WO2023092926A1 - 一类促进组织修复的新多肽及其应用 - Google Patents

一类促进组织修复的新多肽及其应用 Download PDF

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WO2023092926A1
WO2023092926A1 PCT/CN2022/085715 CN2022085715W WO2023092926A1 WO 2023092926 A1 WO2023092926 A1 WO 2023092926A1 CN 2022085715 W CN2022085715 W CN 2022085715W WO 2023092926 A1 WO2023092926 A1 WO 2023092926A1
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ala
tyr
gly
tissue repair
products
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PCT/CN2022/085715
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English (en)
French (fr)
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叶文才
王磊
张冬梅
刘俊珊
范春林
曹佳青
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暨南大学
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Priority to EP22897027.3A priority Critical patent/EP4438616A1/en
Priority to AU2022396332A priority patent/AU2022396332A1/en
Publication of WO2023092926A1 publication Critical patent/WO2023092926A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the fields of medicine, medical equipment and daily chemicals, in particular to a class of novel polypeptides for promoting tissue repair and applications thereof.
  • tissue trauma includes not only trauma such as burns and wounds, but also non-healing wounds and ulcers such as gastric ulcers, oral ulcers, diabetic ulcers, autoimmune skin ulcers, venous stasis ulcers and Bed sores caused by prolonged bed rest.
  • non-healing wounds and ulcers such as gastric ulcers, oral ulcers, diabetic ulcers, autoimmune skin ulcers, venous stasis ulcers and Bed sores caused by prolonged bed rest.
  • the pathogenesis of refractory wounds and ulcers is complicated, the course of the disease is long, the treatment is difficult, and the treatment costs are high, which brings heavy physical and mental pressure and economic burden to patients.
  • growth factor drugs are widely used in the treatment of refractory wounds and ulcers, and have shown good therapeutic effects.
  • epidermal growth factor epidermal growth factor
  • basic fibroblast growth factor and vascular endothelial growth factor are all effective for wound and ulcer healing.
  • high cost and poor stability of these endogenous factors limit their clinical application. Therefore, it is very necessary to find and develop active substances with significant activity, low production cost and good stability for treating tissue wounds, refractory wounds and ulcers.
  • the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and provide a class of new polypeptides that can promote tissue repair and applications thereof.
  • the present invention adopts the following technical solutions:
  • TRF1-TRF12 A new polypeptide that promotes tissue repair, named TRF1-TRF12, its structure is shown in Formula I:
  • R 1 H or Ala or Gly or Ala-Ala- or Val-Ala-;
  • R 2 Asn or Asp
  • R 3 Ser or Tyr or -Gly-Ser-Tyr or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His- Val-Arg or -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg-Glu-Tyr-Pro-Ala-Gly-Val-Ser-Ala-Ala.
  • novel polypeptide compounds include but not limited to TRF1-TRF12 as shown below:
  • TRF1-TRF12 The preparation of the new polypeptides (TRF1-TRF12) can be carried out by known methods in the prior art. It can be chemically synthesized with a polypeptide synthesizer, or the nucleotide sequence can be deduced from the polypeptide sequence, and then cloned into biosynthesis in expression vectors;
  • Another object of the present invention is to disclose the application of the novel polypeptides (TRF1-TRF12) in the preparation of products for promoting tissue repair or improving skin aesthetics.
  • the new polypeptides are used in the preparation of products for treating wounds, scalds, ulcers or improving skin aesthetics.
  • the product is preferably a medicine, a medical device or a daily chemical product.
  • the drug, medical device or daily chemical product contains effective doses of one or more novel polypeptides (TRF1-TRF12), or pharmaceutically acceptable salts or stereoisomers thereof, and the balance As excipients or other compatible drugs.
  • TRF1-TRF12 novel polypeptides
  • pharmaceutically acceptable salts or stereoisomers thereof the balance As excipients or other compatible drugs.
  • the adjuvants refer to conventional excipients, such as solvents, disintegrants, flavoring agents, preservatives, coloring agents and binders.
  • the other compatible drugs refer to other natural drugs, chemical drugs or biological drugs.
  • the medicines, medical devices or daily chemical products can be used as tablets, capsules, injections, liposome nanoparticles, controlled release agents, gel creams, ointments, liniments for external use, patches, creams, detergents , lotion, gel, toner, etc.
  • the present invention has the following advantages and effects:
  • polypeptides (TRF1-TRF12) described in the present invention are a class of polypeptides with new structures, and these new polypeptides all contain disulfide bonds, have stable structures and are not easy to degrade.
  • TRF1-TRF12 The new polypeptides (TRF1-TRF12) provided by the present invention can significantly promote the proliferation of human immortalized epidermal cells (HaCAT), and at the same time significantly promote the migration of human skin fibroblasts (HSF), and have the effect of promoting tissue repair.
  • HaCAT human immortalized epidermal cells
  • HSF human skin fibroblasts
  • the new polypeptides (TRF1-TRF12) provided by the present invention can be used to promote tissue repair, treat digestive tract ulcers and oral ulcers, and have the effect of removing wrinkles, scars and stains.
  • the new polypeptides (TRF1-TRF12) provided by the present invention have low toxicity, and no obvious toxic side effects are observed at an oral dose of 500 mg/kg.
  • novel polypeptides (TRF1-TRF12) provided by the present invention can be chemically or biologically synthesized, and are easy to produce in large quantities.
  • Figure 1 is the mass spectrum of the new polypeptide TRF1, where A is its ESI-MS image; B is its MS 2 image (m/z 50-580); C is its MS 2 image (m/z 580-1110).
  • Figure 2 is the mass spectrum of the new polypeptide TRF2, where A is its ESI-MS image; B is its MS 2 image (m/z 50-420); C is its MS 2 image (m/z 390-810); D is its MS image (m/z 390-810); MS 2 pattern (m/z 810-1170).
  • Figure 3 is the mass spectrum of the new polypeptide TRF3, where A is its ESI-MS image; B is its MS 2 image (m/z 50-600); C is its MS 2 image (m/z 600-1300).
  • Figure 4 is the mass spectrum of the new polypeptide TRF4, where A is its ESI-MS image; B is its MS 2 image (m/z 50-510); C is its MS 2 image (m/z 510-1250).
  • Figure 5 is the mass spectrum of the new polypeptide TRF5, where A is its ESI-MS image; B is its MS 2 image (m/z 50-650); C is its MS 2 image (m/z 620-1200).
  • Figure 6 is the mass spectrum of the new polypeptide TRF6, where A is its ESI-MS image; B is its MS 2 image (m/z 50-400); C is its MS 2 image (m/z 400-900); D is its MS image (m/z 400-900); MS 2 pattern (m/z 850-1800).
  • Figure 7 is the mass spectrum of the new polypeptide TRF7, where A is its ESI-MS image; B is its MS 2 image (m/z 50-400); C is its MS 2 image (m/z 400-970); D is its MS image (m/z 400-970); MS 2 pattern (m/z 970-1700).
  • Figure 8 is the mass spectrum of the new polypeptide TRF8, wherein A is its ESI-MS image; B is its MS 2 image (m/z 50-620); C is its MS 2 image (m/z 620-1150).
  • Figure 9 is the mass spectrum of the new polypeptide TRF9, where A is its ESI-MS image; B is its MS 2 image (m/z 50-420); C is its MS 2 image (m/z 390-810); D is its MS image (m/z 390-810); MS 2 pattern (m/z 800-1200).
  • Figure 10 is the mass spectrum of the new polypeptide TRF10, where A is its ESI-MS image; B is its MS 2 image (m/z 50-500); C is its MS 2 image (m/z 450-1250).
  • Figure 11 is the mass spectrum of the new polypeptide TRF11, where A is its ESI-MS image; B is its MS 2 image (m/z 50-420); C is its MS 2 image (m/z 390-820); D is its MS image (m/z 390-820); MS 2 pattern (m/z 800-1270).
  • Figure 12 is the mass spectrum of the new polypeptide TRF12, where A is its ESI-MS image; B is its MS 2 image (m/z 50-380); C is its MS 2 image (m/z 380-920); D is its MS 2 pattern (m/z 920-1560).
  • Fig. 13 is an analysis diagram of the effect of new polypeptides TRF1-TRF12 on the proliferation of HaCAT cells.
  • Fig. 14 is an analysis diagram of the effect of new polypeptides TRF1-TRF12 on the migration of HSF cells.
  • the above-mentioned crude polypeptide is dissolved in acetonitrile aqueous solution with a volume ratio of 20%, filtered, and purified by high-performance liquid chromatography.
  • the detection wavelengths are 214nm, 254nm, and 280nm, and the mobile phase is acetonitrile-water (volume ratio 10:90 ⁇ 50:50) , the flow rate was 1mL/min, the target peak was collected, concentrated, and freeze-dried to obtain the pure peptide.
  • polypeptide TRF2 ⁇ TRF12 The synthesis process of polypeptide TRF2 ⁇ TRF12 is similar to the above TRF1. Put 1g 0.3 ⁇ 0.5mmol of amino acid with Fmoc and side chain protection (the first carboxyl terminal)-Wang resin in a solid phase synthesizer, after deprotection, activation, Condensation, washing and other steps sequentially connect the amino acids in the sequence to synthesize the peptide connected to the resin, and then cut and purify to obtain the pure peptide.
  • the polypeptides TRF1-TRF12 were weighed and dissolved in deionized water respectively to prepare a sample solution with a concentration of 0.1 mg/mL. Inject the sample solution separately into the AB Sciex 5600+ mass spectrometer for analysis. Among them, mass range (m/z): 50-2000Da; acquisition mode: positive ion mode; scanning mode: selected ion scanning; capillary voltage: 3.0kV; ion source temperature: 110°C; collision energy range: 50-80V. Perform secondary scanning on the parent ion, and analyze the b and y ions according to the fragments of the secondary mass spectrum, so as to verify the amino acid residue sequence of the new polypeptide, as shown in Figures 1-12.
  • Cell inoculation cells in the logarithmic growth phase were selected and counted after digestion. Take the required number of cells and add an appropriate amount of medium to dilute to 1 ⁇ 10 4 /mL. Homogenize the cells by pipetting with a row gun, then add the cell solution to a 96-well plate (100 ⁇ L per well), and incubate in a CO 2 incubator.
  • the test results are shown in Figure 13.
  • the new polypeptides TRF1-TRF12 have a significant pro-proliferation effect on HaCAT cells at a lower concentration (0.08-2.00 ⁇ g/mL) (cell proliferation rate 114 ⁇ 5%-135 ⁇ 3%).
  • Example 4 The promoting effect of new polypeptide TRF1 ⁇ TRF12 on the migration of HSF cells
  • (1) Cell inoculation and dosing Place the Transwell chamber in a clean bench for ultraviolet sterilization for 30 minutes, and then place it in a 24-well plate. Count the cells after digestion, and dilute the cells with culture medium to a density of 2 ⁇ 10 5 cells/mL. Add the drugs TRF1-TRF12 to be tested and the positive control drug into the cell fluid, and dilute them into drug-containing cell fluids with different concentrations. Add 100 ⁇ L of drug-containing cell solution to the upper chamber of the administration group, add 100 ⁇ L of cell solution to the blank group, and then add 600 ⁇ L of blank medium to each small chamber, and place them in an incubator for culture.
  • test results showed that the new polypeptides TRF1-TRF12 (2 ⁇ g/mL) could significantly promote the migration of HSF cells.
  • the average number of migrated cells in the drug group was 578 ⁇ 9-396 ⁇ 14, which was significantly higher than that of the control group (132 ⁇ 10 , with statistical significance (P ⁇ 0.05), the results are shown in Figure 14.
  • Example 5 The repairing effect of the new polypeptide TRF7 on mouse skin wounds
  • the transparent oxygen-permeable dressing was gently lifted off, 0.1 mL of the medicine was dripped onto the wound, and a new dressing was pasted on.
  • test results show that the new polypeptide TRF7 can significantly promote the healing of skin wounds in mice, which has a significant difference compared with the control group, and is better than Kangfuxin Liquid.
  • test results show that the new polypeptide TRF7 can significantly promote the healing of scalded skin in mice, which has a significant difference compared with the control group, and is better than Kangfuxin Liquid.
  • Example 7 The protective effect of the new polypeptide TRF7 on rat stress gastric ulcer
  • Test method 20 male SD rats weighing 180-210g (purchased from Guangdong Animal Center) were randomly divided into blank group, model group, sucralfate group (1g/kg, positive drug group), TRF7 low-dose group (12.5mg/kg) and TRF7 high-dose group (50.0mg/kg).
  • the administration group was intragastrically administered for 7 consecutive days. 24 hours before modeling, no food or water was allowed. Except for the control group, the models were made by restraint water immersion.
  • the rats in each group were fixed on the rat board, and immersed in a constant temperature (20°C) water tank with their heads up for 8 hours. The xiphoids are flush.
  • the rats were sacrificed by cervical dislocation, laparotomy, and pylorus ligation.
  • the stomach was perfused with 2 mL of 10% formaldehyde solution, the cardia was ligated, and the gastric body was taken out and fixed in formaldehyde solution for 15 minutes. They were cut along the greater curvature of the stomach, rinsed with normal saline, and unfolded. The gastric mucosal damage was observed and the gastric ulcer index was calculated.
  • Ulcer index was calculated according to the Guth standard: 1 point for spotting bleeding, 2 points for linear bleeding length ⁇ 1 mm, 3 points for 1-2 mm, 4 points for 2-4 mm, 5 points for >4 mm, 5 points for width >4 mm 1mm hour and minute value ⁇ 2. The results are shown in Table 3.
  • test results show that the new polypeptide TRF7 has a significant protective effect on rat stress gastric ulcer, which is significantly different from the model group, and its protective effect is better than that of the positive drug sucralfate.
  • Example 8 The protective effect of the new polypeptide TRF7 on oral ulcers in rats
  • Test method 15 healthy SD rats weighing 180-210 g (purchased from Guangdong Provincial Animal Center) were randomly divided into model group, positive drug group (Kangfuxin liquid), TRF7 low-dose group (0.5 mg/mL) and high-dose group.
  • the dosage group 2.0 mg/mL was modeled by chemical burning. Take a plastic tube with a diameter of 5 mm, place a small cotton ball soaked in 95% phenol solution on one end of the plastic tube, fix the cotton end on the left buccal mucosa of the anesthetized rat, keep it for 60 seconds, and wash the burning surface with normal saline .
  • Ulcer scoring criteria are as follows: no congestion, edema, ulcer surface diameter ⁇ 1 mm, 1 point; mild congestion, edema, ulcer surface diameter 1-2 mm, 2 points; moderate congestion, edema, ulcer surface diameter 2-3 mm, 3 points; Severe hyperemia, edema, ulcer surface diameter > 3mm, 4 points. The results are shown in Table 4.
  • test results show that the new polypeptide TRF7 can significantly promote the healing of oral ulcers in rats, which has a significant difference compared with the model group, and is better than Kangfuxin Liquid.
  • Embodiment 9 The acute toxicity experiment of new polypeptide TRF1 ⁇ TRF12
  • Test method Take Kunming mice weighing 18-22g (purchased from Guangdong Provincial Animal Center), divide them into random groups, 10 mice in each group, orally administer different doses of TRF1-TRF12, and calculate the median lethal dose LD according to the survival of the mice 50 .
  • Subject population 18 to 55 years old, 10 females, 10 males, 20 people in total. The subjects have healthy skin, no history of skin allergies, and meet the voluntary inclusion criteria.
  • Test method select a qualified patch device, use the closed patch test method, drop about 0.020-0.025mL (2mg/mL) of polypeptide TRF7 into the patch device, and stick the special tape for external use on the subject's back, 24 Remove the test product after 1 hour, observe the skin reaction at 0.5, 6, 12, 24, and 48 hours after removal, and record the results according to the skin reaction grading standard in the "Hygienic Standards for Cosmetics".
  • Test results 20 subjects in this test passed the patch test and observed skin reactions at 0.5, 6, 12, 24, and 48 hours, and no adverse skin reactions were observed, indicating that the new polypeptide TRF7 of the present invention is safe for use on the skin .
  • Polypeptide TRF7 0.1g, propylene glycol 50g, grind, add 100mL water for injection to dilute, mix well, then add 9g sodium chloride, after dissolving, add water for injection to 1000mL, adjust the pH value to 5.5-6.5, filter, pot, extinguish bacteria, and 1000 injections were obtained.
  • Polypeptide TRF7 0.1g add 40g of lactose and 10g of starch slurry in turn, directly load into the rotary granulator/coater to prepare granules, and spray the plasticized ethylcellulose coating agent suspension diluted to 15% solids by mass fraction onto the rotating bed of peptide particles.
  • the granules were film-coated with a dispersion carrier made of poloxamer 188 to form sustained-release granules with an average particle size of approximately 450 [mu]m.
  • a dispersion carrier made of poloxamer 188 to form sustained-release granules with an average particle size of approximately 450 [mu]m.
  • TRF7 Take 0.1g of polypeptide TRF7, dissolve it in 45mL of distilled water, filter, add 5mL of glycerin and 1mL of azone to the filtrate, make up the volume to 50mL with distilled water, and make a liniment for external use containing TRF7 0.2%.
  • polypeptide TRF7 Take 0.1g of polypeptide TRF7 for later use, mix 1g of No. 26 white oil, 1g of stearyl alcohol, 1g of stearic acid, 0.5g of monoglyceride, 0.05g of 350 silicone oil, 0.5g of GTCC, and 0.03g of methylparaben, and heat to 90°C to make the first semi-finished product; add 4g of deionized water, 0.1g of polypeptide TRF7, 0.3g of Pingpingjia-20, and 0.5g of glycerin to the first half of the finished product, and heat it to 80°C to form the second semi-finished product; The semi-finished product was homogenized twice at 80°C (3000 rpm, 10 minutes) and then stirred for 30 minutes. After cooling to 45°C to form a creamy form, 0.005g of Cathone was added and mixed well to obtain 1 % Peptide Cream.
  • TRF7 Take 0.1g TRF7 and prepare it as a 0.2mg/mL aqueous solution; add 5g glycerin to 1g hyaluronic acid to disperse, add water to dissolve, and stir evenly to obtain a hyaluronic acid solution; dissolve 1g trehalose and 1g allantoin in water and mix with Mix the above hyaluronic acid solutions and stir evenly to obtain a mixed solution; add the polypeptide solution, 1g of D-panthenol, 10g of oat ⁇ -glucan, 10g of 1,2-pentanediol and 0.05g of preservatives to the above mixed solution in sequence , add water and stir well, that is.
  • step (3) Pour the product obtained in step (3) into the product obtained in step (4) quickly, homogenize at a constant temperature for 3 to 5 minutes, and cool;
  • TRF7 0.1g TRF7 as 0.2mg/mL aqueous solution; mix the above polypeptide solution, 2.5g glycerin, 3g decyl glucoside, 3g sodium cocoyl apple amino acid, 1.5g sodium laureth sulfate, 0.1g solubilizer Add 0.05g of essence and 0.05g of essence into water and stir in turn, then add 0.5g of thickener, stir until completely swollen to get cleansing gel.
  • Test product the polypeptide emulsion prepared in Example 22 of the present invention
  • Subject population 18 to 55 years old, 18 females, 12 males, 30 people in total.
  • the subjects' skin has wrinkles, stains, dull color, and unsightly factors such as minimally invasive and post-operative scars on the skin.
  • Example 22 After the subject's skin was cleaned, the emulsion prepared in Example 22 was applied to the skin, and the effect was observed and felt.
  • the subjects said that using the new polypeptide TRF7 emulsion product can increase the water content of the skin, enhance the skin's moisturizing power and skin elasticity, and improve the skin's supple and moist feeling.
  • This product has the effects of whitening, removing wrinkles, scars and spots.
  • the subjects showed no allergies.
  • Test product the facial mask prepared in Example 20 of the present invention.
  • Subject population 18-58 years old, 21 females, 9 males, 30 people in total.
  • the subject's facial skin has wrinkles, spots, dull color, and unsightly factors such as minimally invasive and post-operative scars on the skin.
  • Test method After cleansing the subject's face, apply the new polypeptide mask prepared in Example 20 on the face once a day to observe and feel the effect.
  • the subjects said that using the new polypeptide TRF7 mask product can increase the water content of the skin, enhance the skin's moisturizing power and skin elasticity, and improve the skin's supple and moist feeling.
  • the product has the effect of whitening, removing wrinkles, scars and spots.
  • the subjects showed no allergies.

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Abstract

本发明公开了一种促进组织修复的新多肽及其应用，所述新多肽在较低浓度下可促进人永生化表皮细胞的增殖和人表皮成纤维细胞的迁移，能够促进创伤和溃疡的修复，改善皮肤美感，且毒副作用低，具有良好的应用前景，可用于制备治疗创面、烫伤、溃疡的药物和医疗器械或改善皮肤美感的日化用品。

Description

一类促进组织修复的新多肽及其应用 技术领域：
本发明涉及药物、医疗器械及日化领域，具体涉及一类促进组织修复的新多肽及其应用。
背景技术：
随着社会的发展及老龄化进程加速，组织创伤不仅包括烧伤、创伤等外伤，还包括难愈性创面和溃疡如胃溃疡、口腔溃疡、糖尿病溃疡、自身免疫性皮肤溃疡、静脉淤滞性溃疡以及长期卧床所致褥疮等。难愈性创面和溃疡的发病机制复杂、病程较长、治疗难度大、治疗费用高，给患者带来沉重的身心压力和经济负担。目前，生长因子类药物广泛应用于难愈性创面和溃疡的治疗，显示出良好的治疗效果，其中表皮生长因子、碱性成纤维细胞生长因子以及血管内皮生长因子等均对创面和溃疡愈合有重要作用，但是这些内源性因子制备成本高、稳定性差，限制了它们的临床应用。因此，很有必要寻找和开发活性显著、生产成本低、稳定性好的治疗组织创伤、难愈性创面和溃疡的活性物质。
发明内容：
本发明的目的在于，克服上述现有技术的不足，提供一类可以促进组织修复的新多肽及其应用。
为实现上述目的，本发明采用以下技术方案：
一种促进组织修复的新多肽，命名为TRF1～TRF12，其结构如式I所示：
Figure PCTCN2022085715-appb-000001
式I中：R 1＝H或Ala或Gly或Ala-Ala-或Val-Ala-；
R 2＝Asn或Asp；
R 3＝Ser或Tyr或-Gly-Ser-Tyr或-Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr或-Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg或 -Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg-Glu-Tyr-Pro-Ala-Gly-Val-Ser-Ala-Ala。
进一步的，在本发明的优选方案中，所述的新多肽类化合物，包括但不限于如下所示的TRF1～TRF12:
Figure PCTCN2022085715-appb-000002
所述的新多肽(TRF1～TRF12)的制备，可采用现有技术中的公知方法进行，既可以用多肽合成仪进行化学合成，也可以通过将多肽序列推导出核苷酸序列，然后克隆到表达载体中进行生物合成；
本发明的另一目的在于，公开了所述的新多肽(TRF1～TRF12)在制备促进组织修复或改善皮肤美感的产品中的应用。
优选的，所述的新多肽(TRF1～TRF12)在制备治疗创伤、烫伤、溃疡或改善皮肤美感的产品中的应用。
较佳的，所述的产品优选为药品、医疗器械或日化用品。
较佳的，所述的药品、医疗器械或日化用品中含有有效剂量的一种或多种新多肽(TRF1～TRF12)，或其药学上可接受的盐或其立体异构体,余量为辅料或其它可配伍的药物。
所述的辅料是指常规的赋形剂，如溶剂、崩解剂、矫味剂、防腐剂、着色剂和粘合剂等。
所述的其它可配伍的药物，是指其它天然药物、化学药品或生物药物。
所述的药品、医疗器械或日化用品可以采用片剂、胶囊剂、注射剂、脂质体纳米粒、控释剂、凝胶膏剂、软膏剂、外用搽剂、贴剂、霜剂、洗涤剂、乳液、凝胶、爽肤水等。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明所述的多肽(TRF1～TRF12)是一类具有新结构的多肽，这些新多肽均含有二硫键，结构稳定，不易降解。
(2)本发明提供的新多肽(TRF1～TRF12)能显著促进人永生化表皮细胞(HaCAT)的增殖，同时能显著促进人皮肤成纤维细胞(HSF)的迁移，具有促进组织修复作用。
(3)本发明提供的新多肽(TRF1～TRF12)可用于促进组织修复、治疗消化道溃疡、口腔溃疡，并具有去除皱纹、疤痕和色斑作用。
(4)本发明提供的新多肽(TRF1～TRF12)毒性较低，在口服500mg/kg剂量下未观察到明显的毒副作用。
(5)本发明提供的新多肽(TRF1～TRF12)可以化学合成或生物合成，易于大量制备。
附图说明
图1为新多肽TRF1的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～580)；C为MS 2图(m/z 580～1110)。
图2为新多肽TRF2的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～420)；C为MS 2图(m/z 390～810)；D为MS 2图(m/z 810～1170)。
图3为新多肽TRF3的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～600)；C为MS 2图(m/z 600～1300)。
图4为新多肽TRF4的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～510)；C为MS 2图(m/z 510～1250)。
图5为新多肽TRF5的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～650)；C为MS 2图(m/z 620～1200)。
图6为新多肽TRF6的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～400)；C为MS 2图(m/z 400～900)；D为MS 2图(m/z 850～1800)。
图7为新多肽TRF7的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～400)；C为MS 2图(m/z 400～970)；D为MS 2图(m/z 970～1700)。
图8为新多肽TRF8的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～620)；C为MS 2图(m/z 620～1150)。
图9为新多肽TRF9的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～420)；C为MS 2图(m/z 390～810)；D为MS 2图(m/z 800～1200)。
图10为新多肽TRF10的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～500)；C为MS 2图(m/z 450～1250)。
图11为新多肽TRF11的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～420)；C为MS 2图(m/z 390～820)；D为MS 2图(m/z 800～1270)。
图12为新多肽TRF12的质谱图，其中A为其ESI-MS图；B为其MS 2图(m/z 50～380)；C为MS 2图(m/z 380～920)；D为MS 2图(m/z 920～1560)。
图13为新多肽TRF1～TRF12对HaCAT细胞增殖影响的结果分析图。
图14为新多肽TRF1～TRF12对HSF细胞迁移影响的结果分析图。
具体实施方式
下面结合实施例及附图对本发明作进一步的描述，但本发明的实施方式不限于此。
实施例1新多肽TRF1～TRF12的合成
(1)新多肽TRF1的固相合成
将1g 0.3～0.5mmol带Fmoc及侧链保护的酪氨酸-Wang树脂置于固相合成仪中，加入20mL N,N-二甲基甲酰胺使其充分溶胀。将溶剂抽干后，加入20％哌啶(按体积比溶于N,N-二甲基甲酰胺)，搅拌约10min后抽干，重复2次，使Fmoc基团脱除完全，加入20mL N,N-二甲基甲酰胺洗涤，抽干溶剂，重复3次；加入0.5mmol带Fmoc及侧链保护的半胱氨酸，再加入等量的HBTU/N,N-二甲基甲酰胺和N,N-二异丙基乙胺/N,N-二甲基甲酰胺溶液，于N 2保护下反应40～60min；用N,N-二甲基甲酰胺洗脱未反应的药品和试剂，茚三酮检测；后续采用相同的方法连接剩余氨基酸，经过脱保护、活化、缩合、洗涤等步骤合成得到接在树脂上的多肽。向树脂上的多肽加入裂解液(三氟乙酸/三异丙基硅烷/纯水/1,2-乙二硫醇，体积比94:2.5:2.5:1)切割2～3h后，加入冰乙醚，离心后得到下 层沉淀。取出沉淀，除去残留的三氟乙酸，加入一定量的碘溶液，于室温下搅拌反应约1h，即得到目标多肽粗品。
上述多肽粗品用体积比为20％的乙腈水溶液溶解，过滤，采用高效液相色谱仪纯化，检测波长为214nm、254nm、280nm，流动相为乙腈-水(体积比10:90～50:50)，流速为1mL/min，收集目标峰，浓缩、冻干，得到多肽纯品。
(2)新多肽TRF2～TRF12的固相合成
多肽TRF2～TRF12的合成过程与上述TRF1类似，将1g 0.3～0.5mmol带Fmoc及侧链保护的氨基酸(羧基端第一个)-王树脂置于固相合成仪中，经过脱保护、活化、缩合、洗涤等步骤依次连接序列中的氨基酸，合成得到连接在树脂上的多肽，再经切割、纯化得到多肽纯品。
实施例2新多肽TRF1～TRF12的质谱鉴定
称取多肽TRF1～TRF12分别溶解于去离子水中，配置成浓度为0.1mg/mL的样品溶液。将样品溶液分别注入AB Sciex
Figure PCTCN2022085715-appb-000003
5600+质谱仪进行分析。其中质量范围(m/z)：50～2000Da；采集模式：正离子模式；扫描模式：选择离子扫描；毛细管电压：3.0kV；离子源温度：110℃；碰撞能量范围：50～80V。对母离子进行二级扫描，根据二级质谱碎片，对其进行b和y离子分析，从而验证新多肽的氨基酸残基序列，见图1～12。
实施例3新多肽TRF1～TRF12对HaCAT细胞增殖的促进作用
(1)细胞接种：选用对数生长期的细胞，消化后计数。取所需细胞数，加入适量培养基稀释至到1×10 4/mL。用排枪吹打均匀细胞，然后将细胞液加到96孔板中(每孔100μL)，置于CO 2培养箱中孵育。
(2)加药：待细胞贴壁后移除旧培养基，对照组加入空白培养基，阳性对照组加入EGF，药物组分别加入不同浓度的TRF1～TRF12，孔板四周加入PBS，实验设置6个复孔，置于培养箱中孵育48h。
(3)加MTT：吸除旧培养基，加入配置好的MTT(避光)，每孔30μL，然后置于培养箱中继续孵育4h，形成甲瓒晶体。
(4)测定OD值：移除MTT，每孔中加入100μL DMSO溶液，使甲瓒溶解充分，用多功能酶标仪(检测波长：595nm)检测吸光度。计算细胞存活率， 重复实验至少3次以上。
试验结果如图13所示，新多肽TRF1～TRF12在较低浓度(0.08～2.00μg/mL)下对HaCAT细胞具有显著的促增殖作用(细胞增殖率114±5％～135±3％)。
实施例4新多肽TRF1～TRF12对HSF细胞迁移的促进作用
(1)细胞接种及加药：将Transwell小室置于超净台中紫外灭菌30min，然后置于24孔板中。细胞消化后计数，用培养基稀释细胞至密度为2×10 5个/mL。将待测药物TRF1～TRF12及阳性对照药加到细胞液中，稀释成不同浓度的含药细胞液。给药组于上室加入100μL含药细胞液，空白组加入100μL细胞液，然后于各个小室加入600μL空白培养基，置于培养箱中培养。
(2)固定、染色及拍照：孵育结束后，弃去培养基，用PBS清洗，用4％多聚甲醛固定下室细胞(大约30min)。固定后用PBS清洗两次，在每孔下室加入600μL染色液染色20min。回收结晶紫，PBS清洗，用棉签小心抵住上室内膜，擦去没有迁移成功的细胞，风干后用荧光倒置显微镜拍照，每个小室随机拍取5个区域，最后用GraphPad Prism 6.0统计分析细胞平均迁移个数。
试验结果显示，新多肽TRF1～TRF12(2μg/mL)能明显促进HSF细胞的迁移，作用48h后，药物组平均迁移细胞数在578±9～396±14个，明显高于对照组132±10个，具有统计学意义(P＜0.05)，结果如图14所示。
实施例5新多肽TRF7对小鼠皮肤创伤的修复作用
选取15只8～10周雄性昆明小鼠(购自广东省动物中心)分笼饲养。随机分为对照组，阳性药组(康复新液)，TRF7低剂量组(0.5mg/mL)和TRF7高剂量组(2.0mg/mL)。将小鼠麻醉，背部脱毛、消毒后，利用直径为6mm的打孔器于小鼠背部裸露皮肤处行打孔，切除一圆形皮肤，包括表皮和真皮层，将伤口部位贴上透明透氧敷料，起到屏蔽和保护作用。对照组给予生理盐水，给药时轻轻揭起透明透氧敷料，滴加0.1mL药液滴于伤口，再贴上新的敷料。连续给药10d，每天给药两次，每天测量伤口面积大小变化，给药10天后处死小鼠。按公式创伤愈合率％＝(S 1-S n)/S 1(S 1为第一天创伤面积，S n为第n天创伤面积)计算创面愈合率。结果见表1。
表1小鼠创伤愈合率统计结果
Figure PCTCN2022085715-appb-000004
注：与模型组相比，*P＜0.05有显著性差异
试验结果表明，新多肽TRF7能明显促进小鼠皮肤创伤的愈合，与对照组比较具有显著性差异，且优于康复新液。
实施例6新多肽TRF7对小鼠皮肤烫伤的修复作用
选取15只8～10周雄性昆明小鼠(购自广东省动物中心)分笼饲养，自由取食，实验前一天停止进食。用质量分数13％Na 2S溶液涂抹脱毛，将20g的砝码置于水浴锅内一同加热至沸，维持10min，然后用钳子夹住砝码上端，迅速放在小鼠皮肤上5s，稍加压力，形成烫伤面积为2cm×2cm的浅Ⅱ度烫伤模型。次日，伤口滴加不同溶度的TRF7(0.5mg/mL或2.0mg/mL)、生理盐水和康复新液各0.1mL，每日换药一次。于3、5、10天观察创面愈合情况。按实施例5的方法计算创面愈合率。结果见表2。
表2小鼠烫伤愈合率统计结果
Figure PCTCN2022085715-appb-000005
注：与模型组相比，*P＜0.05有显著性差异
试验结果表明，新多肽TRF7能明显促进小鼠皮肤烫伤的愈合，与对照组比较具有显著性差异，且优于康复新液。
实施例7新多肽TRF7对大鼠应激性胃溃疡的保护作用
试验方法：取体重180～210g雄性SD大鼠20只(购自广东省动物中心)，随机分为空白组、模型组、硫糖铝组(1g/kg，阳性药组)、TRF7低剂量组(12.5mg/kg)和TRF7高剂量组(50.0mg/kg)。给药组灌胃给药，连续7天。造模前24h禁食不禁水，除对照组外，采用束缚水浸法造模，将各组大鼠固定于鼠板，头向上垂直浸泡在恒温(20℃)水槽中8h，水面与大鼠剑突齐平。应激造模结束后，将大鼠颈椎脱臼处死，剖腹，结扎幽门。向胃内灌注体积分数10％甲醛溶液2mL，结扎贲门，取出胃体置于甲醛溶液中固定15min。沿胃大弯剪开，生理盐水冲洗后展开，观察胃黏膜损伤并计算胃溃疡指数。按Guth标准计算溃疡指数(UI)：点状出血计为1分，线状出血长度<1mm为2分，1～2mm为3分，2～4mm为4分，>4mm为5分，宽度>1mm时分值×2。结果见表3。
表3大鼠溃疡指数统计结果
Figure PCTCN2022085715-appb-000006
注：与模型组相比，*P＜0.01有显著性差异
试验结果表明，新多肽TRF7对大鼠应激性胃溃疡具有明显保护作用，与模型组比较具有显著性差异，其保护效果优于阳性药硫糖铝。
实施例8新多肽TRF7对大鼠口腔溃疡的保护作用
试验方法：取体重180～210g健康SD大鼠15只(购自广东省动物中心)，随机分为模型组、阳性药组(康复新液)、TRF7低剂量组(0.5mg/mL)和高剂量组(2.0mg/mL)，采用化学灼烧法造模。取一直径为5mm的塑料管，将浸透95％ 苯酚溶液的小棉球置于塑料管一端，将棉花端固定于麻醉大鼠左侧颊黏膜上，保留60秒，用生理盐水清洗灼烧面。24h后观察，左侧实验区形成圆形溃疡，表面有黄白色脓肿，与周边分界明显，周围黏膜红肿充血，溃疡面直径＞3mm者即造模成功。实验组、模型组和阳性药组的溃疡面分别滴加新多肽溶液、生理盐水和康复新液各0.1mL，每日给药两次。于1、3、5天后观察溃疡面愈合情况。溃疡积分标准如下：无充血、水肿溃疡面直径＜1mm记1分；轻度充血、水肿，溃疡面直径1～2mm记2分；中度充血、水肿，溃疡面直径2～3mm记3分；重度充血、水肿溃疡面直径＞3mm记4分。结果见表4。
表4大鼠口腔溃疡愈合积分
Figure PCTCN2022085715-appb-000007
注：与模型组相比，*P＜0.05
试验结果表明，新多肽TRF7能够明显促进大鼠口腔溃疡的愈合，与模型组比较具有显著性差异，且优于康复新液。
实施例9新多肽TRF1～TRF12的急性毒性实验
试验方法：取体重18～22g的昆明种小鼠(购自广东省动物中心)，随机分组，每组10只，灌胃不同剂量的TRF1～TRF12，根据小鼠存活情况，计算半数致死剂量LD 50。半数致死量按如下公式计算：半数致死剂量(mg/kg)＝(小鼠一半死亡时的剂量/对应小鼠的体重)。
试验结果：新多肽TRF1～TRF12在500mg/kg的给药剂量下，均未观察到小鼠死亡和明显不良反应，说明其毒性都较低，LD 50大于500mg/kg(表5)。
表5 TRF1～TRF12对小鼠的急性毒性作用
Figure PCTCN2022085715-appb-000008
Figure PCTCN2022085715-appb-000009
实施例10新多肽TRF7的皮肤安全性评价
受试人群：年龄18～55岁之间，女性10人，男性10人，总计20人。受试者皮肤健康，无皮肤病过敏史，符合受试者志愿入选标准。
试验方法：选用合格的斑贴器，以封闭式斑贴试验方法，将多肽TRF7约0.020～0.025mL(2mg/mL)滴于斑贴器内，外用专用胶带贴覆于受试者背部，24小时后去除试验品，分别于去除后0.5、6、12、24、48小时观察皮肤反应，按照《化妆品卫生规范》中皮肤反应分级标准记录其结果。
试验结果：本试验20名受试者通过斑贴试验，在0.5、6、12、24、48小时观察皮肤反应，均未观察到皮肤不良反应，说明本发明的新多肽TRF7均对皮肤使用安全。
实施例11片剂的制备
取多肽TRF7 0.1g，乳糖40g，淀粉浆60g，硬脂酸镁0.2g，混合，过筛，干燥后压片。每片含TRF7 0.001g。
实施例12注射剂的制备
多肽TRF7 0.1g，丙二醇50g，研磨，再加100mL注射用水稀释，混匀，然后加入氯化钠9g，溶解后再加入注射用水至1000mL，调pH值5.5～6.5，滤过，灌封，灭菌，即得1000支注射用针剂。
实施例13固体脂质纳米颗粒的制备
取多肽TRF7 0.1g，依次加入大豆卵磷脂500mg，溶于25mL乙醇中，另取硬脂酸200mg和大豆卵磷脂500mg溶于25mL环己烷中，混合搅拌均匀；于37℃恒温水浴中减压旋转蒸发除去有机溶剂，使药物及辅料在烧瓶壁形成均匀脂质薄膜，于真空干燥器中放置过夜，除尽有机溶剂；另取聚乙二醇单硬脂酸酯3750mg，搅拌溶解在175mL水中，加入上述薄膜中，超声10min，定容至250mL，得淡黄色透明溶液；将此溶液冷冻干燥可得冻干粉。用球磨机研磨 24小时，制得粒径均匀的纳米粒，混匀并分装。每袋含TRF7 0.001g。
实施例14控释胶囊的制备
多肽TRF7 0.1g，依次加入乳糖40g和淀粉浆10g，直接装到旋转制粒机/包衣器制备颗粒，将稀释到质量分数为15％固体的塑化乙基纤维素包衣剂悬浮液喷雾到多肽颗粒的旋转床上。在喷雾期间，用泊洛沙姆188制成的分散体载体膜包衣颗粒，形成平均颗粒度大约为450μm的持续释放的颗粒。混匀装入胶囊，每个胶囊含TRF7 0.001g。
实施例15外用凝胶膏剂的制备
称取甘油2g，依次加入聚丙烯酸0.75g和氢氧化铝0.1g，充分混合，加入多肽TRF7 0.2g，在真空条件下充分搅拌混合得①；另外称取纯化水5g，将乳酸0.06g、羧甲基纤维素钠0.1g溶解在水得②；将②加入到①中，在真空条件下充分搅拌，经交联反应得到含药膏体，将含药膏体涂布(控制含药膏体层厚度在1.0mm左右)，裁剪成常规贴膏剂的规格，每贴含药物的量为0.02g，晾干，包装。
实施例16软膏剂的制备
硬脂醇40g与白凡士林45g在水浴中熔化，加热至75℃，备用。十二烷基硫酸钠1.46g，羟苯甲酯0.025g，羟苯丙酯0.015g，丙二醇13g，多肽TRF7 0.2g依次溶于水中，加热至75℃，将75℃硬脂醇与白凡士林加入，搅拌至冷，制成含TRF7 0.2％的软膏剂。
实施例17外用搽剂的制备
取多肽TRF7 0.1g，将其溶解于45mL蒸馏水中，过滤，滤液加入甘油5mL，氮酮1mL，蒸馏水补足体积至50mL，制成含TRF7 0.2％的外用搽剂。
实施例18创可贴的制备
称取硬脂酸聚羟氧40酯2g、聚山梨酯80 2g、泊洛沙姆188 1g、磷酸二氢钠0.3g、羟苯乙酯0.1g、纯化水50mL，在130℃条件下灭菌30min～60min， 降温至30～40℃，搅拌均匀得①；另称取轻质液状石蜡10g、十六醇0.5g、十八醇0.5g、白凡士林3g、单硬脂酸甘油酯1g，灭菌后在30～40℃保温，得②；取多肽TRF7 0.1g溶解于50mL蒸馏水中，过滤，加入①中，搅拌，得③。将②缓慢加入③中，加入15％乙醇3g，快速搅拌，调pH 6.0，保持温度在45℃，高速剪切，乳化5min，搅拌冷却至30～35℃，制得类白色均匀乳膏液；将膏液投到加料板上，用涂布机涂布于内垫上，低温烘干(45℃)，按需要尺寸裁剪，得涂药的内垫；将涂药的内垫粘在基布中部，将离型纸附于基布两端的粘接部。
实施例19面霜的制备
取多肽TRF7 0.1g备用，将26号白油1g，十八醇1g，硬脂酸1g，单甘脂0.5g，350硅油0.05g，GTCC 0.5g，尼泊金甲酯0.03g混合，加热到90℃，制成第一半成品；在第一半成品中加入去离子水4g、多肽TRF7 0.1g、平平加-20 0.3g、甘油0.5g，并加热到80℃，形成第二半成品；将第二半成品在80℃下进行2次均质(转速3000转/分钟，时间10分钟)后继续搅拌30分钟，冷却到45℃形成膏霜状后加入0.005g卡松，混匀，分别制得含1％多肽的面霜。
实施例20面膜的制备
多肽TRF7 0.1g溶解于10mL去离子水中，过滤，滤液与甘油润湿的卡波姆0.3g混合，加入三乙醇胺，调节pH值为6～7，均匀涂布于面膜纸，制成每张含有多肽0.01g的面膜。
实施例21爽肤水的制备
取0.1g TRF7，配置为0.2mg/mL的水溶液；将甘油5g加入透明质酸1g中分散，加水溶解，搅拌均匀，得透明质酸溶液；将海藻糖1g和尿囊素1g用水溶解后与上述透明质酸溶液混合，搅拌均匀，得混合溶液；将多肽溶液、D-泛醇1g、燕麦β-葡聚糖10g、1,2-戊二醇10g和防腐剂0.05g依次加入上述混合溶液，加水搅拌均匀，即得。
实施例22乳液的制备
(1)将0.1g TRF7配置为0.2mg/mL的水溶液；
(2)将0.05g透明质酸钠加水溶解分散，搅拌均匀，得透明质酸溶液；
(3)将甘油5g、丁二醇3g、海藻糖2g、尿囊素0.2g、增稠剂0.1g，用水溶解，加热至75℃；
(4)将霍霍巴籽油2g、氢化聚癸烯3g、聚二甲基硅氧烷2g、辛酸/癸酸三甘油酯3g、乳化剂3g加热至75℃，搅拌均匀；
(5)将步骤(3)制得的产物快速倒入步骤(4)制得的产物中，恒温均质3～5min，冷却；
(6)冷却至60℃以下，加入(1)、(2)，均质；冷却至40℃以下，加入防腐剂和香精，即得乳液。
实施例23洁面啫喱的制备
将0.1g TRF7配置为0.2mg/mL的水溶液；将上述多肽溶液、甘油2.5g、癸基葡糖苷3g、椰油酰苹果氨基酸钠3g、月桂醇聚醚硫酸酯钠1.5g、增溶剂0.1g、香精0.05g依次加入水中搅拌，再加入增稠剂0.5g，搅拌至完全溶胀即可得洁面啫喱。
实施例24新多肽乳液的美容效果评价
1.1试验品：本发明实施例22制备的多肽乳液
1.2受试人群：年龄18～55岁之间，女性18人，男性12人，总计30人。受试者皮肤有皱纹，色斑，色泽暗哑，以及皮肤上留有微创、手术后疤痕等不美观因素。
1.3测试方法：受试者皮肤清洁后，将实施例22制备的乳液涂覆到皮肤上，观察和感受使用效果。
1.4测试评估结果如下表6：
表6新多肽TRF7乳液的试用(8周)反馈总结表
Figure PCTCN2022085715-appb-000010
Figure PCTCN2022085715-appb-000011
受试者表示，使用新多肽TRF7乳液产品，可提高肌肤含水量，增强肌肤保湿力和皮肤弹性，提高肌肤水嫩润泽感。该产品具有美白，去皱纹、疤痕和色斑的效果。受试者未出现过敏现象。
实施例25新多肽面膜的美容效果评价
1.1试验品：本发明实施例20制备的面膜。
1.2受试人群：年龄18～58岁之间，女性21人，男性9人，总计30人。受试者脸部皮肤有皱纹，色斑，色泽暗哑，以及皮肤上留有微创、手术后疤痕等不美观因素。
1.3测试方法：受试者脸部清洁后，将实施例20制备的新多肽面膜涂覆于面部，每日一次，观察和感受使用效果。
1.4测试评估结果如下表7：
表7新多肽TRF7面膜的试用(8周)反馈总结表
Figure PCTCN2022085715-appb-000012
受试者表示，使用新多肽TRF7面膜产品，可提高肌肤含水量，增强肌肤保湿力和皮肤弹性，提高肌肤水嫩润泽感。产品具有美白，去皱纹、疤痕和色斑的效果。受试者未出现过敏现象。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、 组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (8)

  1. 一种促进组织修复的新多肽，其特征在于，所述新多肽的结构如下所示：
    Figure PCTCN2022085715-appb-100001
    其中：R 1＝H、Ala、Gly、Ala-Ala-或Val-Ala-；
    R 2＝Asn或Asp；
    R 3＝Ser、Tyr、-Gly-Ser-Tyr、-Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr、-Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg或-Gly-Ser-Tyr-Ala-Pro-Leu-Gly-Tyr-His-Val-Arg-Glu-Tyr-Pro-Ala-Gly-Val-Ser-Ala-Ala。
  2. 根据权利要求1所述的新多肽，其中，所述新多肽的结构如TRF1～TRF12中任一项所示：
    Figure PCTCN2022085715-appb-100002
  3. 权利要求1或2所述的新多肽在制备促进组织修复或改善皮肤美感的产品中的应用。
  4. 根据权利要求3所述的新多肽在制备促进组织修复或改善皮肤美感的产品中的应用，其中，所述的产品为药品、医疗器械或日化用品。
  5. 根据权利要求4所述的新多肽在制备促进组织修复或改善皮肤美感的产品中的应用，其中，所述的药品、医疗器械或日化用品中包括有效剂量的新多肽的一种或多种，或其药学上可接受的盐或其立体异构体，余量为辅料或其它可配伍的药物。
  6. 根据权利要求4所述的新多肽在制备促进组织修复或改善皮肤美感的产品中的应用，其中，所述的药品、医疗器械或日化用品为片剂、胶囊剂、注射剂、脂质体纳米粒、控释剂、凝胶膏剂、软膏剂、外用搽剂、贴剂、霜剂、洗涤剂、乳液、凝胶或爽肤水。
  7. 根据权利要求5所述的新多肽在制备促进组织修复或改善皮肤美感的产品中的应用，其中，所述的辅料为溶剂、崩解剂、矫味剂、防腐剂、着色剂或粘合剂。
  8. 一种药物组合物，其特征在于，含有权利要求1或2所述的新多肽和药学上可接受的载体。
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