Transdermal dipeptide and application thereof

Abstract

The invention relates to a transdermal dipeptide and application thereof, and belongs to the technical field of active polypeptides. A novel transdermal dipeptide Arg-Pro is designed and synthesized, the transdermal dipeptide has obvious penetration enhancing capacity, the penetration enhancing effect of the transdermal dipeptide is better than that of existing penetration enhancers, namely transdermal peptide TD-1, acetyl hexapeptide-1 and menthol, and the transdermal dipeptide is simple in structure, small in molecular weight, high in biocompatibility and free of toxic effects on cells, can be used for preparing penetration enhancing products and has good application prospects. Wide development prospects and application potentials are realized. Meanwhile, transdermal dipeptides Lys-Pro and Asn-Pro are also synthesized, and the transdermal delivery effect of the transdermal dipeptides Lys-Pro and Asn-Pro is slightly lower than that of Arg-Pro, but the transdermal dipeptides Lys-Pro and Asn-Pro still have an obvious penetration promoting effect.

Description

Technical Field

[0001] The invention relates to a transdermal dipeptide and application thereof, belonging to the technical field of active polypeptides. Background Art

[0002] The skin is the largest organ. Whether it is transdermal drug delivery for the treatment of skin diseases or for the purpose of beauty and skin care, how to promote the transdermal absorption of active ingredients is the focus of research by scientific researchers.

[0003] Traditional penetration enhancers are prone to skin allergies and inflammation, are toxic at high concentrations, are ineffective against large molecules and require instrumentation. Physical penetration enhancers are costly and inconvenient to carry. Long-term use can easily lead to thinning of the stratum corneum and skin infections. Biological penetration enhancer technology uses a class of peptide molecules with skin penetrating ability, about 10 to 30 amino acids, which can effectively promote the permeation of large molecules and other active ingredients. Transdermal peptides are non-irritating and will not cause skin allergies or damage. Drugs have good compatibility with transdermal peptides, and have stable physical and chemical properties, fast onset of action, and long duration of action.

[0004] Transdermal peptides are polypeptides with enhanced transdermal function, which can enhance and / or facilitate the penetration of active molecules through the skin, thereby increasing the dose of active molecules entering the systemic circulation or increasing the dose of active molecules reaching target organs, tissues and cells, and can be used for transdermal administration of large molecular weight drugs such as polypeptides, proteins and nucleic acid molecules.

[0005] Menthol, as a widely recognized percutaneous penetration enhancer, can destroy the lamellar structure of the stratum corneum and induce lipid cells to form a single-layer vacuolar structure, but it can cause functional damage to the stratum corneum and cause toxicity to the skin. Wan et al. used osthole as a target drug to conduct an in vitro percutaneous penetration experiment. When the dosage of menthol was as high as 0.5%, the permeation enhancement ratio was only 5.19.

[0006] Acetyl hexapeptide-1 (Ac-Nle-Ala-His-D-Phe-Arg-Trp-NH2) can induce the release of interleukins, and the release of interleukins increases the permeability of capillaries. Therefore, acetyl hexapeptide-1 has a certain permeability-promoting effect.

[0007] The transdermal peptide is transdermal peptide TD-1, with the amino acid sequence of ACSSSPSKHCG. It can overcome the skin barrier, quickly penetrate cells, retain its original structure and function, and enter cells independently of endocytosis. It can not only penetrate cells by itself, but also connect with other peptides to promote their cell penetration, thereby improving the transdermal efficiency of biomacromolecules.

[0008] Chinese patent document CN106084006A provides a polypeptide with transdermal function or transdermal enhancement function and a nucleic acid sequence encoding the polypeptide. The amino acid sequence of the polypeptide is ACSSTKKHCG, which can promote and / or enhance the transdermal penetration of other substances, including small molecule active substances, large molecule active substances such as proteins, nucleic acids, etc.

[0009] It can be seen that the prior art has proposed a technical solution that a polypeptide composed of about 10-30 amino acids can enhance the transdermal effect. However, through the prior art and the inventor's previous research, it was found that when the amino acid sequence changes, including the number and order of amino acids, the transdermal effect will also change significantly, lacking predictability. In order to enrich the product variety, different transdermal polypeptides can be further explored. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a transdermal dipeptide and its application. The present invention designs and synthesizes a new transdermal dipeptide, which has a good permeation-promoting effect, and its permeation-promoting effect is even better than that of transdermal peptide TD-1.

[0011] The technical solution of the present invention is as follows:

[0012] A transdermal dipeptide, the amino acid sequence of which is: Arg-Pro, or Lys-Pro, or Asn-Pro, in order from C-terminus to N-terminus.

[0013] Preferably according to the present invention, the amino acid sequence of the transdermal dipeptide is: Arg-Pro.

[0014] Application of the above transdermal dipeptide in the preparation of transdermal products.

[0015] Preferably according to the present invention, the transdermal products include cosmetic preparations, hair preparations, medicines, and foods; further preferably, the cosmetic preparations include skin care products and cosmetics.

[0016] A cosmetic preparation comprising the above transdermal dipeptide and at least one active ingredient for skin care, skin function improvement or skin color regulation.

[0017] A hairdressing preparation comprises the above transdermal dipeptide and at least one hairdressing active ingredient, wherein the hairdressing active ingredient comprises an ingredient having the effects of promoting hair growth, preventing hair loss, and changing hair structure, composition or color.

[0018] A pharmaceutical composition comprises the transdermal dipeptide and at least one active pharmaceutical ingredient in an effective dose for treating a disease.

[0019] A method for assisting the transdermal penetration of active substances is to mix the above transdermal dipeptide with an effective dose of active substances and apply the mixture to the skin surface.

[0020] Beneficial effects:

[0021] The present invention designs and synthesizes a new transdermal dipeptide Arg-Pro, which has obvious permeation-promoting ability, and its permeation-promoting effect is better than the existing permeation-promoting agents transdermal peptide TD-1, acetyl hexapeptide-1 and menthol, and its structure is simple, molecular weight is small, biocompatibility is high, and it has no toxic effect on cells, and can be used for the preparation of permeation-promoting products, and has broad development prospects and application potential. At the same time, the present invention also synthesizes transdermal dipeptides Lys-Pro and Asn-Pro, and although their transdermal delivery effects are slightly lower than Arg-Pro, they still have obvious permeation-promoting effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the synthetic route of the transdermal dipeptide Arg-Pro; in the figure, Aa represents amino acid.

[0023] Figure 2 This is the infrared spectrum of the transdermal dipeptide Arg-Pro.

[0024] Figure 3 This is the mass spectrum of the transdermal dipeptide Arg-Pro.

[0025] Figure 4 Schematic diagram of the Franz diffusion cell.

[0026] Figure 5 This is the cumulative permeation curve of diclofenac sodium per unit area in different treatment groups.

[0027] Figure 6 The figure is a bar graph showing the cell viability of human skin fibroblasts under different concentrations of transdermal dipeptide.

[0028] Figure 7 This is the cumulative permeation curve of diclofenac sodium per unit area in different transdermal dipeptide treatment groups. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The drugs and reagents involved in the examples, unless otherwise specified, are all common commercially available products; the experimental steps involved in the examples, unless otherwise specified, are all conventional technical operations in the art.

[0030] Example 1: Preparation of transdermal dipeptide

[0031] The synthetic route of the transdermal dipeptide Arg-Pro is as follows Figure 1 As shown, the specific steps are as follows:

[0032] (1) Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM) (the C-terminus is the carboxyl end);

[0033] (2) After 2 hours, wash the resin with 3 times the volume of N,N-dimethylformamide (DMF) and then drain it. Repeat this process four times and drain the resin for later use.

[0034] (3) Add 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin; after removing the protecting group, wash four times with DMF 3 times the volume of the resin and then drain;

[0035] (4) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0036] (5) Weigh 1.5 g of amino acid Arg into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0037] (6) Take 5 mL of 1-hydroxy-benzotriazole (HOBT, condensing agent) in a 50 mL centrifuge tube, add 20 mL of DMF to dissolve it, then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the solution to the above reactor after it becomes clear, and then place the reactor in a shaker at 30 °C for reaction;

[0038] (7) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF, and dried for later use;

[0039] (8) Add 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin; after removing the protecting group, wash four times with DMF 3 times the volume of the resin and then drain;

[0040] (9) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful;

[0041] (10) Weigh 1.5 g of amino acid Pro into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0042] (11) Take 5 mL of 1-hydroxy-benzotriazole (HOBT, condensing agent) in a 50 mL centrifuge tube, add 20 mL of DMF to dissolve it, then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the solution to the above reactor after it becomes clear, and then place the reactor in a shaker at 30 °C for reaction;

[0043] (12) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF, and dried for later use;

[0044] (13) 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) was added to the reactor and the resin was shaken on a decolorization shaker for 20 min to remove the Fmoc protecting group on the resin. After the protecting group was removed, the resin was washed four times with DMF 3 times the volume of the resin and then dried.

[0045] (14) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0046] (15) The peptide was cleaved from the resin using 95% cleavage solution (trifluoroacetic acid: 1,2-ethanedithiol: triisopropylsilane: water = 95:2:2:1, volume ratio) (10 mL of cleavage solution per gram of resin) and centrifuged four times with icy ether (cleavage solution: ether = 1:9, volume ratio); finally, it was separated and purified by HPLC and then freeze-dried to obtain the transdermal dipeptide Arg-Pro.

[0047] The infrared spectrum results of the transdermal dipeptide Arg-Pro obtained above are as follows Figure 2 As shown in the figure, the characteristic absorption peak of the transdermal dipeptide Ala-Pro appears at 3394 cm -1 、3197cm -1 、1670cm -1 、1515cm -1 、1469cm -1 、1368cm -1 、1195cm -1 、1135cm -1 、915cm -1 They belong to the NH bond in -CO=NH- and NH 2 The stretching vibration of NH in the bond, the stretching vibration of the associated -NH- bond, the amide I band of -CO=NH-, the stretching vibration of -C=N, the stretching vibration of -CH 3 - deformation vibration absorption peak, -CH 2-Deformation vibration absorption peak of -, stretching vibration of -CO-, stretching vibration of -CN- bond, bending vibration of -OH in -COOH.

[0048] The mass spectrometry results of the transdermal dipeptide Arg-Pro obtained above are as follows: Figure 3 As shown in the figure, the simulated mass-to-charge ratio of the transdermal dipeptide Arg-Pro is 271.18, and the measured mass-to-charge ratio is 271.27.

[0049] The above results prove that the transdermal dipeptide Arg-Pro has been successfully synthesized.

[0050] Example 2: Transdermal delivery study of transdermal dipeptide Arg-Pro

[0051] The diffusion cell experiment was conducted using mouse skin as the substrate. The specific steps are as follows:

[0052] 1. Preparation of ex vivo mouse skin

[0053] Take healthy mice, anesthetize them with 10% urethane solution (dosage: 8 mL / kg), first shave the coarse hair on the back of the mice, then patiently cut the hair so as not to affect the contact between the drug and the skin, then inject an overdose of urethane solution to kill the mice, cut the back skin, and finally carefully remove the subcutaneous fat and mucous tissue, clean the mouse skin with PBS buffer, spread it flat on aluminum foil, and store it in a -20℃ refrigerator for later use.

[0054] 2. Construction of in vitro percutaneous absorption experimental device

[0055] The Franz diffusion cell used in the experiment is as follows Figure 4 As shown in the figure, the speed of the star-shaped stirrer in the experiment is 500 r / min, the volume of the receiving chamber is 6.5 mL, and the effective diffusion area is 0.785 cm 2 In the experiment, PBS buffer (pH 7.4) was used as the receiving medium and the experiment was carried out in a constant temperature water bath at (32±0.5)℃.

[0056] Before the experiment, first heat the PBS buffer to be used to (32±0.5)℃, and turn on the constant temperature water bath system. Take out the prepared mouse skin, rinse it carefully with PBS buffer, wipe the liquid on the surface of the skin dry and place it on the diffusion cell, with the mouse skin stratum corneum facing up, and discard the receiving solution after balancing for 30 minutes. Add fresh PBS buffer to the receiving chamber again, place the mouse skin on the diffusion cell, drain the bubbles under the skin, and add PBS buffer dissolved with diclofenac sodium to the supply chamber. Take samples of 200μL at the 2nd, 4th, 6th, and 8th hours, and add fresh receiving solution of the same volume and temperature. After filtering the sample through a 0.45μm microporous filter membrane, dilute it to a certain multiple, and determine its concentration using a liquid chromatograph.

[0057] 3. HPLC column preparation

[0058] Chromatographic column: Amethyst C18-H (250mm×4.6mm, 5μm), mobile phase: methanol-0.4% glacial acetic acid solution (80:20), detection wavelength is 254nm, flow rate is 1.0mL / min, column temperature: 30℃, injection volume is 10μL.

[0059] 4. Drug concentration screening

[0060] Take appropriate amount of diclofenac sodium in PBS buffer to prepare 0.1, 0.2, 0.3, 0.4, 0.5, 0.6% (g / mL) diclofenac sodium test solution, and measure its 8h permeability. The results show that when the drug concentration is greater than 0.2%, its 8h permeability stops rising, so the drug concentration of diclofenac sodium solution is specified to be 0.1%.

[0061] 5. In vitro percutaneous penetration test

[0062] This experiment was divided into five groups, namely, transdermal dipeptide group, transdermal peptide TD-1 group, acetyl hexapeptide-1 group, menthol group, and blank control group. The above-mentioned permeation enhancers were dissolved in PBS buffer containing 0.1% diclofenac sodium, wherein the concentration of transdermal dipeptide was 0.08%, and the concentrations of menthol, transdermal peptide TD-1, and acetyl hexapeptide-1 were 0.5%, respectively. First, the PBS buffer to be used was heated to (32±0.5)℃, and the constant temperature water bath system was turned on. Take out the prepared mouse skin, rinse it carefully with PBS buffer, wipe the liquid on the surface of the skin dry and place it on the diffusion cell, with the mouse skin stratum corneum facing up, and discard the receiving solution after balancing for 30 minutes. Add fresh PBS buffer to the receiving chamber again, place the mouse skin on the diffusion cell, drain the bubbles under the skin, and add PBS buffer dissolved with the permeation enhancer to the supply chamber. Take samples of 200μL at 2, 4, 6, and 8h, and supplement with fresh receiving solution of the same volume and temperature at the same time. The sample was filtered through a 0.45 μm microporous filter membrane and then diluted to a certain multiple, and the concentration of diclofenac sodium was determined by liquid chromatography.

[0063] Among them, the cumulative permeation amount of diclofenac sodium per unit area (Q n ) is calculated by formula (1):

[0064]

[0065] In formula (1), Q n is the cumulative permeation per unit area at the nth time point (μg / cm 2 ), C n is the drug concentration measured at the nth time point (μg / mL), V is the volume of the receptor chamber (mL), C iis the drug concentration measured at each sampling (μg / mL), V i is the volume of each sampling (mL), A is the effective diffusion area (cm 2 ).

[0066] Steady state flux J s , Permeability coefficient K p , permeability enhancement ratio ER, and permeability are calculated by formulas (2), (3), (4), and (5), respectively:

[0067]

[0068]

[0069]

[0070] Permeability (%) = Q n / Initial drug loading in the supply chamber × 100% (5)

[0071] In the formula, Q p is the total amount of permeated drug (μg), A is the effective diffusion area (cm 2 ), t is the corresponding diffusion time (h), C d is the initial drug loading concentration in the supply chamber (μg / cm 3 ), K Pi is the permeability coefficient of the sample (cm / h), K P0 is the permeability coefficient of the blank control group (cm / h), Q n is the cumulative permeation per unit area at the nth time point (μg / cm 2 ).

[0072] The cumulative permeation amount of diclofenac sodium per unit area in different treatment groups is as follows Figure 5 As shown by Figure 5 It can be seen that after adding various penetration enhancers for 8 hours, the order of the cumulative permeation amount of diclofenac sodium per unit area is: Q 透皮二肽 >Q 透皮肽TD-1 >Q 乙酰基六肽-1 >Q 薄荷醇 >Q 空白对照组 .

[0073] The main transdermal permeation parameters of drugs in different treatment groups are shown in Table 1. The data in Table 1 show that the transdermal dipeptide has the best transdermal permeation effect and the highest drug permeation rate.

[0074] Table 1. Main percutaneous permeation parameters of drugs in different treatment groups

[0075] Permeation enhancers <![CDATA[J s (μg / h cm 2 )]]> <![CDATA[K p ×10 -2 (cm / h)]]> ER Permeability Transdermal dipeptide 66.92 6.69 4.81 21.1％ Transdermal Peptide TD-1 65.11 6.51 4.68 20.4％ Acetyl Hexapeptide-1 48.93 4.89 3.5 15.3％ Menthol 20.59 2.05 1.47 6.4％ Blank control group 13.90 1.39 1 4.3％

[0076] According to the research results, the transdermal dipeptide Arg-Pro has the best transdermal delivery rate, which is significantly better than the existing permeation enhancers (transdermal peptide TD-1, acetyl hexapeptide-1, menthol). The order of transdermal delivery rate from large to small is: transdermal dipeptide Arg-Pro, transdermal peptide, acetyl hexapeptide-1, menthol.

[0077] Example 3: Biocompatibility test

[0078] Cytotoxicity experiments are conducted in vitro to simulate the growth environment of organisms. They can screen out the cytotoxicity of batches of samples in a short period of time and are an important basis for other cell experiments. This experiment uses human skin fibroblasts as a cell model, and the concentrations of the samples to be tested are set at 60μg / mL, 80μg / mL, 100μg / mL, 120μg / mL, and 140μg / mL, and the CCK-8 method is used for determination. The specific steps are as follows:

[0079] (1) Cell Counting Kit-8 (CCK-8) reagent can be used to quickly and conveniently identify the toxicity of analytical substances. The working principle is as follows: it contains WTS-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt], which has multiple nitro groups (-NO 2 ) and aromatic sulfonic acid group (-SO 3 H) groups, which have strong light absorption, photolysis occurs to produce active substances such as free radicals or ions. These active substances further undergo redox reactions, and under the action of 1-Methoxy PMS, they are easily reduced to yellow formazan substances by dehydrogenases in cells, and the amount of the product is proportional to the cell survival rate. The yellow formazan product is characterized by high water solubility and special absorption at 450nm, and the cytotoxicity of the sample to be tested can be determined by detecting its absorbance.

[0080] Human skin fibroblasts were cultured to an appropriate density, digested with trypsin, and then prepared into a cell suspension, which was inoculated in a 96-well plate. 100 μL of the cell suspension was inoculated in each well, and the number of cells in each well was 6-8×10 3 The cells were incubated at 37°C with 5% CO 2 , incubate for 24h.

[0081] (2) This experiment set up a blank control group and an experimental group. The concentration of the test sample (i.e., transdermal dipeptide) in the experimental group was set to 5 concentration gradients, and at least three replicates were set for each concentration. No reagent was added to the blank control group. After the cells were firmly attached to the wall overnight, the culture medium was discarded and fresh culture medium containing different amounts of the test sample was added. After the operation was completed, the cells were incubated at 37°C and 5% CO. 2 , incubate for 24h.

[0082] (3) Take the 96-well plate out of the incubator and add 10 μL of CCK-8 detection solution to each well. Incubate the plate at 37°C and 5% CO 2 Incubate in an incubator for 3 h.

[0083] (4) After incubation, take out the sample and measure the absorbance at 450 nm.

[0084] (5) Result analysis: Using the measured absorbance value, the cell viability was calculated according to the following formula:

[0085]

[0086] In the formula, A x represents the absorbance of the experimental group at 450 nm, A 0 Represents the absorbance of the blank control group at 450 nm.

[0087] The results are as follows Figure 6 As shown, analysis shows that the cell survival rate of human skin fibroblasts treated with transdermal dipeptide exceeds 85%, and has basically no toxic effect on cells.

[0088] Embodiment 4:

[0089] In addition to the above-mentioned transdermal dipeptide Arg-Pro, two other transdermal dipeptides were designed: Lys-Pro and Asn-Pro.

[0090] The transdermal dipeptides Lys-Pro and Asn-Pro were synthesized according to the method of Example 1, and their transdermal effects were studied according to the experimental method of Example 2.

[0091] The cumulative permeation amount of diclofenac sodium per unit area in different transdermal dipeptide treatment groups is as follows Figure 7 As shown by Figure 7 It can be seen that after adding different transdermal dipeptides for 8 hours, the order of the cumulative permeation of diclofenac sodium per unit area is: Q Arg-Pro >Q Lys-Pro >Q Asn-Pro The results showed that although the transdermal delivery effects of the transdermal dipeptides Lys-Pro and Asn-Pro were slightly lower than that of Arg-Pro, they still had significant penetration-enhancing effects.

[0092] It is speculated that the reason why the above three dipeptides have a permeation-enhancing effect is that arginine (Arg), lysine (Lys), and asparagine (Asn) all contain aminoimino groups, and proline (Pro) contains n-pentyl nitro groups. Therefore, it is possible that when the dipeptide chain has a combination of aminoimino and n-pentyl nitro groups, the dipeptide has a certain permeation-enhancing effect.

[0093] According to the experimental method of Example 3, it was verified that the above two transdermal dipeptides Lys-Pro and Asn-Pro also have basically no toxic effect on cells.

Claims

1. A transdermal dipeptide, characterized in that: Its amino acid sequence is: Arg-Pro, or Lys-Pro, or Asn-Pro, in order from C-terminus to N-terminus.

2. The transdermal dipeptide according to claim 1, characterized in that Its amino acid sequence is: Arg-Pro.

3. Use of the transdermal dipeptide according to claim 1 or 2 in the preparation of transdermal products.

4. The use according to claim 3, characterized in that The transdermal products include cosmetic preparations, hairdressing preparations, medicines, and foods.

5. The use according to claim 4, characterized in that The cosmetic preparations include skin care products and cosmetics.

6. A cosmetic preparation, characterized in that The invention comprises the transdermal dipeptide according to claim 1 or 2, and at least one active ingredient for skin care, skin function improvement or skin color regulation.

7. A hairdressing preparation, characterized in that: The invention comprises the transdermal dipeptide according to claim 1 or 2, and at least one hair-beautifying active ingredient, wherein the hair-beautifying active ingredient comprises an ingredient having the effects of promoting hair growth, preventing hair loss, and changing hair structure, composition or color.

8. A pharmaceutical composition, characterized in that The invention comprises the transdermal dipeptide according to claim 1 or 2, and at least one active pharmaceutical ingredient in an effective dose for treating a disease.

9. A method for assisting the transdermal delivery of an active substance, characterized in that: The transdermal dipeptide according to claim 1 or 2 is mixed with an effective dose of active substance and applied to the skin surface.

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