Polypeptide and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to polypeptide and application thereof in preparation of medicine for treating oral mucosa diseases. The polypeptide disclosed by the invention can be used for treating recurrent aphthous oral ulceration and oral mucositis caused by anti-tumor treatment. The polypeptide disclosed by the invention has a remarkable treatment effect, can promote ulcer healing, and is dose-dependent and time-dependent; the polypeptide is high in safety, a degradation product is natural amino acid, and the polypeptide has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to polypeptides and their application in the preparation of drugs for treating oral mucosal diseases. Background Art

[0002] The oral mucosa refers to the pink, moist and soft tissue covering the inside of the oral cavity. Oral mucosal diseases refer to the general term for various types of numerous diseases occurring on the oral mucosa, including oral ulcers, oral mucositis, etc. The causes of most oral mucosal diseases are relatively complex, the course of the disease is chronic and protracted, the oral cavity will ulcer and ache, and there is a risk of canceration.

[0003] Recurrent aphthous ulcer (RAU), also known as recurrent oral ulcer, is the most common oral mucosal disease. An epidemiological survey of RAU among residents aged 20 - 69 in Zhejiang Province showed that among the 11,212 people surveyed, the prevalence rate of RAU was as high as 9.99% ( Wang Xiaodong, Zheng Weijun, Du Yu, etc. Epidemiological investigation of recurrent aphthous ulcer in residents aged 20 - 69 years in Zhejiang Province [J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2018, 33(06): 2687 - 2689. ). The clinical manifestations of RAU are recurrent round or oval ulcers, characterized by a yellow false membrane covering the lesion surface, a congested red halo around, a central depression, and obvious burning pain (yellow, red, concave, pain), with the characteristics of recurrence, periodicity, and self - limitation. The onset of ulcers hinders diet and speech, affecting the quality of life of patients. At present, the etiology and pathogenesis of recurrent aphthous ulcer are still unclear, but the academic community generally believes that it is the result of the combined action of multiple factors, including immunity, genetics, local trauma, mental stress and anxiety, etc. ( Shi Linjun, Zhou Zengtong. Diagnosis and disease management of recurrent aphthous ulcer [J]. Chinese Journal of Stomatology Magazine, 2022, 57(3): 314 - 318. ). Recent research progress suggests that abnormal immune function may be the most important potential pathogenesis of RAU, especially abnormal cellular immune function. A Nature sub - journal reported the role of T - cell regulation in the etiology of RAU ( Dudding T, Haworth S, Lind PA, et al. Genome wide analysis for mouth ulcers identifies associations at immune regulatory loci [J]. Nat Commun, 2019, 10(1): 1052. ). Some studies have also found that the formation of RAU mainly depends on the activation of local Th1 - type immune responses ( Lewkowicz N, Kur B, Kurnatowska A, et al. Expression of Thl / Th2 / Th3 / Thl7 - related genes in recurrent aphthous ulcers [J]. Arch Immunol Ther Exp (Warsz), 2011, 59(5): 399-406. ).

[0004] During the treatment of malignant tumors, especially in the treatment of head and neck tumors, oral mucositis (OM) is a common complication. It is generally believed that its occurrence is closely related to chemotherapy, molecular targeted therapy, immunotherapy, radiotherapy (field of irradiation, dose, and technique), and the patient's own risk factors. Chemotherapy-related oral mucositis, also known as chemotherapy-induced oral mucositis (CTOM), is the most common oral complication during chemotherapy, usually occurring within 4 to 7 days after the start of chemotherapy and reaching its peak at 10 to 14 days. Radiotherapy-related oral mucositis (RTOM) refers to the inflammatory or ulcerative lesions of the oral mucosa caused by radiotherapy. OM caused by anti-tumor treatment mainly presents as oral mucosal congestion, erythema, edema, erosion, and ulcers of varying degrees. Patients often show local pain, difficulty in eating, dry mouth, and taste disorders, which affect the quality of life of patients. It may also affect the tolerance and compliance of anti-tumor treatment and reduce the anti-tumor efficacy ( Chinese Society of Clinical Oncology Antitumor Drug Safety Management Expert Committee, Chinese Society of Clinical Oncology Tumor Support and Rehabilitation Therapy Expert Committee. Expert consensus on the diagnosis, prevention and treatment of acute oral mucositis caused by 449-459. ).

[0005] Currently, clinical treatment mainly focuses on local symptomatic treatment, and the main purpose is to reduce inflammation, relieve pain, and promote ulcer healing, such as oral rinses and oral gels made from ethanol, glucocorticoids, traditional Chinese medicine, etc. Although there are many treatment methods, each has its limitations. For example, mouthwash products mainly clean the oral cavity, and the efficacy is limited; long-term use of glucocorticoid drugs may cause adverse reactions that exceed the benefits of patients; traditional Chinese medicine has a poor taste, etc. Generally, the overall treatment effect is not ideal and it is difficult to cure completely. In summary, there is no specific medicine for RAU and OM caused by anti-tumor treatment, which seriously affects the health level and quality of life of patients.

[0006] Polypeptides are compounds formed by the connection of three or more α-amino acids through peptide bonds, generally composed of about 12 to 50 amino acids, and have a variety of biological activities. Studies have found that certain polypeptides have inhibitory and killing effects on bacteria, fungi, viruses, protozoa, cancer cells, etc., and can promote the wound healing process and improve immunity. For example, Melitiin has antiviral effects ( antitumor therapy [J]. Chinese Clinical Oncology Journal, 2021, 26(05): Memariani H, et al. Melittin: a venom - derived peptide with (1):5-17. ), and Cathelicidin LL-37 may play a protective role in cardiomyocyte apoptosis and myocardial I / R injury by activating AKT, ERK, and the phosphorylation and nuclear export of FoxO3a ( promising anti - viral properties. Eur J Clin Microbiol Infect Dis. 2020 Jan;39 Bei Y, et al. Cathelicidin - related antimicrobial peptide protects against myocardial ischemia / reperfusion ). We also found that some polypeptides have antiviral effects (injury. BMC Med. 2019 Feb 20;17(1):42. Ling Jianqun, etc. Application of a polypeptide in the preparation of drugs for treating enterovirus infections, Chinese Patent Application No. 201710201536.3; Ling Jianqun, etc. Application of a polypeptide in the preparation of drugs for preventing and treating influenza virus infections, Chinese Patent Application No. 202010925748.8） Anti - pneumonia effect (Ling Jianqun, etc. Application of a polypeptide in the preparation of drugs for preventing and treating pneumonia, Chinese Patent Application No. 202010925707.9 and anti - gastric ulcer effect ( Ling Jianqun, etc. Application of a polypeptide in the preparation of drugs for preventing and treating gastric diseases, Chinese Invention Application No. 202011228345.4 ).

[0007] Through comprehensive and in - depth research, the applicant surprisingly found that several polypeptides described in the present invention have good effects on anti - oral ulcer and anti - oral mucositis, and can provide new treatment methods and drugs for the clinical treatment of oral mucosal diseases such as RAU and OM caused by anti - tumor treatment. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the first aspect of the present invention is to provide polypeptides, and the amino acid sequences of the polypeptides are: SEQ ID No:1, SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

[0009] The second aspect of the present invention is to provide the use of polypeptides in the preparation of drugs for treating oral mucosal diseases.

[0010] In particular, the amino acid sequences of the polypeptides are: SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

[0011] In particular, the oral mucosal diseases include recurrent aphthous ulcer or oral mucositis caused by anti - tumor treatment.

[0012] In particular, the oral mucositis caused by anti - tumor treatment includes chemotherapy - related oral mucositis or radiotherapy - related oral mucositis.

[0013] In particular, the drug further includes other pharmaceutically acceptable excipients, carriers or auxiliary components.

[0014] In particular, the polypeptides can be used in combination with one or more of other therapeutic drugs such as vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, proprietary Chinese medicines, local anesthetic and analgesic drugs, tissue - promoting repair drugs or immune drugs.

[0015] The third aspect of the present invention is to provide a class of drugs, and the drugs contain polypeptides with amino acid sequences: SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

[0016] In particular, the drug further comprises other pharmaceutically acceptable excipients, carriers or auxiliary components.

[0017] The fourth aspect of the present invention lies in providing the use of a class of drugs in the preparation of drugs for treating oral mucosal diseases, wherein the drug contains polypeptides with amino acid sequences of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, or SEQ ID No: 6.

[0018] In particular, the oral mucosal diseases are preferably recurrent aphthous ulcers and oral mucositis caused by anti-tumor treatment.

[0019] In particular, the oral mucositis caused by anti-tumor treatment includes chemotherapy-related oral mucositis or radiotherapy-related oral mucositis.

[0020] In particular, the drug further comprises other pharmaceutically acceptable excipients, carriers or auxiliary components.

[0021] In particular, the drug can be used in combination with one or more of other therapeutic drugs such as vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, proprietary Chinese medicines, local anesthetic and analgesic drugs, tissue repair-promoting drugs or immune drugs.

[0022] The present invention has the following advantages and effects compared with the prior art: The present invention provides several polypeptides and their use in the preparation of drugs for treating oral mucosal diseases. At the same time, it provides a class of drugs with polypeptides as the active ingredient and their use in the preparation of drugs for treating oral mucosal diseases. Compared with the prior art, in the application of the polypeptides of the present invention in the treatment of oral mucosal diseases, the therapeutic effect is equivalent to that of commercially available drugs, and even better than that of commercially available drugs. It can be widely used in the treatment of oral mucosal diseases, and the polypeptides of the present invention have high safety. The final degradation product is amino acid and there are no adverse reactions. It has broad application prospects. Description of the Drawings

[0023] Figure 1 : Effect diagram of the reduction of ulcer area in each drug administration group Detailed Embodiments

[0024] One embodiment of the present invention lies in providing polypeptides; the polypeptides can be obtained according to the preparation method of the polypeptides described in the authorized patent ( Ling Jianqun, etc. A Preparation method and application of antibacterial peptides, Chinese Patent Application No. 201310245373.0 ) or can be synthesized, prepared and purified by solid-phase synthesis. The amino acid sequences of the polypeptides are SEQ ID NO: 1, SEQ ID No: 4, SEQ ID No: 5 or SEQ ID No: 6.

[0025] Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art. In the examples, all raw materials added are commercially available unless otherwise specified.

[0026] One embodiment of the present invention is to provide the use of a polypeptide in the preparation of a drug for treating oral mucosal diseases, and the amino acid sequence of the polypeptide is: SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

[0027] Preferably, the oral mucosal diseases include recurrent aphthous ulcer or oral mucositis caused by anti-tumor treatment.

[0028] Preferably, the oral mucositis caused by anti-tumor treatment includes chemotherapy-related oral mucositis or radiotherapy-related oral mucositis.

[0029] Preferably, the drug further comprises other pharmaceutically acceptable excipients, carriers or adjuvant components.

[0030] Pharmaceutically acceptable excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. Except for the active ingredients, they have been reasonably evaluated in terms of safety and are substances included in pharmaceutical preparations. The same pharmaceutical excipient can be used in pharmaceutical preparations for different administration routes and has different functions and uses. Suitable pharmaceutically acceptable excipients are well-known to those of ordinary skill in the art.

[0031] Pharmaceutically acceptable carriers refer to carriers used for the administration of therapeutic agents, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily the active ingredients themselves and have no excessive toxicity after administration. Suitable carriers are well-known to those of ordinary skill in the art.

[0032] Pharmaceutically acceptable adjuvant components have certain physiological activities, but the addition of this component will not change the dominant position of the above drug composition in the treatment of diseases, but only play an auxiliary effect. These auxiliary effects are only the utilization of the known activities of this component and are common auxiliary treatment methods in the medical field.

[0033] The pharmaceutically acceptable excipients, carriers or adjuvant components added to the drugs provided by the present invention can play the roles of shaping, acting as carriers or improving stability. In addition, they also have important functions such as solubilization, cosolubilization or sustained and controlled release.

[0034] There are no special restrictions on the above-mentioned pharmaceutical excipients, carriers or auxiliary components, and appropriate pharmaceutical preparations can be selected according to the choice of specific dosage forms. Available pharmaceutical excipients, carriers or auxiliary components include excipients (such as sugar derivatives like lactose, sucrose, glucose, honey, mannitol and sorbitol; starch derivatives like corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives like crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose; gum arabic; dextran; silicate derivatives like magnesium aluminum metasilicate; phosphate derivatives like calcium phosphate; carbonate derivatives like calcium carbonate; sulfate derivatives like calcium sulfate, etc.), binders (such as gelatin, polyvinylpyrrolidone and polyethylene glycol), disintegrants (such as cellulose derivatives like sodium carboxymethyl cellulose, polyvinylpyrrolidone), lubricants (such as talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, leucine), stabilizers (methyl paraben, propyl paraben, etc.), flavoring agents (such as common sweeteners, acidulants and spices), etc. For sprays, additional agents such as solvents, cosolvents, antioxidants, bacteriostatic agents, surfactants, etc. can also be added as needed.

[0035] There is no need to list all the pharmaceutically acceptable carriers or excipients one by one, and those of ordinary skill in the art can make specific selections according to the common general knowledge they have mastered.

[0036] Preferably, the polypeptide can be used in combination with one or more of other therapeutic drugs such as vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, Chinese patent medicines, local anesthetic and analgesic drugs, tissue repair promoting drugs or immune drugs, etc. When used in combination with these drugs, it can not only enhance the therapeutic effect, but also improve the comfort of patients with oral mucosal diseases. Among them, vitamin drugs include drugs mainly composed of compound vitamin B group, vitamin C or vitamin E; antibiotic drugs include metronidazole, tinidazole, gentamicin, ciprofloxacin, ofloxacin or tetracycline; corticosteroid drugs include prednisone, dexamethasone or hydrocortisone; traditional Chinese medicines such as dandelion, honeysuckle vine, etc.; Chinese patent medicines such as aloe vera gel containing active substances such as anthraquinones, vitamins, sugars, lipids, etc.; local anesthetic and analgesic drugs such as lidocaine, etc.; tissue repair promoting drugs such as epidermal growth factor and fibroblast growth factor, etc.; immune drugs include cyclosporine, thymosin, thymopeptide enteric-coated tablets or α-peptide mannan, etc.

[0037] One embodiment of the present invention is to provide a class of drugs, which contain a polypeptide, and the amino acid sequence of the polypeptide is: SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5 or SEQ ID No:6.

[0038] Preferably, the drug further comprises other pharmaceutically acceptable excipients, carriers or adjuvants.

[0039] One embodiment of the present invention is to provide the use of a class of drugs in the preparation of drugs for treating oral mucosal diseases, wherein the drugs contain polypeptides with amino acid sequences of SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, or SEQ ID No: 6.

[0040] Preferably, the oral mucosal diseases are recurrent aphthous ulcer and oral mucositis caused by anti-tumor treatment.

[0041] Preferably, the oral mucositis caused by anti-tumor treatment includes chemotherapy-related oral mucositis or radiotherapy-related oral mucositis.

[0042] Preferably, the drug further comprises other pharmaceutically acceptable excipients, carriers or adjuvants.

[0043] Preferably, the drug can be used in combination with one or more of other therapeutic drugs such as vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, Chinese patent medicines, local anesthetic and analgesic drugs, tissue repair promoting drugs or immune drugs.

[0044] In one embodiment, the present invention relates to a method for treating oral mucosal diseases, especially recurrent aphthous ulcer and oral mucositis caused by anti-tumor treatment, which comprises the following steps: applying an aqueous solution containing the polypeptide of the present invention to the oral mucosal lesion site, wherein the amino acid sequence of the polypeptide is: SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5 or SEQ ID No: 6, filling the aqueous polypeptide solution into a spray bottle, shaking the spray bottle well before use, and spraying it onto the oral mucosal lesion site of the oral mucosal disease 1-6 times a day.

[0045] As used in the context of the present invention, the term "treatment" refers to obtaining a desired pharmacological or physiological effect. Such effect can be preventive of a disease in the following aspects: preventing or partially preventing a disease, symptom or pathological condition, and / or can be therapeutic of a disease in the following aspects: partially or completely curing a disease, disorder, symptom or reaction caused by a pathological condition. Thus, "treatment" encompasses any treatment of a disease in mammals, especially humans, including: (a) preventing the emergence of a pathological condition in an individual who may be predisposed to develop the pathological condition but has not been diagnosed as having such pathological condition, i.e., for a subject who may be predisposed to develop a pathological condition but has not experienced or shown symptoms of the condition, preventing the clinical symptoms of the pathological condition from occurring in the body; (b) inhibiting, i.e., preventing or alleviating the development of a pathological condition or its clinical symptoms; or (c) relieving the symptoms associated with a pathological condition. For the purposes of the present invention, this includes, but is not limited to, symptom relief, reduction in the degree of the disease, stabilization of the disease state (i.e., not worsening), delay or slowdown in the progression of the disease, improvement or alleviation of the disease state, and remission (whether partial or total), whether detectable or not. The term "treatment" includes an increase in the severity of inhibition or reduction of a pathological condition or symptom (e.g., improvement or alleviation of the symptoms of an oral mucosal disease, shortening of the duration of the symptoms, non-worsening of the disease state. For RAU, alleviating pain, promoting ulcer healing, shortening the course of the disease, prolonging the recurrence interval, etc. For CTOM / RTOM, controlling oral inflammation, relieving pain, protecting the ulcer surface, promoting early healing, preventing further deterioration and development of mucositis, etc.) relative to the absence of treatment, and does not necessarily mean complete cessation of the relevant disease, disorder or condition.

[0046] The present invention discloses for the first time the use of the polypeptide described in the present invention as a pharmaceutically active ingredient in the preparation of drugs for treating and preventing oral mucosal diseases, especially recurrent aphthous ulcer and oral mucositis caused by anti-tumor treatment. Therefore, any medicament prepared by combining the polypeptide described in the present invention alone or in combination with other substances as a pharmaceutically active ingredient with excipients, as long as it is indicated or suggested on its packaging or instructions or on any other promotional materials that it has the function of treating and preventing oral mucosal diseases, especially recurrent aphthous ulcer and oral mucositis caused by anti-tumor treatment, falls within the scope of protection of the present invention.

[0047] Example 1 Solid-phase synthesis, preparation and purification method of the polypeptide described in the present invention Synthesis method: In the synthesis of the linear peptides of SEQ ID No: 1 - 6 according to the present invention, 9-fluorenylmethyloxycarbonyl (FMOC) is used as the protecting group at the amino terminus. 4-methyl-benzhydrylamine resin (HCl, MBHA resin) is selected as the solid-phase carrier for polypeptide synthesis. HOBt / DCC (1-hydroxybenzotriazole / N,N'-dicyclohexylcarbodiimide) is used as the condensing agent for the reaction, and the peptide chain is extended from the carboxyl terminus to the amino terminus (C-terminus → N-terminus). A mixed solution (by mass percentage) of 90.0% trifluoroacetic acid, 4.0% water, and 6.0% triisopropylsilane (TIA) is used to cleave the polypeptide sequence containing 15 - 20 amino acid residues from the MBHA resin. After repeated precipitation with ether several times, it is purified by preparative reverse-phase high-performance liquid chromatography RP-HPLC.

[0048] Purification process: Use a C18 reverse-phase preparative column (250 mm × 20 mm, 5 μm); mobile phase: 1‰ trifluoroacetic acid, 0% - 65% (by volume percentage) acetonitrile as the mobile phase, and gradient elution is carried out at a flow rate of 1.5 mL / min. The main peak eluate is collected, frozen at -80°C for 6 hours, then placed in a freeze dryer for freeze-drying. After freeze-drying into a loose solid powder, it is stored in a -20°C refrigerator for standby. Analyzed by HPLC, the purity is 92% - 99%; identified by ESI-Q-TOF-MS / MS, its molecular weight and amino acid sequence are consistent with the polypeptide to be prepared.

[0049] The polypeptides obtained by preparation are shown in Table 1.

[0050] Table 1 Polypeptides obtained by preparation and detection results Polypeptide serial number Weight (g) Purity (%) Molecular weight (Da) Amino acid sequence AA SEQ ID No:1 5.6 92.5 2571.26 MGRFKRFRKKFKKLFKKLSP 20 SEQ ID No:2 6.2 92.3 2474.11 MGRFKRFRKKFKKLFKKLS 19 SEQ ID No:3 5.7 95.6 2342.95 GRFKRFRKKFKKLFKKLS 18 SEQ ID No:4 9.6 97.6 2255.87 GRFKRFRKKFKKLFKKL 17 SEQ ID No:5 8.2 95.1 2142.71 GRFKRFRKKFKKLFKK 16 SEQ ID No:6 8.1 95.8 2085.66 RFKRFRKKFKKLFKK 15 Example 2 Therapeutic effects of the polypeptides according to the present invention at different doses on a rat model of recurrent aphthous ulcer 1. Method SPF-grade SD rats, weighing 150 - 180 g, male. After 1 week of adaptive feeding, on the day before immunization, all rats were depilated with 8% sodium sulfide. Two points were selected at the depilated area, and the antigen injection area was 2 cm on each side of the spine 2, Immunize with the intradermal injection of an antigen emulsifier, 0.1 mL per point, once a week for 8 consecutive weeks. On the 54th day after immunization, the model group (administered with normal saline) and the three dose groups of the test substance (the polypeptide SEQ ID No: 4 of the present invention, with low, medium, and high doses being 0.2, 2, and 20 mg / kg / d respectively) were anesthetized with propofol and fixed on the operating table. The mucosal side of the lower lip of the rats was dried with a sterile cotton ball, isolated from moisture, and a 3 mm × 3 mm square filter paper containing 50% glacial acetic acid was placed on the dried lower lip mucosa. After 30 s, the filter paper was removed for burning the oral mucosa.

[0051] At 48 h after the glacial acetic acid treatment, the presence of ulcer lesions with the clinical characteristics of "yellow, red, concave, and painful" in the oral mucosa was used as a sign of successful modeling. After successful modeling, the SD rats were randomly divided into 4 groups, with 5 rats in each group. The freeze-dried powder of the polypeptide of the present invention was directly dissolved in sterile water to prepare different concentrations of 0.13 mg / mL, 0.6 mg / mL, and 3 mg / mL, which were filled into a spray bottle, and oral spray administration was started every other day for 7 consecutive days. The specific dosing scheme is shown in Table 2. At the end of the experiment, all 4 groups of rats were anesthetized by intraperitoneal injection of 10% chloral hydrate and sacrificed.

[0052] Table 2 Dosing Scheme Group Administration method Administration dose (mg / kg) Administration concentration (mg / mL) Administration frequency Model group Oral spray 0 0 6 times a day, 3 sprays each time SEQ ID No:4 low-dose group Oral spray 0.2 0.13 1 time a day, 2 sprays each time SEQ ID No:4 medium-dose group Oral spray 2 0.6 3 times a day, 3 sprays each time SEQ ID No:4 high-dose group Oral spray 20 3 6 times a day, 3 sprays each time Note: The modeling period is relatively long. During this period, the body weight of the rats increases. When dosing, the body weight is calculated as 0.4 kg; 0.15 mL is sprayed each time, and less than 1 spray is recorded as 1 spray. 2. Observe and measure the oral ulcer area of the rats, and evaluate the healing changes in the ulcer area: Before dosing, on the 3rd day, the 5th day, and 7 days after dosing, measure the ulcer area; measure the diameter of the ulcer surface with a vernier caliper, and measure it once a day after dosing, and calculate the ulcer area according to the formula.

[0053] Ulcer area = π × [(maximum transverse diameter value / 2) × (maximum longitudinal diameter value / 2)].

[0054] 3. Statistical Processing Statistical method: Use Microsoft Excel 2019 to process the data. For measurement data, the t-test is used. P < 0.05 indicates that the difference is statistically significant.

[0055] 4. Results As shown in Table 3, the polypeptides of the present invention at different doses all have a certain therapeutic effect on rats with recurrent aphthous oral ulcers, can promote ulcer healing, and show a dose- and time-dependent manner.

[0056] Table 3 Results of the changes in the ulcer area of the test animals in each group

[0057] Example 3 Therapeutic effect of the polypeptide of the present invention on a recurrent aphthous ulcer rat model 1. Method SPF-grade male SD rats, weighing 150 - 180 g, were adaptively fed for 1 week. Six rats were randomly selected as the blank group and raised normally without modeling treatment. The remaining animals were modeled according to the method of Example 2. The successfully modeled rats were randomly divided into 5 groups: the model group, the positive drug group, and 3 test substance groups (SEQ ID No:1, SEQ ID No:2, SEQ ID No:3), with 6 rats in each group. Referring to Example 2, the polypeptide aqueous solution was administered by oral spraying, with an actual use concentration of 0.36 mg / mL, 5 times a day, 3 sprays each time. The rat body weight was calculated as 0.4 kg, and the final dose was 2 mg / kg. The positive drug group was given triamcinolone acetonide ointment (0.1%, w / w), 5 times a day, applied to the ulcer site. The detection indexes were the same as those in Example 2.

[0058] 2. Results The observation effects of the control group and each administration observation group are shown in Table 4. The results in Table 4 show that compared with the model group, the administration groups had a significant therapeutic effect on the aphthous ulcer model. Compared with the positive drug, the polypeptide of the present invention had obvious advantages in terms of the effective rate and the shortening time of the ulcer area.

[0059] Table 4 Results of the changes in the ulcer area of the experimental animals in each group

[0060] Example 4 Therapeutic effect of the polypeptide of the present invention on a recurrent aphthous ulcer rat model 1. Method SPF-grade male SD rats, weighing 150 - 180 g, were adaptively fed for 1 week. Six rats were randomly selected as the blank group and raised normally without modeling treatment. The remaining animals were modeled in the same way as in Example 2. The successfully modeled rats were randomly divided into 5 groups: the model group, the positive drug group, and 3 test substance groups (SEQ ID No:4, SEQ ID No:5, SEQ ID No:6), with 6 rats in each group. Referring to Example 2, the polypeptide aqueous solution was administered by oral spraying, with an actual use concentration of 0.36 mg / mL, 5 times a day, 3 sprays each time. The rat body weight was calculated as 0.4 kg, and the final dose was 2 mg / kg. The positive drug group was given triamcinolone acetonide ointment (0.1%, w / w), 5 times a day, applied to the ulcer site. The detection indexes were the same as those in Example 2.

[0061] 2. Results The observation effects of each group of animal models are shown in Table 5. The results in Table 5 show that compared with the model group, the administration group has a significant therapeutic effect in promoting ulcer healing in the aphthous oral ulcer model.

[0062] Table 5 Results of the changes in ulcer area of experimental animals in each group

[0063] Example 5 The alleviating effect of the polypeptide of the present invention on a rat model evaluating oral mucositis induced by the chemotherapeutic drug 5-fluorouracil (5-FU) 1. Method Forty-eight male SD rats were randomly selected, and 6 of them were used as the blank control group, which were normally raised without modeling treatment. The remaining animals were intraperitoneally injected with 5-fluorouracil (5-FU, 0.05 mg / g) for 2 consecutive days. The first day of modeling was recorded as D1. On D3, an oral ulcer model was established by contacting the buccal mucosa with NaOH particles. They were randomly divided into 7 groups, namely the model group and 6 test substance groups, that is, the polypeptides SEQ ID No: 1-6 described in this patent. On D5, the rats in the experiment were given the polypeptides SEQ ID No: 1-6 described in this patent. Referring to Example 2, the aqueous polypeptide solution was administered by oral spraying, and the actual use concentration was 0.72 mg / mL, 2 times a day, 2 sprays each time. The body weight of the rats was calculated as 0.22 kg, and the final dose was 2 mg / kg. The animals were sacrificed on D10 for sample collection.

[0064] 2. Evaluation of the changes in the healing of rat oral ulcers The damage condition of the oral mucosa was observed, and the ulcer area was measured using Image J software.

[0065] 3. Statistical processing Statistical method: Microsoft Excel 2019 was used to process the data. The measurement data were analyzed by t-test, and P < 0.05 indicated that the difference was statistically significant.

[0066] 4. Results The observation effects of the control group and each administration observation group are shown in Table 6. Table 6 and Figure 1 The results show that compared with the model group, the ulcer damage area of the animals in the polypeptide SEQ ID No: 1-6 groups on D10 was significantly reduced, and the difference was statistically significant (P < 0.05). It has a significant therapeutic effect in treating the oral ulcer model induced by 5-fluorouracil (5-FU) combined with sodium hydroxide.

[0067] Table 6 Observation and scoring results of experimental animals in each administration group - individual results

[0068] Example 6 Evaluation of the alleviating effect of the polypeptide of the present invention on a mouse model of oral mucous ulcer induced by radiation 1. Method Male C57BL / 6J mice at 6 - 8 weeks old were irradiated with 8 Gy per day for 3 consecutive days on the head and neck. One week later, oral ulcers occurred in about 80% of the mice. The successfully modeled mice were randomly divided into 4 groups, with 6 mice in each group. Another 6 healthy mice were used as blank controls. The blank control group and the model group were given normal saline by oral spraying. The administration group was given the aqueous solution of the polypeptide of the present invention by oral spraying every day. The actual concentration used was 1.44 mg / mL, 2 times a day, 1 spray each time, for 7 consecutive days. The experiment was carried out 2 times. The polypeptide SEQ ID No:1, SEQ ID No:2, SEQ ID No:3 of the present invention was given in the first time, and the polypeptide SEQ ID No:4, SEQ ID No:5, SEQ ID No:6 of the present invention was given in the second time.

[0069] 2. Observe the changes of oral mucosal injury in mice and score By observing the oral mucosa of mice, a comprehensive score was made according to the degree of mucosal injury (redness, erosion, ulceration, etc.), the number / size of ulcers, etc.

[0070] The scoring criteria are as follows, and the scores of each item are added up to the final score:

[0071] Scoring example: If an animal has 1 area of redness and 2 ulcers in the oral mucosa, and the major diameters of the ulcers are about 0.4 and 0.8 mm respectively, the score is 1 + 2×2 + 1 = 6 points 3. Results As shown in Table 7 and Table 8, compared with the model group, the degree of oral mucosal injury in the administration group was significantly reduced. The polypeptides SEQ ID No:1 - 6 of the present invention have a good therapeutic effect on the mouse model of oral mucous ulcer induced by radiation, and there is a significant difference in the therapeutic effect compared with the model group.

[0072] Table 7 Results of oral ulcer scores of animals in each group

[0073] Table 8 Results of oral ulcer scores of animals in each group

[0074] This part of the examples further illustrates the content of the present invention, but should not be construed as a limitation of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

2. Use of a polypeptide in the preparation of a medicament for treating oral mucosal diseases, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

3. The application according to claim 2, characterized in that: The oral mucosal diseases include recurrent aphthous ulcer or oral mucositis caused by anti-tumor therapy.

4. The application according to claim 3, characterized in that: The oral mucositis caused by anti-tumor therapy includes chemotherapy-related oral mucositis or radiotherapy-related oral mucositis.

5. The application according to any one of claims 2-4, characterized in that: The drug also includes other pharmaceutically acceptable excipients, carriers or auxiliary components.

6. The application according to any one of claims 2-5, characterized in that: The polypeptide can be used in combination with one or more of vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, Chinese patent medicines, local anesthetic and analgesic drugs, tissue repair-promoting drugs or immune drugs.

7. A drug, characterized in that: Containing any one of the polypeptides recited in claim 1.

8. The drug according to claim 7, characterized in that: The drug also includes other pharmaceutically acceptable excipients, carriers or auxiliary components.

9. Use of any one of the drugs according to claims 7 and 8 in the preparation of a drug for treating oral mucosal diseases.

10. Use of a drug containing a polypeptide with an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 in the preparation of a drug for treating oral mucosal diseases.

11. The application according to any one of claims 9 and 10, characterized in that: The oral mucosal diseases include recurrent aphthous ulcer or oral mucositis caused by anti-tumor therapy.

12. The application according to claim 11, wherein: The oral mucositis caused by anti-tumor therapy includes chemotherapy-related oral mucositis or radiotherapy-related oral mucositis.

13. Any of the applications according to claims 8-12, characterized in that: The drug can be used in combination with one or more of vitamin drugs, antibiotic drugs, corticosteroid drugs, traditional Chinese medicines, Chinese patent medicines, local anesthetic and analgesic drugs, tissue repair-promoting drugs or immune drugs.

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