Spray for preventing and treating alopecia as well as preparation method and application thereof

Abstract

The invention discloses a spray for preventing and treating alopecia as well as a preparation method and application thereof, and the spray comprises the following components in parts by weight: 0.5-3 parts of polypeptide HHH001, 500-700 parts of absolute ethyl alcohol, 900-1100 parts of propylene glycol, 2-5 parts of a pH regulator and 350-450 parts of water, the amino acid sequence of the polypeptide HHH001 is as shown in SEQ ID NO. 1. By optimizing the formula, the problem of poor stability of the polypeptide is solved, and the polypeptide can keep activity in the spray and is effectively absorbed by scalp, so that the treatment effect is improved. The spray can directly act on scalp, avoids side effects of systemic administration, and is convenient to use and easy for long-term self-management of patients.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a spray for preventing and treating hair loss, and a preparation method and application thereof. Background Art

[0002] Currently, the drugs used to treat hair loss on the market are mainly small molecule drugs, of which minoxidil and finasteride are the two most representative ones. Minoxidil promotes hair growth by dilating the blood vessels of the hair follicles, while finasteride inhibits 5α-reductase, reduces the level of dihydrotestosterone (DHT), and delays hair loss. However, these small molecule drugs may be accompanied by side effects during long-term use. Therefore, finding safe and effective new treatment options is of great clinical significance.

[0003] In recent years, peptide drugs have received extensive attention due to their high efficiency and targeting. Regulating GPCR receptors through peptides to achieve therapeutic effects has become a new research hotspot. Peptide drugs have the advantages of strong specificity and low side effects, especially in physiological regulation involving complex signaling pathways. However, the traditional route of peptide administration is mainly through injection, such as intravenous injection, subcutaneous injection or intramuscular injection, and local use is relatively limited. In addition, due to their complex structure, poor stability, and easy enzymatic hydrolysis in the body, peptides have low bioavailability. Therefore, it is urgent to develop a peptide preparation for local medication that can be stored stably for a long time to ensure the effectiveness of long-term medication. Summary of the invention

[0004] In view of the defects in the prior art, the present invention proposes a spray for preventing and treating hair loss, and a preparation method and application thereof. The main active ingredient in the spray of the present invention is a novel polypeptide HHH001, whose amino acid sequence is shown in SEQ ID No.1. The polypeptide can target a specific GPCR receptor and play a role in preventing and treating hair loss. In order to make the polypeptide more suitable for the application scenario of local treatment of hair loss, the present invention innovatively develops a polypeptide HHH001 spray. Compared with the injection form, the spray can act directly on the scalp, avoiding the side effects of systemic administration, and is easy to use and easy for patients to manage themselves for a long time. The problem of poor stability of the polypeptide is solved by optimizing the formula, so that the polypeptide can remain active in the spray and be effectively absorbed by the scalp, thereby improving the therapeutic effect.

[0005] The present invention provides a spray for preventing and / or treating hair loss, which comprises the following components in parts by weight: 0.5 to 3 parts of polypeptide HHH001, 500 to 700 parts of anhydrous ethanol, 900 to 1100 parts of propylene glycol, 2 to 5 parts of a pH regulator and 350 to 450 parts of water;

[0006] The mixed use of anhydrous ethanol and propylene glycol is beneficial to improve the solubility of peptide HHH001. The combination of the two can provide a more balanced dissolution environment for the peptide, reduce the risk of peptide precipitation or aggregation, and thus improve the stability of the solution. In addition, peptides are sensitive to pH, and the combination of anhydrous ethanol and propylene glycol can better cooperate with pH regulators to maintain the pH of the solution within a stable range, thereby improving the chemical stability of the peptide.

[0007] The amino acid sequence of the polypeptide HHH001 is shown in SEQ ID NO.1.

[0008] In some embodiments, the pH of the spray is 7.5-8.0.

[0009] In some embodiments, the mass ratio of the polypeptide HHH001 to anhydrous ethanol is 1:(550-650), preferably 1:600.

[0010] In some embodiments, the mass ratio of the polypeptide HHH001 to propylene glycol is 1:(950-1100), preferably 1:1000.

[0011] In some embodiments, the pH adjuster is any one of phosphate buffer, Tris-hydrochloric acid buffer, acetate, sodium citrate, citric acid and tartaric acid; and the phosphate buffer is sodium phosphate buffer or potassium phosphate buffer.

[0012] The present invention also provides a method for preparing the spray, comprising the following steps:

[0013] Anhydrous ethanol, propylene glycol and water are weighed in parts by weight, mixed evenly, added with polypeptide HHH001 and stirred, added with a pH adjuster and continued to stir to obtain a mixed solution, which is then packaged and filled to obtain the spray of the present application.

[0014] In some embodiments, the stirring time after adding polypeptide HHH001 is 1.5 to 3.0 hours.

[0015] The present invention also provides the use of the spray in preparing medical products for preventing and / or treating hair loss.

[0016] The present invention also provides the use of the spray in preparing medical products or cosmetics for promoting hair growth.

[0017] In some embodiments, the hair includes head hair, eyebrows and eyelashes, preferably head hair.

[0018] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0019] 1. The present invention solves the problem of poor stability of polypeptides through optimized formula, so that the polypeptides can remain active in the spray and be effectively absorbed by the scalp, thereby improving the therapeutic effect. The spray can act directly on the scalp, avoiding the side effects of systemic administration, and is easy to use and easy for patients to manage themselves for a long time.

[0020] 2. The spray of the present invention not only performs well in preventing hair loss, but also has the advantages of being safe and non-toxic, suitable for long-term use, and can provide patients with an efficient and convenient hair loss treatment plan. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The changes in the appearance of the peptide solution after adding different volumes of anhydrous ethanol;

[0023] Figure 2 The changes in the appearance of the peptide solution after adding different volumes of propylene glycol;

[0024] Figure 3 The changes in the appearance of the peptide solution after adding different volumes of pure water;

[0025] Figure 4 The changes in the appearance of the peptide solution after adding different pH buffers;

[0026] Figure 5 The following figure shows the changes in the appearance of the peptide solution after adding different buffers. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0029] Source of raw materials:

[0030] Peptide HHH001, Zhongke Haohanhang;

[0031] Anhydrous ethanol: Nanjing Chemical Reagent Co., Ltd.;

[0032] Propylene glycol: Hubei Gedian Renfu Pharmaceutical Excipients Co., Ltd.;

[0033] Buffer preparation:

[0034] Sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer (Na 2 HPO 4 -NaH 2 PO 4 Buffer solution, pH = 8.0): Solution A: weigh 3.5904 g Na 2 HPO 4 Add water to dissolve and dilute to 50mL; Solution B: weigh NaH 2 PO 4 0.2760g was dissolved in water and diluted to 10mL. 45.75mL of solution A and 4.25mL of solution B were mixed evenly.

[0035] in,

[0036] Preparation of potassium dihydrogen phosphate buffer: Dissolve 27.22g potassium dihydrogen phosphate in water and dilute to 1000mL to obtain 0.2mol / L potassium dihydrogen phosphate solution. Take 50mL of 0.2mol / L potassium dihydrogen phosphate solution into a 200mL volumetric flask, add 46.1mL NaOH (0.2mol / L), and add water to the mark to obtain.

[0037] Potassium hydrogen phthalate-hydrochloric acid buffer (pH=3.0): Dissolve 40.85g potassium hydrogen phthalate in water and dilute to 1000mL to obtain 0.2mol / L potassium hydrogen phthalate solution. Take 50mL of 0.2mol / L potassium hydrogen phthalate solution into a 200mL volumetric flask, add 22.3mL HCL (0.2mol / L), and add water to the mark to obtain.

[0038] Preparation of Tris-hydrochloric acid buffer: Dissolve 1.214 g of tris(hydroxymethyl)aminomethane in water and dilute to 10 mL, and adjust the pH to 8.020 with 6 mol / L hydrochloric acid.

[0039] equipment:

[0040] New improved small rotating arm spray head (press-on bottle): 0.1mL / spray, Shenzhen Bona Precision Drug Delivery Co., Ltd.;

[0041] New improved small rotating arm spray head (screw bottle): 0.1mL / spray, Shenzhen Bona Precision Drug Delivery Co., Ltd.;

[0042] External liquid medicine high-density polyethylene bottle (squeeze bottle): 10mL, Shenzhen Bona Precision Drug Delivery Co., Ltd.;

[0043] PET brown screw-mouth bottle: 10 mL, Shenzhen Bona Precision Drug Delivery Co., Ltd.

[0044] Electronic balance: AS220.X2, RADWAG;

[0045] Electronic scale: BJ6100D, Precisa;

[0046] 1. Study on the solubility characteristics of peptide HHH001:

[0047] (1) Anhydrous ethanol: To determine the solubility characteristics of polypeptide HHH001 in anhydrous ethanol, 10 mg of polypeptide was dissolved in 5 mL, 10 mL, and 20 mL of anhydrous ethanol, respectively, and sonicated for 1 min to observe the dissolution of polypeptide HHH001.

[0048] The results are as follows Figure 1 As shown, the solution remained turbid, indicating that none of the polypeptides were completely dissolved.

[0049] (2) Propylene glycol: To determine the solubility characteristics of polypeptide HHH001 in propylene glycol, 10 mg of polypeptide was dissolved in 5 mL, 10 mL, and 20 mL of propylene glycol, respectively, and sonicated for 1 min to observe the solubility of polypeptide HHH001.

[0050] The results are as follows Figure 2 As shown, the solution remained turbid, indicating that none of the polypeptides were completely dissolved.

[0051] (3) Pure water: To determine the solubility characteristics of polypeptide HHH001 in pure water, 10 mg of polypeptide was dissolved in 5 mL, 10 mL, and 20 mL of pure water, respectively, by ultrasonic treatment for 1 min, and the dissolution of polypeptide HHH001 was observed.

[0052] The results are as follows Figure 3 As shown, the solution remained turbid, indicating that none of the polypeptides were completely dissolved.

[0053] (4) Adjust pH to investigate solubility characteristics. Dissolve the peptide in water to prepare a 0.5 mg / mL peptide solution, and add potassium dihydrogen phosphate buffer (KH 2 PO 4 The pH of the solution was adjusted to 7.8 and 4.8 respectively by adding potassium hydrogen phthalate-hydrochloric acid buffer to adjust the pH to 3.347.

[0054] The results are as follows Figure 4As shown, when the pH is 7.8, the insoluble matter in the polypeptide solution is significantly reduced, but there are still insoluble matter. When the pH is 4.8 and 3.347, the polypeptide solution has more insoluble matter, and the insoluble matter does not decrease significantly after lowering the pH, indicating that acidic conditions are not conducive to the dissolution of polypeptides, while neutral and weakly alkaline conditions are conducive to the dissolution of polypeptides.

[0055] (5) Different buffer types: The polypeptide was dissolved in water to prepare a 0.5 mg / mL polypeptide solution, and the following treatments were performed: using disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH of the polypeptide aqueous solution to 7.8; using potassium dihydrogen phosphate buffer to adjust the pH of the solution to 7.8; using Tris-hydrochloric acid buffer to adjust the pH of the polypeptide aqueous solution to 7.6, and observing the dissolution of the solutions.

[0056] The results are as follows Figure 5 As shown, from left to right are potassium dihydrogen phosphate buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and Tris-hydrochloric acid buffer. By comparing the dissolution of the solutions by the three different ion type buffers, it can be seen that disodium hydrogen phosphate-sodium dihydrogen phosphate and Tris-hydrochloric acid buffers have better solubilization effects on polypeptides after adjusting the pH. Therefore, in subsequent experiments, disodium hydrogen phosphate-sodium dihydrogen phosphate is used as the pH regulator to prepare polypeptide solutions.

[0057] 2. Study on the stability of peptide solutions for preventing and / or treating hair loss:

[0058] Example 1

[0059] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0060] Weigh 79.6g of anhydrous ethanol, 174.3g of propylene glycol, and 66.5g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.1683g of polypeptide and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.8 with 0.67 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0061] Example 2

[0062] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0063] Weigh 200g of anhydrous ethanol, 360g of propylene glycol, and 140g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.2g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO4 -NaH 2 PO 4 The pH value was adjusted to 7.9 with 0.80 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0064] Example 3

[0065] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0066] Weigh 46.67g of anhydrous ethanol, 73.33g of propylene glycol, and 30g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.2g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.5 with 0.33 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0067] Example 4

[0068] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0069] Weigh 79.6g of anhydrous ethanol, 174.3g of propylene glycol, and 66.5g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.1683g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.8 with 0.42 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0070] Example 5

[0071] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0072] Weigh 200g of anhydrous ethanol, 360g of propylene glycol, and 140g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.2g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 8.0 with 0.90 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0073] Example 6

[0074] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0075] Weigh 120g of anhydrous ethanol, 360g of propylene glycol, and 140g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.2g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.9 with 0.72 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0076] Example 7

[0077] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this embodiment is as follows:

[0078] Weigh 200g of anhydrous ethanol, 200g of propylene glycol, and 140g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.2g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.9 with 0.68 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0079] Comparative Example 1

[0080] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this comparative example is as follows:

[0081] Weigh 253.9g propylene glycol and 66.5g purified water in a 1000mL beaker and stir for 3min. The solution is clear and transparent. Weigh 0.1683g polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.8 with 0.54 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0082] Comparative Example 2

[0083] The preparation method of the polypeptide solution for preventing and / or treating hair loss in this comparative example is as follows:

[0084] Weigh 253.9g of anhydrous ethanol and 66.5g of purified water in a 1000mL beaker and stir for 3min. The solution is now clear and transparent. Weigh 0.1683g of polypeptide HHH001 and add it to the above solution and stir for 1.5h. Then add Na 2 HPO 4 -NaH 2 PO 4 The pH value was adjusted to 7.8 with 0.43 mL of buffer solution and stirring was continued for 1.5 h to finally form a clear transparent solution.

[0085] Half of the polypeptide solutions prepared in the examples and comparative examples were placed in high-density polyethylene bottles and crimped using a crimping device. The other half of the spray was placed in a brown PET screw-mouth bottle and the cap was screwed manually. The spray was placed at 2-8°C for 0 days, 15 days and 30 days, and the appearance of the polypeptide solution, the content of polypeptide HHH001 and related substances were tested to observe the stability of the solution.

[0086] Detection method: After a series of liquid chromatography method development (after screening of chromatographic columns, mobile phase systems, additive concentrations, gradient change rates, flow rates and column temperatures), the following (Table 1) analysis method for the content of polypeptide HHH001 and related substances was established.

[0087] Table 1 Parameters of content and related substances determination method

[0088]

[0089] Note: TFA is trifluoroacetic acid, ACN is acetonitrile.

[0090] The test results are shown in Table 2:

[0091] Table 2 Stability test results of sprays prepared in Examples and Comparative Examples

[0092]

[0093]

[0094] As shown in Table 2, the spray prepared in the embodiment of the present invention has good stability, can be stored for more than one month at 2-8°C, has low impurity content, and maintains stable polypeptide content, thus ensuring the effectiveness of medication. The spray prepared in Comparative Examples 1 and 2 has poor stability, and after being stored for a period of time, the polypeptide aggregates and forms insoluble precipitates, making the originally clear solution turbid, and the total impurity content increases significantly.

[0095] In summary, the present invention provides a spray for preventing and treating hair loss, which avoids the problem of poor long-term storage stability of polypeptide HHH001 by optimizing the formula of the polypeptide solution and compounding it with anhydrous ethanol and propylene glycol in a specific ratio, thereby ensuring the long-term effectiveness of the spray in treating hair loss and improving the treatment effect. The spray of the present application can act directly on the scalp, avoiding the side effects of systemic administration, and is easy to use and easy for patients to manage themselves in the long term.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0097] Sequence Listing SEQ ID NO.1MPRLPPAGGGGSYLYQWLGAPVPYPDPLEP.

Claims

1. A spray for preventing and / or treating hair loss, characterized in that: The composition comprises the following components in parts by weight: 0.5 to 3 parts of polypeptide HHH001, 500 to 700 parts of anhydrous ethanol, 900 to 1100 parts of propylene glycol, 2 to 5 parts of pH regulator and 350 to 450 parts of water; The amino acid sequence of the polypeptide HHH001 is shown in SEQ ID NO.

1.

2. The spray according to claim 1, characterized in that The pH of the spray is 7.5-8.

0.

3. The spray according to claim 1, characterized in that The mass ratio of the polypeptide HHH001 to anhydrous ethanol is 1:(550-650).

4. The spray according to claim 1, characterized in that The mass ratio of the polypeptide HHH001 to propylene glycol is 1:(950-1100).

5. The spray according to claim 1, characterized in that The pH regulator is any one of phosphate buffer, Tris-hydrochloric acid buffer, acetate, sodium citrate, citric acid and tartaric acid.

6. The method for preparing the spray according to any one of claims 1 to 5, characterized in that: The steps include: Anhydrous ethanol, propylene glycol and water are weighed in parts by weight, mixed evenly, added with polypeptide HHH001 and stirred, added with a pH adjuster and continued to stir to obtain a mixed solution, which is then packaged and filled to obtain the spray of the present application.

7. The preparation method according to claim 6, characterized in that: The stirring time after adding polypeptide HHH001 is 1.5 to 3.0 hours.

8. Use of the spray according to any one of claims 1 to 5 in the preparation of medical products for preventing and / or treating hair loss.

9. Use of the spray according to any one of claims 1 to 5 in the preparation of medical products or cosmetics for promoting hair growth.

10. The use according to claim 9, characterized in that: The hair includes head hair, eyebrows and eyelashes.

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