Application of tripeptides in the preparation of drugs for treating and / or preventing mucosal or skin damage

Abstract

The present invention belongs to the field of biopharmaceutical technology, and specifically relates to the use of tripeptides in the preparation of drugs for treating and / or preventing mucosal or skin damage. The present invention provides the use of a peptide or a salt thereof having an amino acid sequence of valine-proline-leucine in the preparation of drugs for treating and / or preventing diseases caused by mucosal damage or skin damage. The peptides of the present invention can effectively promote the repair of digestive tract mucosal damage and reduce the development of gastrointestinal disease pathology of acute and chronic gastritis and digestive tract ulcers. Pharmacological experimental results show that the peptides of the present invention have significant preventive and therapeutic effects on ethanol-induced mouse gastric ulcer models, and the application of the present invention provides a new idea and strategy for clinical diseases caused by mucosal damage or skin damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to the application of tripeptides in the preparation of drugs for treating and / or preventing mucosal or skin injuries. Background Art

[0002] Mucosal damage is relatively common in life. Infectious diseases, allergic diseases, local irritation, etc. can cause oral mucosal damage; chemical factors (smoking, drinking, tea, coffee and irritating drugs such as aspirin, indomethacin, etc.), physical factors (too cold, too hot, too rough food or overeating, etc.), bacteria or their toxin stimulation and other factors may cause gastric mucosal damage; constipation and other factors may cause intestinal damage.

[0003] The gastric mucosa is a thin and fragile layer of mucosal tissue that exists on the inner wall of the stomach. It acts as a natural "barrier" to protect the safety of the stomach wall. It has a dynamic balance mechanism of damage and self-repair, which protects the normal functioning of the stomach. Once the outside world puts too much burden on the stomach or stimulates it too strongly, the dynamic balance will be broken, and gastric acid will begin to "self-digest" the stomach wall, and then form damage to the concave surface. Once the gastric mucosa is damaged, it is difficult to restore it to its original state. What follows is a series of symptoms of stomach discomfort, such as upper abdominal discomfort or pain, nausea, vomiting, diarrhea, and loss of appetite.

[0004] Chronic gastritis is a chronic inflammation of the gastric mucosa. It is a common and frequently occurring disease in gastroenterology. Clinically, chronic inflammation of the gastric mucosa caused by different reasons (i.e., pathologically manifested as infiltration of monocytes and lymphocytes) and / or glandular atrophic lesions are referred to as chronic gastritis. Damage to the mucosal tissue can clinically lead to gastrointestinal diseases such as chronic gastritis and peptic ulcers. Clinically, the main drugs for the treatment of acute and chronic gastritis and peptic ulcers are small molecule compounds such as gastric acid inhibitors, gastric mucosal protectants, and antibiotics. Existing gastric mucosal protective drugs have limited therapeutic effects, poor effectiveness, long courses of treatment, and high recurrence rates during clinical application, and cannot meet the clinical treatment needs of acute and chronic gastritis and peptic ulcers caused by various reasons.

[0005] Skin trauma or skin injury refers to the damage to normal skin (tissue) caused by external injury factors such as surgery, external force, heat, electric current, chemicals, low temperature, and internal factors of the body such as local blood supply disorders. Skin damage is often accompanied by the destruction of skin integrity and the loss of a certain amount of normal tissue. At the same time, the normal function of the skin is impaired. It is also called a wound or trauma. At present, there are protein / peptide drugs including basic fibroblast growth factor, epidermal growth factor, platelet growth factor, granulocyte-macrophage colony stimulating factor and growth hormone, which have obvious effects on wound repair, skin care, anti-wrinkle and anti-aging. However, the amino acid sequences of these protein / peptide drugs are relatively long, resulting in high preparation costs and poor stability, so their application is subject to certain restrictions. Therefore, it is very necessary to develop drugs for the repair of digestive system mucosal damage or skin damage. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention aims to provide the use of a tripeptide in the preparation of a drug for treating and / or preventing mucosal or skin damage.

[0007] The present invention provides the use of a peptide having an amino acid sequence of valine-proline-leucine or a salt thereof in preparing a drug for treating and / or preventing diseases caused by mucosal damage or skin damage.

[0008] Furthermore, the mucosal damage is digestive system mucosal damage.

[0009] Digestive system mucosal damage is caused by irritants or drugs or stress. The irritants are, for example, hydrochloric acid, ethanol, etc. The drugs are, for example, non-steroidal anti-inflammatory drugs such as aspirin or indomethacin, etc.

[0010] The peptide or its salt having the amino acid sequence of valine-proline-leucine (VPL) of the present invention can protect the digestive system mucosa or repair the damage of the digestive system mucosa, thereby preventing, alleviating or treating gastrointestinal diseases.

[0011] Furthermore, the diseases caused by mucosal damage include acute gastritis, chronic gastritis, chronic atrophic gastritis, gastroduodenal ulcers, functional gastrointestinal diseases, dyspepsia, precancerous lesions, digestive system tumors, gastrointestinal bleeding, gastroesophageal reflux disease, oral ulcers, stomatitis, gingivitis, periodontitis, esophagitis, esophageal ulcers, acute and chronic enteritis, ulcerative colitis, Crohn's disease and mucosal damage diseases caused by radiotherapy and chemotherapy.

[0012] Furthermore, the diseases caused by skin damage include epidermal inflammation, mechanical and surgical wounds, burns and scalds, ulcers, fistulas, bedsores, and skin damage diseases caused by radiotherapy and chemotherapy.

[0013] Furthermore, the peptide with the amino acid sequence of valine-proline-leucine is prepared by chemical synthesis, and is prepared by the steps of linking resin, linking amino acids, cutting, purifying, salt exchange and freeze drying.

[0014] Furthermore, the drug is a preparation prepared by using a peptide with an amino acid sequence of valine-proline-leucine or a salt thereof as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0015] Furthermore, the preparation is an oral preparation or an external preparation.

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides the use of a peptide or a salt thereof having an amino acid sequence of valine-proline-leucine (VPL) in the preparation of a drug for treating and / or preventing diseases caused by mucosal or skin damage. By establishing an in vivo model, the results show that the peptide (VPL) of the present invention has a significant therapeutic effect on an ethanol-induced mouse gastric ulcer model, and the ulcer inhibition rate is significantly higher than that of the teprenone group, confirming that the peptide (VPL) of the present invention can effectively promote the repair of gastrointestinal mucosal damage, reduce the development of acute and chronic gastritis and gastrointestinal ulcer pathology, and can prevent and treat diseases caused by mucosal damage. The application of the present invention provides a new idea and strategy for clinical diseases caused by mucosal damage or skin damage. DETAILED DESCRIPTION

[0018] Example 1 Chemical Synthesis of Peptide VPL

[0019] 1. Linking resin

[0020] (1) Swelling: Weigh 8.34 g of 2-chlorotrityl chloride resin (degree of substitution S = 0.75 mmol / g), add it to a polypeptide solid phase synthesis tube, add 90 mL of dichloromethane and bubbling nitrogen for 30 minutes.

[0021] (2) Linking: 1.80 mL of N,N-diisopropylethylamine and 3.21 g of Fmoc-L-Leu-OH were added in sequence, and nitrogen was bubbled to react at room temperature for 2.5 hours. 7 mL of methanol was added, and the reaction was continued for 30 minutes, and the mixture was drained. The mixture was then washed three times with 60 mL of dimethylformamide, and the resin was drained.

[0022] (3) Removal of Fmoc protecting group: Add 70 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution to the synthesis tube at room temperature, bubbling nitrogen for 10 min, and draining; add 70 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution again, bubbling nitrogen for 10 min, and draining. Then wash with 70 mL of N,N-dimethylformamide 5 times, 3-5 minutes each time. The Fmoc removal result was positive when tested by the ninhydrin method.

[0023] 2. Linking amino acids

[0024] 2.1 Link 2 Pro:

[0025] (1) Amino acid preactivation: 6.46 g of Fmoc-L-Pro-OH and 3.03 g of 1-hydroxybenzotriazole were added to a reaction bottle in sequence, followed by 75 mL of N,N-dimethylformamide / dichloromethane (1:1), stirred to dissolve, and 2.34 g of N,N-diisopropylethylamine and 6 mL of N,N'-diisopropylcarbodiimide were added at -5 to 0°C, and the mixture was reacted for 50 minutes.

[0026] (2) Amino acid connection: Add the activated protected amino acid solution into the synthesis tube, and add an appropriate amount of dichloromethane to clean the equipment. Bubble nitrogen at room temperature for 90 minutes, and test with ninhydrin method, negative. Drain and wash the resin with 75 ml of N,N-dimethylformamide 3 times, 3-5 minutes each time.

[0027] (3) Removal of Fmoc protecting group: Add 75 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution to the synthesis tube at room temperature, bubbling nitrogen for 10 min, and draining; add 75 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution again, bubbling nitrogen for 10 min, and draining. Then wash with 75 mL of N,N-dimethylformamide 5 times, 3-5 minutes each time. The Fmoc removal result was positive when tested by the ninhydrin method.

[0028] 2.2 Link 1 Val:

[0029] (1) Amino acid preactivation: 7.02 g of Fmoc-L-Val-OH and 3.28 g of 1-hydroxybenzotriazole were added to a reaction bottle in sequence, and then 75 mL of N,N-dimethylformamide / dichloromethane (1:1) was added and stirred to dissolve. 2.75 g of N,N-diisopropylethylamine and 3 mL of N,N'-diisopropylcarbodiimide were added at -5 to 0°C and the mixture was reacted for 40 minutes.

[0030] (2) Amino acid connection: Add the activated protected amino acid solution into the synthesis tube, and add an appropriate amount of dichloromethane to clean the equipment. Bubble nitrogen at room temperature for 2 hours, and test with ninhydrin method, negative. Drain and wash the resin with 75 mL of N,N-dimethylformamide 3 times, 3-5 minutes each time.

[0031] (3) Removal of Fmoc protecting group: Add 75 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution to the synthesis tube at room temperature, bubbling nitrogen for 10 min, and draining; add 75 mL of 20% 1,8-diazabicycloundec-7-ene-N,N-dimethylformamide (v / v) solution again, bubbling nitrogen for 10 min, and draining. Then wash with 75 mL of N,N-dimethylformamide 3 times and 75 mL of dichloromethane 6 times, each for 3-5 minutes. The Fmoc removal result was detected by the ninhydrin method, which was positive. Drain and proceed to the next step of cutting.

[0032] 3. Cutting

[0033] Add 70mL of trifluoroacetic acid, 2.5mL of H2O, and 2.5mL of triisopropylsilane to the synthesis tube in sequence, and react for 3 hours with nitrogen bubbling. Filter by suction, wash the resin once with 20mL of trifluoroacetic acid, combine the filtrate, concentrate the trifluoroacetic acid under reduced pressure at 35℃, add 50mL of methyl tert-butyl ether, and 150mL of n-hexane to precipitate an oily substance, pour out the supernatant, concentrate under reduced pressure at 35℃ to obtain an oily substance, and transfer it to purification without weighing. LCMS was sent for inspection, and the purity was 97.65%, [M+H]+328.20, and the result was correct.

[0034] 4. Purification, salt exchange and freeze-drying

[0035] Chromatographic parameters:

[0036] Chromatographic column: YMC-Actus Triart C 18 250x30 mm(S-5um,12nm)

[0037] Eluent A1: 0.1 M acetic acid

[0038] Eluent A2: 0.025M acetic acid-0.1M ammonium acetate

[0039] Eluent B: acetonitrile

[0040] Flow rate: 25mL / min

[0041] UV detection wavelength: 220nm

[0042] Steps:

[0043] a) 95% eluent A1 + 5% eluent B to balance the chromatographic column;

[0044] b) Sample injection: 0.6446 g sample, dissolved in 5 mL purified water and filtered through a filter with a pore size of 0.45 μm;

[0045] c) equilibrate the chromatographic column with 95% eluent A2 + 5% eluent B;

[0046] d) Gradient elution of A1 and B, the gradient is as follows:

[0047] Table 1 Gradient elution program of eluent A1 and eluent B

[0048] Time / min Eluent A1 (0.1M acetic acid) / % Eluent B (acetonitrile) / % 0 95 5 5 95 5 35 70 30 40 50 50

[0049] e) collecting the target peptide eluate;

[0050] f) Rotary evaporation concentration: water bath temperature 40°C;

[0051] g) Freeze-dried to obtain 0.1273 g of pure product, purity: 99.85%, yield: 19.74%, MS (ESI), m / z: 328.30 ([M+H]+).

[0052] The identification results of the peptide Val-Pro-Leu are as follows:

[0053] High resolution mass spectrometry (TOF-HRMS), molecular formula: C 16 H 29 N3O4; m / z: 328.22346 ([M+H] + ).

[0054] 1H NMR(600MHz,D2O)δ4.39(dd,J=8.4,6.1Hz,1H),4.09(dd,J=9.7,5.6Hz,2H), 3.69–3.61(m,1H),3.56–3.48(m,1H),2.25–2.16(m,2H),2.02–1.91(m,3H,A cOH),1.90–1.82(m,2H),1.63–1.53(m,1H),1.52–1.43(m,2H),1.00(d,J=7. 0Hz, 3H), 0.90 (d, J = 6.9Hz, 3H), 0.82 (d, J = 6.6Hz, 3H), 0.78 (d, J = 6.5Hz, 3H).

[0055] Example 2 Anti-ulcer effect of the peptide sample obtained in Example 1 on the ethanol-induced gastric ulcer model in mice

[0056] Experimental animals: 5-week-old SPF-grade C57BL / 6JGPt male mice, obtained from Chengdu Yaokang Biotechnology Co., Ltd., animal license number: SCXK (Chuan) 2020-034.

[0057] Experimental methods: After the experimental animals were adaptively fed, all animals were fasted but not watered for 24 hours after administration one day before the experiment. Before modeling, the experimental mice were randomly divided into: 5 blank groups, 10 model groups, 10 teprenone groups, and 10 VPL administration groups. Except for the blank group and the model group, which were gavaged with 200uL purified water, the teprenone group was gavaged with 160mg / kg, and the VPL administration group was gavaged with 0.1mg / kg. One hour after administration, each group of mice was orally gavaged with 0.9mL / kg of anhydrous ethanol for modeling. One hour later, the animals were killed by cervical dislocation, the gastric cardia was ligated and the pylorus was clamped, and the whole stomach was removed. 1mL of 1% formaldehyde solution was injected into the stomach, the cardia was ligated, and the stomach was taken out and placed in 1% formaldehyde solution. After soaking for 30 minutes, the stomach tissue was taken out, cut open along the greater curvature of the stomach, and the stomach contents were washed clean with saline. After flattening, the damage of the gastric mucosa of the mice was observed and measured, and the ulcer index and ulcer inhibition rate were calculated. The results are shown in Table 2.

[0058] Calculation method of ulcer index: if the length of the cord-like lesion is greater than 1 mm, measure its length and score 1 point for each millimeter; if its width is greater than 1 mm, double the score according to the number of millimeters of the width; if the length is less than 1 mm, score 0.5 points. Add up the scores to get the ulcer index of the animal.

[0059] Ulcer inhibition rate % = (ulcer index of model group - ulcer index of drug administration group) / ulcer index of model group × 100%.

[0060] Table 2 Anti-ulcer effect of VPL on ethanol-induced gastric ulcer model in mice

[0061] Sample name Test dose / mg / kg Ulcer Index Ulcer inhibition rate / % VPL 0.1 12.80±1.99 62.35 Teprenone group 160 22.30±2.22 34.41 Model Group - 34.00±7.25 -

[0062] In summary, the peptide (VPL) of the present invention has a good therapeutic effect on the ethanol-induced gastric ulcer model in mice, and the ulcer inhibition rate is significantly higher than that of the teprenone group. This shows that the peptide (VPL) of the present invention can effectively promote the repair of mucosa, alleviate the development of gastrointestinal disease pathology of acute and chronic gastritis and peptic ulcer, and can prevent and treat diseases caused by mucosal damage. The application of the present invention provides a new idea and strategy for diseases caused by mucosal or skin damage in clinical practice.

Claims

1. Use of a peptide having an amino acid sequence of valine-proline-leucine or a salt thereof in the preparation of a drug for preventing diseases caused by gastric mucosal damage induced by ethanol.

2. The use according to claim 1, characterized in that: The peptide is prepared by chemical synthesis.

3. The use according to claim 1, characterized in that: The medicine is a preparation prepared by taking a peptide with an amino acid sequence of valine-proline-leucine or a salt thereof as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

4. The use according to claim 1, characterized in that: The preparation is an oral preparation.