Polypeptide capable of selectively inhibiting COX-2 and having IGF-1 function and hair growth application

Abstract

The invention provides a polypeptide capable of selectively inhibiting COX-2 and having an IGF-1 function and application of the polypeptide, and belongs to the technical field of hair growth. The invention provides a polypeptide capable of selectively inhibiting COX-2 and having an IGF-1 function. The polypeptide comprises an end-capping group, a self-assembly group and an insulin-like growth factor C structural domain polypeptide IGF-1C which are connected in sequence, the end-capping group is naproxen; the self-assembly group is gffy; the amino acid sequence of the insulin-like growth factor C structural domain polypeptide IGF-1C is GYGSSSRRAPQT (insulin-like growth factor C structural domain polypeptide). The polypeptide capable of selectively inhibiting COX-2 and having the IGF-1 function, provided by the invention, has double functions of promoting hair follicle cell proliferation and reducing the expression level of inflammatory cells COX-2 so as to regulate an immune microenvironment, and can effectively promote hair regeneration.

Description

Technical Field

[0001] The present invention relates to the technical field of hair growth, and in particular to a polypeptide that selectively inhibits COX-2 and has IGF-1 function and its application in promoting hair growth. Background Art

[0002] Although hair loss is not a life-threatening disease, it has a significant impact on the patient's mental health and quality of life. Hair loss is usually divided into two categories: scarring alopecia and non-scarring alopecia. Non-scarring alopecia is more common in clinical practice. Non-scarring alopecia includes androgenic alopecia, alopecia areata, telogen effluvium, trichotillomania, etc. The most common types of hair loss are alopecia areata (AA) and androgenic alopecia (AGA). Alopecia areata is an autoimmune disease in which the immune system attacks hair follicles, causing hair loss. The key pathogenic factor of AGA is androgen. Testosterone is converted into DHT under the action of type II 5α-reductase. DHT binds to the androgen receptor (AR) overexpressed in the hair follicles, resulting in hair follicle atrophy, hair loss and even baldness. Clinically, the preferred treatment for alopecia areata and AGA is drug therapy. However, the drugs currently approved for the treatment of alopecia areata are rituximab tosylate and baricitinib; the drugs for the treatment of AGA are minoxidil and finasteride. The therapeutic effects of these drugs vary from person to person, and are accompanied by certain side effects, such as skin irritation, allergic reactions, or sexual dysfunction. Therefore, the development of new effective and safe therapeutic drugs is of great significance.

[0003] Insulin-like growth factor 1 (IGF-1) plays a key role in regulating hair follicle stem cell function and hair growth cycle. IGF-1 activates downstream signaling pathways by binding to its receptor IGF-1R, thereby promoting the proliferation of hair follicle cells. In addition, IGF-1 has been identified as an important mitogenic and morphogenic regulator in hair follicle biology, affecting hair follicle proliferation, tissue remodeling, hair growth cycle, and hair follicle differentiation. In AGA, IGF-1 is regulated by androgens. Studies have shown that the IGF-1 secreted by the dermal papilla cells of bald hair follicles is much less than that of non-bald hair follicles. Therefore, IGF-1 has broad application prospects in the treatment of hair loss diseases. However, the commercial application of IGF-1 is subject to multiple limitations. First, the recombinant expression of IGF-1 protein faces the challenge of low expression levels in a variety of expression systems, which is difficult to meet clinical and scientific research needs. Second, the production cost of IGF-1 is high, which is related to its complex production process and purification process. In addition, IGF-1 has poor stability and is easily affected by environmental factors such as temperature and pH value. In order to maintain the biological activity of the protein, the transportation and storage conditions are relatively harsh. These factors together limit the application of IGF-1.

[0004] In the pathological process of AGA and AA, there is an inflammatory response in the local microenvironment of the hair follicles, especially in AA, where there is a large infiltration of inflammatory cells around the hair follicles. Cyclooxygenase-2 (COX-2) is an inducible enzyme that is low in expression in normal tissues but highly expressed under inflammatory stimulation. The prostaglandin E2 (PGE2) catalyzed by it is a key mediator in the inflammatory response and has the function of promoting the chemotaxis and aggregation of inflammatory cells. COX-2 is an important target for anti-inflammatory treatment. Currently, COX-2 selective inhibitors such as naproxen have been used for anti-inflammatory treatment. Therefore, the purpose of inhibiting the chemotaxis and aggregation of inflammatory cells and improving the immune microenvironment of hair follicles can be achieved by inhibiting or degrading COX-2.

[0005] In summary, new therapeutic materials that can simultaneously promote hair follicle cell proliferation and inhibit inflammatory responses in the hair follicle microenvironment show greater therapeutic potential in the clinical treatment of hair loss diseases. A material that can inhibit or degrade COX-2 protein to inhibit inflammatory responses and mimic IGF-1 protein to promote hair follicle cell proliferation may provide a more effective strategy for hair loss treatment, especially in regulating the hair follicle immune microenvironment and promoting hair regeneration. Summary of the invention

[0006] The purpose of the present invention is to provide a hair growth product for AGA and AA types of hair loss, which has both the effects of selectively inhibiting COX-2 and promoting hair growth.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a polypeptide that selectively inhibits COX-2 and has the function of IGF-1, wherein the self-assembling polypeptide comprises a capping group, a self-assembling group and an insulin-like growth factor C domain polypeptide IGF-1C that are connected in sequence;

[0009] The blocking group is naproxen; the self-assembling group is gffy (SEQ ID NO: 1); and the amino acid sequence of the insulin-like growth factor C domain polypeptide IGF-1C is GYGSSSRRAPQT (SEQ ID NO: 2).

[0010] Preferably, the self-assembling group gffy is an L-type or D-type amino acid.

[0011] Preferably, the structural formula of the polypeptide is as shown in Formula I:

[0012]

[0013] The present invention also provides a hair growth solution prepared by the polypeptide that selectively inhibits COX-2 and has IGF-1 function. The hair growth solution uses water as a solvent and contains 0.1 to 100 μM of the self-assembling polypeptide.

[0014] Preferably, the hair growth solution is administered by skin application.

[0015] The present invention also provides the use of the self-assembling polypeptide or the hair growth solution in preparing a product promoting hair growth.

[0016] Preferably, the hair growth product targets androgenic alopecia and alopecia areata.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The polypeptides provided by the present invention that selectively inhibit COX-2 and have IGF-1 functions can be prepared into aqueous solutions and used by skin application.

[0019] (2) The polypeptides provided by the present invention that selectively inhibit COX-2 and have IGF-1 function have the dual functions of anti-inflammation and promoting hair follicle cell proliferation, and effectively promote hair regeneration.

[0020] (3) The polypeptides provided by the present invention that selectively inhibit COX-2 and have IGF-1 functions have small molecular weight and high transdermal absorption efficiency.

[0021] (4) The hair growth solution provided by the present invention that selectively inhibits COX-2 and has the function of IGF-1 has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the high performance liquid chromatogram of the polypeptide Npx-gffy-IGF-1C detected in Experimental Example 1. The ordinates of the upper and lower figures are 0.00, 0.25, 0.50, 0.75, 1.00 and 1.25 mv (x1000) from bottom to top, and the abscissas are 0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 min from left to right; the upper figure is Npx-gffy-IGF-1C CH1 (220 nm), and the lower figure is Npx-gffy-IGF-1C.lcd CH2 (254 nm);

[0023] Figure 2 It is the mass spectrum of the polypeptide Npx-gffy-IGF-1C detected in Experimental Example 3; the ordinates in the figure are 0.00, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50Inten(x1,000,000) from bottom to top, and the abscissas are 600.0, 625.0, 650.0, 675.0, 700.0, 725.0, 750.0, 775.0, 800.0, 825.0, 850.0, 875.0, 900.0, 925.0, 950.0, 975.0, 1000.0, 1025.0, 1050 .0, 1075.0, 1100.0, 1125.0, 1150.0, 1175.0, 1200.0, 1225.0, 1250.0, 1275.0, 1300.0 m / z. The ion peaks from left to right in the figure are 610.3000, 633.4500, 664.6000, 693.6500, 788.2500, 861.6000, 914.5000, 952.5500, 995.8000, 1039.1500, 1079.2500, 1113.5500, 1271.9500, 1307.4000;

[0024] Figure 3 The dose-response diagram of the polypeptide Npx-gffy-IGF-1C promoting cell proliferation in Experimental Example 3;

[0025] Figure 4 It is the protein immunoblot and quantitative graph of the polypeptide Npx-gffy in Experimental Example 4;

[0026] Figure 5 This is a graph of hair growth monitoring in mice. DETAILED DESCRIPTION

[0027] 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.

[0028] The sources of the preparations involved in the embodiments of the present invention are as follows:

[0029] 2-Chlorotrityl resin (substitution rate 1.158 mmol / g), Fmoc-amino acid, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (hereinafter referred to as HATU) were purchased from Jier Biochemical (Shanghai) Co., Ltd.;

[0030] N,N-diisopropylethylamine (DIPEA) and triisopropylsilane (TIS) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0031] Trifluoroacetic acid (TFA) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0032] Anhydrous dichloromethane (DCM), N,N-dimethylformamide (DMF), and piperidine were purchased from Tianjin Chemical Reagent Company;

[0033] Cell proliferation-toxicity detection kit Cell Counting Kit-8 (CCK8) was purchased from Langbolide;

[0034] Cell culture medium (Dulbecco's Modified Eagle Medium, DMEM) and fetal bovine serum were purchased from Thermo Fisher Scientific;

[0035] C57BL / 6 mice, male, were purchased from Weitonglihua Biotechnology Co., Ltd.

[0036] The devices involved in the embodiments of the present invention are:

[0037] Electronic balance (arturious, Germany, BS124S);

[0038] Freeze dryer (Beijing Yatai Kelong, LGJ-1-50).

[0039] Example 1

[0040] Synthesis of peptide Npx-gffy-GYGSSSRRAPQT

[0041] This embodiment provides a polypeptide that selectively inhibits COX-2 and has IGF-1 function, whose structural formula is shown in Formula I. It is synthesized using the classic Fmoc-solid phase synthesis method. The obtained polypeptide is referred to as Npx-gffy-IGF-1C. The synthesis of the polypeptide is coupled sequentially from the C-terminus to the N-terminus.

[0042]

[0043] The specific steps are as follows:

[0044] (1) Swelling: Weigh 0.5 mmol of dichlororesin (1.158 mmol / g) as a solid carrier and place it in a solid phase tube for peptide synthesis. Add dichloromethane (DCM) and place it on a shaker for reaction and swelling for 10 min. Then squeeze out the solution in the solid phase tube.

[0045] (2) Adding the first amino acid: Weigh 1 mmol of threonine with an Fmoc protecting group and 1.5 mmol of N,N-diisopropylethylamine (DIEA) and dissolve them in 10 ml of DCM. After ultrasonic dissolution, add them to the solid phase tube and place it on a shaker for reaction for 1 hour.

[0046] (3) Blocking: squeeze out the solution in the solid phase tube, then add the prepared blocking solution (1 ml DIEA, 17 ml DCM, 2 ml MeOH) and place on a shaker to react for 15 min to protect the active sites on the dichlororesin that are not connected to amino acids to avoid connection errors.

[0047] (4) Solvent replacement: After sealing, squeeze out the solution in the solid phase tube, wash it once with DCM, and then wash it three times with N, N-dimethylformamide (DMF), each time for 1 min. The last time the solution was squeezed out, no precipitation was found in the water test, indicating that it was clean.

[0048] (5) Deprotection: Add 15 mL of 20% piperidine solution (piperidine:DMF=1:4) to cleave the Fmoc protecting group of the first amino acid. React for 0.5 h. Extrude the solution and check for precipitation, indicating successful cleavage. Add appropriate amount of DMF for 4 washes to remove excess piperidine and Fmoc protecting group.

[0049] (6) Adding amino acids: Weigh 1 mmol of the next amino acid and 1 mmol of the peptide condensation reagent (HATU), fully dissolve them in 10 ml of DMF, add 0.5 ml of DIEA, mix well, and add them to the solid phase tube. Place it on a shaker to react for 1 h, then wash it with DMF 4 times and squeeze out the solution in the solid phase tube.

[0050] (7) Repeat steps (5) to (6) until the target peptide is synthesized with the last capping group naproxen. Weigh 1 mmol naproxen and 1 mmol peptide condensation reagent (HBTU), fully dissolve them in 10 ml DMF, add 0.5 ml DIEA, mix well, add to the solid phase tube, and place on a shaker to react overnight.

[0051] (8) Cutting: squeeze out the solution in the solid phase tube and wash it with DCM for 5 times. After the last wash, squeeze out the solution and check if there is any precipitation. Add the prepared cutting solution (9.5 ml trifluoroacetic acid, 0.25 ml triisopropylsilane, 0.25 ml ddH2O) and react for 60 min.

[0052] (9) Collection: Collect the completely reacted solution in the solid phase tube into a round-bottom flask, and wash the solid phase tube three times with DCM, returning the solution to the round-bottom flask each time.

[0053] (10) Crude extraction: The collected solution was evaporated by rotary evaporation to remove the organic solvents DCM and TFA, and then anhydrous ether was added to precipitate a solid crude product.

[0054] (11) Recovery: Filter with a vacuum pump, drain the crude product, weigh it, and place it at -20°C for later use.

[0055] (12) Purification: Separate the crude product by high performance liquid chromatography (HPLC) to purify the target product.

[0056] (13) Inspection: Collect the solution corresponding to the HPLC absorption peak and detect it by liquid chromatography-mass spectrometry (LC-MS) to determine the molecular weight of the target product.

[0057] (14) Freeze drying: The target product solution was collected and the organic phase was removed by rotary evaporation. The remaining solution was sealed with a sealing film with a few pores and placed in a -80°C refrigerator to freeze into a solid. Finally, it was placed in a vacuum freeze dryer for freeze drying to obtain the polypeptide Npx-gffy-IGF-1C of Example 1 that selectively inhibits COX-2 and has the function of IGF-1.

[0058] Example 2

[0059] Synthesis of peptide Npx-gffy

[0060] This example provides a self-assembling polypeptide that selectively inhibits COX-2, the structural formula of which is shown in Formula II, and which is prepared according to the Fmoc-solid phase synthesis method of Example 1.

[0061]

[0062] Comparative Example 1

[0063] Synthesis of peptide Npx-ffg-IGF-1C

[0064] This comparative example provides a polypeptide material with a different polypeptide self-assembly group from that in Example 1 but the same remaining sequence. Its structural formula is shown in Formula III and it is prepared according to the Fmoc-solid phase synthesis method of Example 1.

[0065]

[0066] Experimental Example 1

[0067] HPLC detection of peptide purity

[0068] In this experiment, water containing 0.1% trifluoroacetic acid and acetonitrile containing 0.1% trifluoroacetic acid were used as mobile phase A and mobile phase B, respectively. The purity of Npx-gffy-IGF-1C polypeptide was detected using a C18 reverse phase column at wavelengths of 220 nm and 254 nm. Figure 1 As shown, HPLC had only one single peak at both wavelengths, and the purity was greater than 95%.

[0069] Experimental Example 2

[0070] Mass spectrometry detection

[0071] Figure 2 This is the mass spectrum of the peptide Npx-gffy-IGF-1C. The molecular weight of Npx-gffy-IGF-1C was characterized by mass spectrometry, and [M+2H] 2+ =995.8 doublet peak of the target molecule, indicating successful peptide synthesis.

[0072] Experimental Example 3

[0073] Cell Counting Kit-8 (CCK-8) was used to detect the proliferation-promoting effect of peptides at the cell level

[0074] Mouse primary dermal papilla cells were cultured at 2 × 10 3 The density of cells was set at 100 μL and inoculated into a 96-well culture plate. The inoculated culture plate was placed in a 37°C incubator containing 5% CO2 and incubated for 24 hours. After the incubation, the culture medium was removed, and 100 μL of DMEM culture medium containing different concentrations of the polypeptide Npx-gffy-IGF-1C was added to each well and incubated for another 48 hours. After the incubation was completed again, the supernatant was removed. 100 μL of DMEM culture medium containing 10% CCK-8 solution was added to each well and incubated for 1 hour. The absorbance of each well was measured at a wavelength of 450 nanometers using an enzyme reader. The obtained data was plotted as shown in the figure. Figure 3 As shown, the polypeptide Npx-gffy-IGF-1C has a good effect of promoting the proliferation of dermal papilla cells at a concentration of 25-100 nM.

[0075] Experimental Example 4

[0076] Western blotting to determine the effect of peptide Npx-gffy on COX-2 degradation

[0077] This experiment was divided into three groups: LPS group, Npx group and Npx-gffy group. 5 RAW264.7 cells were inoculated into six-well plates at a density of 1 μg / mL / well. After the cells were completely attached to the wall, the culture medium was discarded. 2 mL of pure DMEM culture medium was added to the LPS group, and 2 mL of naproxen and Npx-gffy peptide were added to the Npx group and Npx-gffy group for 16 h. Then, 20 μL of LPS (final concentration of 1 μg / mL) with a concentration of 100 μg / mL was added to each well to induce macrophage inflammation for 8 h. The culture medium was discarded, and the cells were washed 3 times with PBS. 100 μL of RIPA lysis buffer containing 1× protease inhibitor mixture and 1× phosphatase inhibitor was added to each well. The cells were lysed on ice for 10 min, and the cell lysate was collected in a 1.5 mL EP tube and centrifuged at 14000 rpm for 15 min at 4°C. The supernatant was taken and protein quantification was performed using a BCA kit. The remaining samples were added with 4× loading buffer at a volume ratio of 3:1 between the cell lysate and 4× loading buffer, and then heated at 95°C for 8 min to obtain protein samples. SDS-PAGE polyacrylamide gel electrophoresis was performed using Biyuntian precast gel, with a protein loading amount of 20μg, an electrophoresis voltage of 160V, and a time of 35min. Stop the electrophoresis, cut the gel, transfer the membrane, and transfer the protein from the gel to the PVDF membrane. Wash the PVDF membrane twice with 1×TBST, add the rapid blocking solution and incubate at room temperature for 15min, and wash it 3 times with 1×TBST, 10min each time. Cut the membrane according to the instructions of the marker, add the primary antibody and incubate overnight at 4℃, wash it 3 times with 1×TBST, 10min each time, add the secondary antibody and incubate it at room temperature for 2h, wash it 3 times with 1×TBST, 10min each time, and expose and photograph it after washing. The results are as follows Figure 4 shown.

[0078] from Figure 4 It can be seen that compared with the LPS group and the Npx group, the peptide Npx-gffy can significantly reduce the level of COX-2 protein induced by LPS but has no effect on the protein expression level of COX-1, indicating that the Npx-gffy peptide can selectively reduce the expression of COX-2 protein in LPS-induced macrophages, thereby inhibiting the inflammatory response of macrophages, suggesting that the Npx-gffy-IGF-1C peptide with the Npx-gffy structure can also have anti-inflammatory function by reducing the expression level of COX-2 in inflammatory cells.

[0079] Experimental Example 5

[0080] Physiological hair loss model in mice

[0081] 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 with pink back skin were randomly divided into 3 groups, 3 mice in each group, recorded as Control group, Npx-ffg-IGF-1C group and Npx-gffy-IGF-1C group. Among them, the backs of the mice in the Control group were smeared with water, and the backs of the mice in the Npx-ffg-IGF-1C group and the Npx-gffy-IGF-1C group were smeared with 2μM Npx-ffg-IGF-1C and Npx-gffy-IGF-1C polypeptide aqueous solutions on their backs once a day. Photographs were taken on the 1st, 7th and 10th days of administration to record the growth of the hair on the back of the mice. The results are shown in Figure 5 On the 10th day, only one mouse in the Control group had its back completely blackened, two mice in the Npx-ffg-IGF-1C group had their backs completely gray-blackened, and the backs of all three mice in the Npx-gffy-IGF-1C group had their backs completely blackened, indicating that Npx-gffy-IGF-1C has a good hair growth promoting effect, and the hair growth effect is better than that of the Npx-ffg-IGF-1C polypeptide, proving that compared with the assembled fragment ffg, the use of the assembled fragment gffy coupled with the IGF-1C polypeptide can further improve the polypeptide's hair growth promoting effect.

[0082] 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 polypeptide that selectively inhibits COX-2 and has IGF-1 function, characterized in that: The self-assembling polypeptide comprises a capping group, a self-assembling group and an insulin-like growth factor C domain polypeptide IGF-1C connected in sequence; The blocking group is naproxen; the self-assembling group is gffy; and the amino acid sequence of the insulin-like growth factor C domain polypeptide IGF-1C is GYGSSSRRAPQT.

2. A polypeptide that selectively inhibits COX-2 and has IGF-1 function according to claim 1, characterized in that: The self-assembly group gffy is L-type or D-type amino acid.

3. A polypeptide that selectively inhibits COX-2 and has IGF-1 function according to claim 2, characterized in that: The structural formula of the polypeptide is shown in Formula I:

4. A hair growth solution prepared from the polypeptide that selectively inhibits COX-2 and has IGF-1 function according to any one of claims 1 to 3, characterized in that: The hair growth solution uses water as solvent and contains 0.1-100 μM of the self-assembling polypeptide.

5. The hair growth solution according to claim 4, characterized in that The administration mode of the hair growth solution is skin application.

6. Use of the self-assembling polypeptide according to any one of claims 1 to 3 or the hair growth solution according to claim 4 or 5 in the preparation of a product promoting hair growth.

7. The use according to claim 6, characterized in that The hair growth product targets androgenic alopecia and alopecia areata.

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