ankle diameter and calculate swelling

[0066] The diameter from the inside to the outside of the left and right ankles and the ankle thickness were measured with a vernier caliper before modeling (0 day) and on days 4, 7, 10, 14, 17, 20, 23, and 25 after modeling to calculate the foot volume. The foot swelling was calculated and the results were recorded. The swelling calculation formula is (Vt-V0) / V0, where V0 is the foot volume before modeling (mm 3 ), Vt is the foot volume after modeling (mm 3 The foot volume was measured once before modeling (0 day), and then once on the 4th, 7th, 10th, 14th, 17th, 20th, 23rd, and 25th days.

[0067] The quantitative data of the test results were expressed as the mean plus or minus the standard deviation (mean ± SD). SPSS11.0 software was used to perform t-tests between the treatment groups and the control group. * express p < 0.05, ** express p < 0.01.

[0068] Results: Compared with normal rats, after injection of complete Freund's adjuvant containing inactivated Mycobacterium tuberculosis into the left hind foot of the rats after modeling, primary arthritis will occur rapidly in the left hind foot, with obvious swelling and ulceration; secondary arthritis will begin to occur in the right hind foot about 4 days later, and the score will gradually increase; at the same time, the ear blood vessels will proliferate significantly, with obvious redness and swelling; and the tail joint will swell. The results of the in vivo immune protection of the peptide in the adjuvant rat arthritis animal model are shown in Table 2. The data in the table are the results of the left and right hind feet and joint scores of rats in each group at 25 days. The peptide AL-8(0) can play an in vivo immune protection role in the adjuvant rat arthritis animal model. The specific results are as follows:

[0069] Effect of polypeptide AL-8(0) on swelling of left paw of rats with primary arthritis Figure 4 The swelling degree of the left hind foot in the rat positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) were significantly different from those in the model group ( p <0.01), the experimental results were statistically significant. The effect of polypeptide AL-8(0) on the swelling of the right hind foot of rats with secondary arthritis is shown in Figure 5 The swelling degree of the right hind foot of rats in the positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) was significantly different from that in the model group ( p <0.01). The effect of polypeptide AL-8(0) on the joint score of rats with adjuvant arthritis is shown in Figure 6 The limb scores of the rat positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) were significantly lower than those of the model control group ( p<0.01), and the differences were statistically significant compared with the model control group.

[0070] Table 2 Immunoprotective effect of polypeptide AL-8(0) on adjuvant arthritis rat model

[0071]

[0072] * express p < 0.05, ** express p < 0.01.

[0073] Conclusion: Peptide AL-8(0) has therapeutic effect on adjuvant arthritis in rats.

[0074] Example 4 Therapeutic effect of polypeptide AL-8(0) in MSU crystal-induced gouty arthritis animal model

[0075] MSU (Monosodium urate) crystal-induced gouty arthritis rat model was established to study the therapeutic effect of peptide on gouty arthritis (Gout) rats. SD rats were used as test animals. Thirty SPF SD rats (provided by SPF (Beijing) biotechnology co., Ltd., animal production license number: SCXK (Beijing) 2024-0001), female, 7-8 weeks old, weighing 180-220 g, were used as test animals. They were randomly divided into 6 groups, namely normal control group, model control group, positive drug control group (colchicine 10 mg / kg), and 3 low, medium and high dose groups of peptide AL-8(0) (5, 10, 15 mg / kg). Except for the normal group, the rat gout model was established in each experimental group on day 0 by injecting 200ul of PBS buffer suspension containing MSU crystals (tlrl-msu, InvivoGen, 25 mg / mL) into the left hind foot of the rats, and adding potassium oxonate (1.5g / kg / day) to the drinking water of the rats until the end of the experiment. Subcutaneous injection of drugs was performed from day 10 of modeling: three dose groups of peptide AL-8(0) (5, 10, 15 mg / kg): twice a day for 15 consecutive days; positive drug control group (colchicine Col 10 mg / kg): once every 5 days, for 3 consecutive times for 15 days; normal control group and model control group: physiological saline twice a day for 15 consecutive days. The joint scores of all rats were measured and the swelling of the left and right hind feet was measured on the 4th, 7th, 10th, 14th, 17th, 20th, 23rd and 25th days after modeling to observe the effect of drugs on MSU crystal-induced gouty arthritis in rats. On the 25th day after modeling, the rats were killed by dislocation.

[0076] The evaluation indicators of arthritis are as follows:

[0077] 1) Joint scoring

[0078] Limbs: Scored on a five-point scale of 0-4: 0 = no erythema or swelling; 1 = mild erythema or swelling, with erythema or swelling in one of the anterior / posterior toe joints; 2 = erythema or swelling in more than one toe; 3 = swelling of the paw under the ankle or wrist joint; 4 = swelling of all paws including the ankle joint. The four paws of the rats were scored separately, with the highest score being 16 points. Joint scores were performed on all rats before modeling (0 day) and on days 4, 7, 10, 14, 17, 20, 23, and 25 after modeling, and the results were recorded.

[0079] 2) Measure ankle diameter and calculate swelling

[0080] The diameter from the inside to the outside of the left and right ankles and the ankle thickness were measured with a vernier caliper before modeling (0 day) and on days 4, 7, 10, 14, 17, 20, 23, and 25 after modeling to calculate the foot volume. The foot swelling was calculated and the results were recorded. The swelling calculation formula is (Vt-V0) / V0, where V0 is the foot volume before modeling (mm 3 ), Vt is the foot volume after modeling (mm 3 The foot volume was measured once before modeling (0 day), and then once on the 4th, 7th, 10th, 14th, 17th, 20th, 23rd, and 25th days.

[0081] The quantitative data of the test results were expressed as the mean plus or minus the standard deviation (mean ± SD). SPSS11.0 software was used to perform t-tests between the treatment groups and the control group. * express p < 0.05, ** express p < 0.01.

[0082] Results: Compared with normal rats, after the rats were injected with PBS buffer suspension containing MSU crystals at the left hind foot, primary arthritis quickly occurred in the left hind foot, with obvious swelling and ulceration; secondary arthritis began to occur in the right hind foot about 4 days later, and the joint score gradually increased; at the same time, the ear blood vessels proliferated significantly, and the redness and swelling were obvious; the tail joints were swollen. The results of the in vivo immune protection effect of the peptide in the MSU crystal-induced gouty arthritis rat model are shown in Table 3. The data in the table are the results of the left and right hind feet and joint scores of rats in each group at 25 days. The peptide AL-8(0) can play an in vivo immune protection role in the MSU crystal-induced gouty arthritis rat model. The specific results are as follows:

[0083] Effect of polypeptide AL-8(0) on swelling of left paw in rats with primary gouty arthritis Figure 7 The swelling degree of the left hind foot of the rats in the positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) were significantly different from those in the model group ( p <0.01), the experimental results were statistically significant. The effect of polypeptide AL-8(0) on the swelling of the right hind foot of rats with secondary gouty arthritis is shown in Figure 8 The swelling degree of the right hind foot of rats in the positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) was significantly different from that in the model group ( p <0.01). The effect of polypeptide AL-8(0) on the joint score of rats with gouty arthritis is shown in Fig. 9 The limb scores of rats in the positive control group, low-dose, medium-dose and high-dose groups of peptide AL-8(0) were significantly lower than those in the model control group ( p<0.01), and the differences were statistically significant compared with the model control group.

[0084] Table 3 Immunoprotective effect of polypeptide AL-8(0) on MSU crystal-induced gouty arthritis rat model

[0085]

[0086] * express p < 0.05, ** express p < 0.01.

[0087] Conclusion: Peptide AL-8(0) has a therapeutic effect on MSU crystal-induced gouty arthritis in rats.

[0088] Example 5 Qualitative detection experiment of specific binding of polypeptide AL-8(0) to target at the cellular level - fluorescence imaging experiment

[0089] IL17RA-deficient human keratinocytes HaCaT were constructed by Genema Biotechnology using siRNA. The concentration of IL17RA-deficient HaCaT cells and normal HaCaT cells was adjusted to 1×10 5 cells / ml, and spread them on 24-well plates. Wash twice with ice-cold PBS. Add 1 ml of 1% BSA solution and mix at 4°C for 30 min. Add FITC-AL-8(0) peptide solution with a final concentration of 30ug / ml and mix at 4°C in the dark for 1h. After the drug incubation is completed, wash once with ice-cold PBS. Add Dil dye and wash twice with ice-cold PBS. Use a fluorescence microscope to detect the binding of the peptide to IL17RA, fix the parameters, shoot in the appropriate channels, superimpose the fluorescence images of different channels, and judge the binding of the peptide to IL17RA.

[0090] result: Fig.10 -A shows the immunofluorescence results of normal HaCaT cells at a 20x objective lens and a peptide concentration of 30ug / ml. From the figure we can see that the AL-8(0) peptide can bind to normal HaCaT cells. Fig.10 -B shows the immunofluorescence results of IL17RA-deficient HaCaT cells at a 20x objective lens and a peptide concentration of 30ug / ml. From the figure, we can see that the AL-8(0) peptide cannot bind to the IL17RA-deficient HaCaT cells.

[0091] Fluorescence microscopy confirmed that the anti-IL17RA peptide can bind to cells with high IL17RA expression and bind to the cell membrane surface, but cannot bind to cells with defective IL17RA expression.

Claims

1. A polypeptide AL-8(0), characterized in that Its amino acid sequence is ASPVIYHL-NH2.

2. A pharmaceutical composition, characterized in that It contains the polypeptide AL-8(0) according to claim 1 and pharmaceutically acceptable excipients.

3. Use of the polypeptide AL-8(0) according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a drug for treating autoimmune diseases; the autoimmune disease is rheumatoid arthritis or gouty arthritis.

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