Palmitoylated IGF-1 polypeptide derivative and application thereof in promoting hair growth

Abstract

The invention provides a palmitoylated IGF-1 polypeptide derivative and application of the palmitoylated IGF-1 polypeptide derivative in promoting hair growth, and belongs to the technical field of hair growth products. The palmitoylated IGF-1 polypeptide derivative provided by the invention comprises an end-capping group and an IGF-1 targeting peptide, and the end-capping group is palmitic acid. According to the IGF-1 polypeptide derivative and the preparation method thereof, the hydrophobicity of the IGF-1 polypeptide derivative is enhanced through palmitoylated end capping, the membrane binding property of the IGF-1 targeting peptide is facilitated, the IGF-1 targeting peptide can be helped to better pass through a biological barrier and a lipid membrane, and the stability and the transdermal absorption rate of the IGF-1 targeting peptide are improved. The palmitoylated IGF-1 polypeptide derivative is used for hair follicle cells, and it is found that the palmitoylated IGF-1 polypeptide derivative can promote hair follicle cell proliferation. In addition, through a skin smearing administration mode, it is found that the palmitoylated IGF-1 polypeptide derivative can achieve hair regeneration of unhaired mice.

Description

Technical Field

[0001] The present invention relates to the technical field of hair growth products, and in particular to a palmitoylated IGF-1 polypeptide derivative and an application thereof in promoting hair growth. Background Art

[0002] Hair loss is a condition in which hair loses excessively due to a variety of factors. It is a chronic disease that places a serious psychological burden on patients. At present, the treatment strategies for hair loss mainly include drug therapy and hair transplantation. However, the efficacy and safety of these treatment options are still controversial. Among them, drug therapy is the first choice for the treatment of hair loss. Clinically approved drugs for the treatment of hair loss include minoxidil and finasteride. However, both drugs are accompanied by certain side effects during the treatment process, and minoxidil requires long-term maintenance. Once the drug is stopped, the hair will fall out again within a few months. Hair transplantation is limited by insufficient donors and low transplant survival rate. Therefore, it is urgent to develop a safe and effective strategy for the treatment of hair loss.

[0003] So far, the direct use of growth factors for hair regeneration has broad application prospects. Among them, IGF-1 and its receptor IGF-1R play multiple roles in hair growth and hair follicle biology, including promoting hair follicle cell proliferation, regulating hair growth cycle, and anti-apoptosis. These functions make IGF-1R a potential target for the treatment of hair loss diseases. However, the recombinant expression level of commercial IGF-1 protein is low, which limits its possibility of large-scale production and leads to high production costs. In addition, IGF-1 has poor stability. In order to maintain its biological activity, the transportation and storage conditions are extremely harsh. In addition, the molecular weight of IGF-1 protein is large, and its transdermal absorption efficiency is limited, making it difficult to exert its effect through skin application. These factors jointly affect the application prospects of IGF-1 protein. Summary of the invention

[0004] IGF-1 protein is a protein composed of 70 amino acids and has a structure similar to insulin. The structural domains of IGF-1 are mainly divided into four regions: B, C, A, and D. These domains play an important role in the binding of IGF-1 to its receptor IGF-1R. Among them, the C region consisting of 12 residues connecting the A region and the B region is crucial for the binding of the IGF-1 polypeptide to the receptor (IGF-1R). Its amino acid sequence is GYGSSSRRAPQT. We selected these 12 peptides as the active peptide segments of IGF-1 (i.e., IGF-1 targeting peptides) and performed palmitoylation modification. The purpose is to improve the skin permeability of the IGF-1 polypeptide derivative (GYGSSSRRAPQT) with hair growth promoting effect, which can be directly applied through the skin for administration, with small side effects, high stability, and good hair growth promoting effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a palmitoylated IGF-1 polypeptide derivative, comprising a capping group and an IGF-1 targeting peptide, wherein the capping group is palmitic acid.

[0007] Preferably, the amino acid sequence of the IGF-1 targeting peptide is as shown in SEQ ID NO:1.

[0008] Preferably, the structural formula of the palmitoylated IGF-1 polypeptide derivative is as shown in Formula I:

[0009]

[0010] The present invention also provides a method for preparing the palmitoylated IGF-1 polypeptide derivative, which uses an Fmoc-solid phase synthesis method to synthesize the IGF-1 targeting peptide, and finally couples the end-capping group palmitic acid.

[0011] The present invention also provides the palmitoylated IGF-1 polypeptide derivative, or the use of the palmitoylated IGF-1 polypeptide derivative synthesized by the preparation method in the preparation of products promoting hair growth.

[0012] The present invention also provides a hair growth promoting product containing the palmitoylated IGF-1 polypeptide derivative.

[0013] Preferably, the product uses water as solvent and contains 1 to 1000 μM of palmitoylated IGF-1 polypeptide derivative.

[0014] Preferably, the product is a spread.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The palmitoylated IGF-1 polypeptide derivative provided by the present invention has a small molecular weight and high permeability. The modification of palmitic acid enhances the hydrophobicity of the IGF-1 targeting peptide, helps the membrane binding of the IGF-1 targeting peptide, and can help the IGF-1 targeting peptide to better pass through biological barriers and lipid membranes, thereby improving the stability and transdermal absorption rate of the IGF-1 targeting peptide.

[0017] (2) Palmitoylated IGF-1 polypeptide derivatives were applied to hair follicle cells and found to promote hair follicle cell proliferation.

[0018] (3) The palmitoylated IGF-1 polypeptide derivative provided by the present invention can be prepared into an aqueous solution and applied to the skin for administration, which has a good effect of promoting hair growth. Through the method of administration by skin application, it was found that the palmitoylated IGF-1 polypeptide derivative can achieve hair regeneration in hair-depleted mice.

[0019] (4) The palmitoylated IGF-1 polypeptide derivative solution provided by the present invention has good stability and can be stored at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HPLC of PA-IGF synthesized in Example 1;

[0021] Figure 2 is the mass spectrum of PA-IGF synthesized in Example 1;

[0022] Figure 3 This is a diagram showing the effect of PA-IGF on promoting cell proliferation in Experimental Example 3;

[0023] Figure 4 This is the hair growth monitoring chart of the mouse hair loss model in Experimental Example 4. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1

[0026] The palmitoylated IGF-1 peptide derivative (PA-GYGSSSRRAPQT, referred to as PA-IGF) was synthesized by the classical Fmoc-solid phase synthesis method.

[0027] The structural formula is shown in Formula I:

[0028]

[0029] The specific steps are as follows:

[0030] 2-Chlorotrityl resin was selected as a synthetic carrier, and Fmoc-amino acid (GYGSSSRRAPQT; SEQ ID NO: 1) and palmitic acid were selected as raw materials.

[0031] 1) Weigh 0.5 mmol of dichlororesin into a peptide synthesis tube and swell it with dichloromethane (DCM) at room temperature for 10 min, then blow out the DCM with an ear bulb.

[0032] 2) 0.75 mmol of the first amino acid threonine, 1.5 mmol of N,N-diisopropylethylamine (DIEA) and 10 mL of DCM reagent were mixed until completely dissolved and then added to the reaction system in step 1) and reacted at room temperature (25° C.) for 1 h.

[0033] 3) Blow out the reaction solution with an ear bulb, add 10 mL of blocking solution prepared in the ratio (DIEA:MeOH:DCM=1:2:17, volume ratio) for blocking, and react at room temperature (25°C) for 15 min.

[0034] 4) The reaction solution was blown out with an ear bulb, rinsed with N,N-dimethylformamide (DMF) five times, and then 20% piperidine solution (piperidine:DMF=1:4, volume ratio) was added to remove the Fmoc protecting group and reacted at room temperature for 30 min.

[0035] 5) Use an ear bulb to blow out the reaction solution into water. If a large amount of white precipitate is produced, it means that the amino acid is successfully deprotected. Rinse with DMF 5 times to ensure that the piperidine is completely removed.

[0036] 6) 1 mmol of the next amino acid, 1 mmol of condensing agent HATU, 2 mmol of DIEA and 10 mL of DMF were mixed until completely dissolved, then added to the reaction system and reacted at room temperature for 1 h.

[0037] 7) Repeat steps 4) to 6) until the last amino acid of the target polypeptide sequence is synthesized, and finally add 1 mmol of the end-capping group palmitic acid. (If the synthesis is performed overnight, the condensation agent HBTU needs to be added).

[0038] 8) Blow out the reaction solution with an ear bulb, rinse with DCM 5 times, and add trifluoroacetic acid: triisopropylsilane: ddH 2 The polypeptide was cleaved from the dichlororesin and the side chain protecting groups of the amino acids were removed, and the reaction was carried out at room temperature for 1 h.

[0039] 9) Use a round-bottom flask to collect the reaction liquid blown down by the ear bulb and the liquid after washing with DCM three times, and use a water pump to evaporate the liquid in the flask until it becomes viscous. Replace the oil pump and continue to evaporate until the substance is completely precipitated.

[0040] 10) Add 20 mL of anhydrous ether to the round-bottom flask, transfer the mixture for suction filtration, and finally obtain a dry crude product, which is then separated and purified by preparative liquid chromatography and freeze-dried by a freeze dryer to obtain a palmitoylated IGF-1 polypeptide derivative PA-IGF. The structural formula of PA-IGF is shown in Formula I after identification by mass spectrometry.

[0041] Experimental Example 1

[0042] HPLC detection of the purity of the peptide PA-IGF

[0043] In this experiment, water containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid were used as mobile phases, and the purity of the peptide PA-IGF was detected using a C18 chromatographic column at a detection wavelength of 214 nm. Figure 1 As shown, there is only one single peak in HPLC, and the purity is greater than 95%, which meets the use requirements.

[0044] Experimental Example 2

[0045] Mass spectrometry detection

[0046] Figure 2 This is the mass spectrum of the polypeptide PA-IGF. The molecular weight of PA-IGF was characterized by mass spectrometry, and [M+2H] 2+ =753.35, indicating that the peptide was successfully synthesized.

[0047] Experimental Example 3

[0048] CCK-8 assay to detect cell proliferation

[0049] Mouse dermal papilla cells were cultured at 2×10 3 The cells were seeded into 96-well culture plates at a density of 1.5 × 10 cells / well and placed in a 37°C, 5% CO 2 After the cells adhered to the wall, the culture medium was removed and DMEM culture medium containing different concentrations (0, 1, 5, 25, 50, 100, 500 nM) of polypeptide PA-IGF was added at 100 μL / well and culture was continued for 48 hours. After that, the supernatant was removed and DMEM culture medium containing 10% CCK-8 was added and culture was continued for 2 hours. Finally, the absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader. The results are shown in Figure 3 .from Figure 3 It can be seen that PA-IGF polypeptide can promote the proliferation of mouse hair papilla cells in the concentration range of 5 to 100 nM (the dosage converted to human body can be selected in the range of 1 to 1000 μM).

[0050] Experimental Example 4

[0051] Hair loss treatment in mice

[0052] The mice used in this experiment were male C57BL / 6. The hair on the back of the mice was removed with a shaver and depilatory cream, and the mice were randomly divided into 3 groups, with 3 mice in each group, namely the Control group, the IGF peptide group without palmitoyl modification, and the PA-IGF peptide group. A 10μM PA-IGF aqueous solution was applied to the back of the mice in the PA-IGF group, a 10μM IGF aqueous solution was applied to the back of the mice in the IGF group, and a solvent water was applied to the back of the mice in the Control group, once a day. Photos were taken and recorded on the 1st, 10th, 13th, and 16th days of administration. The results are shown in Figure 4 As shown. Figure 4It can be seen that the skin of mice in the PA-IGF group began to turn gray from the 10th day, began to turn black and grow new hair on the 13th day, and had grown a large amount of new hair on the 16th day, while the skin of mice in the Control group and the IGF group did not begin to show local black color until the 16th day, indicating that the polypeptide PA-IGF significantly promoted the growth of mouse hair.

[0053] It can be seen from the above examples that the present invention selects palmitic acid as the capping group, which can enhance the hydrophobicity of the IGF-1 targeting peptide, help enhance its interaction with the biological membrane, and improve the stability and transdermal absorption rate of the IGF-1 targeting peptide.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A palmitoylated IGF-1 polypeptide derivative, characterized in that: The invention comprises a capping group and an IGF-1 targeting peptide, wherein the capping group is palmitic acid.

2. The palmitoylated IGF-1 polypeptide derivative according to claim 1, characterized in that: The amino acid sequence of the IGF-1 targeting peptide is shown in SEQ ID NO:

1.

3. The palmitoylated IGF-1 polypeptide derivative according to claim 2, characterized in that: The structural formula of the palmitoylated IGF-1 polypeptide derivative is shown in Formula I:

4. A method for preparing the palmitoylated IGF-1 polypeptide derivative according to any one of claims 1 to 3, characterized in that: The IGF-1 targeting peptide was synthesized by Fmoc-solid phase synthesis method, and finally coupled with the capping group palmitic acid.

5. Use of the palmitoylated IGF-1 polypeptide derivative according to any one of claims 1 to 3, or the palmitoylated IGF-1 polypeptide derivative synthesized by the preparation method according to claim 4, in the preparation of a product promoting hair growth.

6. A product for promoting hair growth containing the palmitoylated IGF-1 polypeptide derivative according to any one of claims 1 to 3, or the palmitoylated IGF-1 polypeptide derivative synthesized by the preparation method according to claim 4.

7. The hair growth promoting product according to claim 6, characterized in that: The product uses water as solvent and contains 1 to 1000 μM of palmitoylated IGF-1 polypeptide derivative.

8. The hair growth promoting product according to claim 7, characterized in that: The product described is a spread.

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