Crystalline form of hexapeptide-9 and methods of preparation and use thereof

Abstract

The application provides two different crystal forms of hexapeptide-9, a preparation method and application thereof. The crystal form of hexapeptide-9 provided by the application has good temperature and humidity stability. In addition, the application obtains two new crystal forms of hexapeptide-9 through screening and optimization of different crystallization solvents, temperatures and crystallization conditions, and the method is simple and has the advantages of large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of crystal form technology, specifically relating to the crystal form of hexapeptide-9, its preparation method, and its application. Background Technology

[0002] Hexapeptide-9 is a novel synthetic hexapeptide with significant anti-allergic, repairing, wrinkle-reducing, and acne scar-repairing effects. Its amino acid sequence (Gly-Pro-Gln-Gly-Pro-Gln) is identical to specific regions of human collagen IV and XVII, enabling it to activate skin repair and regeneration mechanisms by mimicking naturally occurring signaling molecules in the skin, thus exhibiting significant anti-aging and repairing effects.

[0003] Hexapeptide-9 has been demonstrated in several patents for its application in cosmetics. CN116077366B describes an anti-aging combination peptide and its application, which adds hexapeptide-9 to a combination of palmitoyl tripeptide-5, palmitoyl pentapeptide-4, and acetyl hexapeptide-8. Under appropriate ratios, this significantly increases collagen content, reduces dynamic wrinkles, and achieves better anti-aging and wrinkle-reducing effects. CN110833514B describes a lyophilized peptide powder and its application in the preparation of anti-aging cosmetics, which uses a composite lyophilized powder containing multiple peptides, including hexapeptide-9, in cosmetics with anti-aging effects. CN117695167B describes a matrix composition, its preparation method, and its application, which combines hexapeptide-9 with multiple peptides to achieve a synergistic skincare effect.

[0004] Besides its application in cosmetics, CN117137830B also has numerous patented applications in medicine and cosmetology. CN117137830B discloses a skin repair composition and its application, which utilizes hexapeptide-9 and hyaluronic acid-based reagents to form a skin repair composition with good skin repair effects. CN119868194B discloses a highly permeable polypeptide combination wound healing agent, specifically involving a polypeptide combination of tripeptide-1, hexapeptide-9, and palmitoyl-modified peptides, for the novel use in treating wounds, ulcers, burns, scars, bumps, and other skin diseases. CN119606786B discloses a cell-active peptide composition that promotes the regeneration of various collagen structural proteins, its preparation method, and its application in medicine and cosmetology. This composition can accelerate the regeneration of skin collagen and other structural proteins, repair skin damage, reduce wrinkles such as crow's feet, tear troughs, nasolabial folds, and under-eye wrinkles, and address skin aging problems caused by the loss of collagen and other structural proteins.

[0005] Crystal form influences the physicochemical properties of drugs: specific crystal forms may indicate greater stability, less decomposition or deterioration, thus extending the drug's shelf life; they also affect the drug's dissolution rate and solubility, with highly soluble crystal forms contributing to improved bioavailability, ensuring rapid absorption and efficacy in the body. Crystal form research helps optimize drug synthesis and purification processes, improving production efficiency and product quality. Crystal form is a crucial factor in drug development and production, directly impacting drug quality, efficacy, and market competitiveness.

[0006] Existing literature on hexapeptide-9 mainly focuses on its synthetic pathway, detection methods, and synergistic effects with other active ingredients. However, a systematic study of its crystal form remains lacking. Furthermore, existing hexapeptide-9 exhibits poor temperature and humidity stability, and since the crystal form of an active substance is closely related to its stability, research into this gap in the understanding of its potential applications is essential. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a crystal form of hexapeptide-9, its preparation method and application. The crystal form of hexapeptide-9 provided by the present invention has good temperature and humidity stability.

[0008] The present invention provides a crystal form of hexapeptide-9, the X-ray powder diffraction pattern of which has diffraction peaks at 2θ±0.2°, wherein 2θ is 5.804°, 9.493°, 11.566°, 18.475°, 23.393° and 25.877°.

[0009] Furthermore, the 2θ is 5.804°, 9.493°, 11.566°, 18.475°, 18.786°, 19.012°, 19.442°, 20.278°, 20.84°, 20.916°, 22.034°, 22.932°, 23.393°, 25.237°, 25.877°, 26.25°, 29.44°, 29.93°, 30.282°, 30.805°, 32.556°, 33.712°, 34.183°, and 34.778°.

[0010] Specifically, the X-ray powder diffraction pattern of the crystal form of hexapeptide-9 provided by this invention is as follows: Figure 1 , 3 Or as shown in Figure 4.

[0011] In this invention, the 2nd X-ray powder diffraction pattern θValues ​​may vary slightly between machines or samples, with differences of approximately 0.2 units or 0.1 units. Therefore, the values ​​cited should not be interpreted as absolute values. Similarly, peak heights may vary by approximately 5 units, 4 units, 3 units, 2 units, or 1 unit. Therefore, the XRPD trace intensity included in this invention is illustrative and not intended for absolute comparison.

[0012] The present invention also provides a method for preparing the crystal form of the above-mentioned hexapeptide-9, comprising the following steps: An aqueous solution of hexapeptide-9 was mixed with an antisolvent, and crystallization was performed to obtain the crystal form of hexapeptide-9. The antisolvent is selected from any one of isopropanol, dimethylformamide (DMF), and 1,4-dioxane.

[0013] Specifically, the present invention first prepares an aqueous solution of hexapeptide-9, wherein the preparation conditions are 15~45℃, which can be any value between 15, 20, 25, 30, 35, 40, 45, or 15~45℃.

[0014] The concentration of the aqueous solution of hexapeptide-9 is 300~500 g / L, and can be any value between 300, 350, 400, 450, 500, or 300~500 g / L.

[0015] The pH of the aqueous solution of hexapeptide-9 is 4.0 to 7.0, and can be any value between 4, 4.5, 5, 5.5, 6, 6.5, 7, or 4.0 to 7.0.

[0016] Preferably, the antisolvent is added to the aqueous solution of hexapeptide-9. The method of addition is not particularly limited in this invention, and includes, but is not limited to, dropwise addition, flow addition, etc. Preferably, it is added dropwise at a rate of 40-50 mL / h, which can be 40, 45, 50, or 40-50 mL / h.

[0017] In some specific embodiments of the present invention, while adding the mixture dropwise, a low-speed stirring is maintained at a rotation speed of 50-250 rpm, which can be any value between 50, 100, 150, 200, 250, or 50-250 rpm.

[0018] The volume ratio of the aqueous solution of hexapeptide-9 to the antisolvent is 1:(1~3), which can be any value between 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or 1:(1~3).

[0019] After the addition is complete, maintain the temperature and stir for 5-10 hours to grow crystals. Then, reduce the temperature to 5-14℃ at a rate of 4-6℃ / h and continue crystallization for 10-24 hours. The crystallization time can be any value between 5, 6, 7, 8, 9, 10, or 5-10 hours. The cooling rate can be any value between 4, 5, 6, or 4-6℃ / h. The cooling temperature can be any value between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 5-14℃. The continued crystallization time can be any value between 10, 12, 14, 16, 18, 20, 22, 24, or 10-24 hours.

[0020] After crystallization, the powder is filtered, then washed with an appropriate amount of anti-solvent, and then vacuum dried at 45°C until the moisture content is ≤8%.

[0021] The present invention also provides a crystal form of hexapeptide-9, wherein the X-ray powder diffraction pattern shows diffraction peaks at 2θ±0.2°, wherein 2θ is 10.6658°, 19.097°, 20.215° and 21.587°.

[0022] Furthermore, the 2θ is 10.6658°, 11.218°, 12.3787°, 18.325°, 19.097°, 19.536°, 19.740°, 20.215°, 21.291°, 21.587°, 22.503°, 23.072°, 24.361°, 24.962°, 25.748°, 26.941°, 27.453°, 28.083°, 29.173°, 31.397°, 33.433°, and 41.974°.

[0023] Specifically, the X-ray powder diffraction pattern of hexapeptide-9 provided by this invention is as follows: Figure 2 As shown.

[0024] The present invention also provides a method for preparing the crystal form of the above-mentioned hexapeptide-9, comprising the following steps: An aqueous solution of hexapeptide-9 was mixed with an antisolvent, and crystallization was performed to obtain the crystal form of hexapeptide-9. The antisolvent is selected from methanol.

[0025] Specifically, the present invention first prepares an aqueous solution of hexapeptide-9, wherein the preparation conditions are 15~45℃, which can be any value between 15, 20, 25, 30, 35, 40, 45, or 15~45℃.

[0026] The concentration of the aqueous solution of hexapeptide-9 is 300~500 g / L, and can be any value between 300, 350, 400, 450, 500, or 300~500 g / L.

[0027] The pH of the aqueous solution of hexapeptide-9 is 4.0 to 7.0, and can be any value between 4, 4.5, 5, 5.5, 6, 6.5, 7, or 4.0 to 7.0.

[0028] Preferably, the antisolvent is added to the aqueous solution of hexapeptide-9. The method of addition is not particularly limited in this invention, and includes, but is not limited to, dropwise addition, flow addition, etc. Preferably, it is added dropwise at a rate of 40-50 mL / h, which can be 40, 45, 50, or 40-50 mL / h.

[0029] In some specific embodiments of the present invention, while adding the mixture dropwise, a low-speed stirring is maintained at a rotation speed of 50-250 rpm, which can be any value between 50, 100, 150, 200, 250, or 50-250 rpm.

[0030] The volume ratio of the aqueous solution of hexapeptide-9 to the antisolvent is 1:(1~3), which can be any value between 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or 1:(1~3).

[0031] After the addition is complete, maintain the temperature and stir for 5-10 hours to grow crystals. Then, reduce the temperature to 5-14℃ at a rate of 4-6℃ / h and continue crystallization for 10-24 hours. The crystallization time can be any value between 5, 6, 7, 8, 9, 10, or 5-10 hours. The cooling rate can be any value between 4, 5, 6, or 4-6℃ / h. The cooling temperature can be any value between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 5-14℃. The continued crystallization time can be any value between 10, 12, 14, 16, 18, 20, 22, 24, or 10-24 hours.

[0032] After crystallization, the powder is filtered, then washed with an appropriate amount of anti-solvent, and then vacuum dried at 45°C until the moisture content is ≤8%.

[0033] The present invention also provides the application of the crystal form of the above-mentioned hexapeptide-9 or the crystal form of hexapeptide-9 prepared by the above preparation method in the preparation of cosmetics.

[0034] Compared with existing technologies, this invention provides two different crystal forms of hexapeptide-9, their preparation methods, and applications. The crystal forms of hexapeptide-9 provided by this invention exhibit good temperature and humidity stability. Furthermore, this invention obtains two new crystal forms of hexapeptide-9 through screening and optimization of different crystallization solvents, temperatures, and crystallization conditions. The methods are simple and have the advantage of large-scale industrial production. Attached Figure Description

[0035] Figure 1X-ray powder diffraction pattern of the crystal form of hexapeptide-9 prepared in Example 1; Figure 2 X-ray powder diffraction pattern of the crystal form of hexapeptide-9 prepared in Example 2; Figure 3 X-ray powder diffraction pattern of the crystal form of hexapeptide-9 prepared in Example 3; Figure 4 X-ray powder diffraction pattern of the crystal form of hexapeptide-9 prepared in Example 4; Figure 5 Liquid chromatograms of the crystal forms of hexapeptide-9 prepared in Examples 1-4; Figure 6 The X-ray powder diffraction pattern of hexapeptide-9 prepared in Comparative Example 1 is shown. Detailed Implementation

[0036] To further understand the present invention, the following examples illustrate the crystal form of hexapeptide-9 provided by the present invention, its preparation method, and its application. The scope of protection of the present invention is not limited by the following examples.

[0037] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0038] In the following examples, hexapeptide-9 is a commercially available amorphous lyophilized powder.

[0039] Example 1

[0040] At 25℃, 100 mL of a 500 g / L hexapeptide-9 aqueous solution was prepared and stirred continuously at 100 rpm. Two volumes of isopropanol were added dropwise at a rate of 45 mL / h. After the addition was complete, the temperature was maintained and stirred for 6 h to allow crystal growth. The temperature was then lowered to 10℃ at a rate of 6℃ per hour, and crystallization continued for 8 h. The mixture was filtered, the powder was washed with isopropanol, and then vacuum dried at 45℃ until the moisture content met the requirements. The yield was 93%, and the purity was 99.7%. Figure 1 The powder X-ray diffraction pattern shown has characteristic peaks at 2θ = 5.804°, 9.493°, 11.566°, 18.475°, 23.393° and 25.877°. Figure 1 The X-ray powder diffraction pattern of the crystal form of hexapeptide-9 prepared in Example 1. Figure 5 The liquid chromatograms are of the crystal forms of hexapeptide-9 prepared in Examples 1-4.

[0041] The crystal form diffraction results of the hexapeptide-9 prepared in Example 1 of this invention are shown in Table 1: Table 1

[0042] Example 2

[0043] At 20℃, 100 mL of a 500 g / L hexapeptide-9 aqueous solution was prepared and stirred continuously at 150 rpm. Methanol (1.5 times its volume) was added dropwise at a rate of 35 mL / h. After the addition was complete, the temperature was maintained and stirred for 6 h to allow crystal growth. The temperature was then lowered to 10℃ at a rate of 5℃ per hour, and crystallization continued for 14 h. The mixture was filtered, and the powder was washed with an appropriate amount of methanol and then vacuum dried at 45℃ until the moisture content met the requirements. The yield was 90%, and the purity was 99.2%. Figure 2 The powder X-ray diffraction pattern shown is obtained using Cu-Kα rays. Characteristic peaks are observed at 2θ = 10.6658°, 19.097°, 20.215°, and 21.587°.

[0044] The crystal form diffraction results of the hexapeptide-9 prepared in Example 2 of this invention are as follows: Table 2

[0045] Example 3

[0046] At 35℃, a 500 g / L hexapeptide-9 solution was prepared in 100 mL. The mixture was stirred continuously at 150 rpm, and 1.8 times its volume of dimethylformamide was added dropwise at a rate of 45 mL / h. After the addition was complete, the temperature was maintained and stirred for 10 h to allow crystal growth. The temperature was then lowered to 10℃ at a rate of 5℃ per hour, and crystallization continued for 24 h. The mixture was filtered, and the powder was washed with an appropriate amount of dimethylformamide. It was then vacuum dried at 45℃ until the moisture content met the requirements. The yield was 92%, and the purity was 99.3%. Figure 3 The powder X-ray diffraction pattern shown is the same as that in Example 1, obtained by measuring Cu-Kα rays.

[0047] Example 4

[0048] At 35℃, a 500 g / L hexapeptide-9 solution was prepared in 100 mL. The mixture was stirred continuously at 150 rpm, and 2 times its volume of 1,4-dioxane was added dropwise at a rate of 45 mL / h. After the addition was complete, the temperature was maintained and stirred for 8 h to allow crystal growth. The temperature was then lowered to 10℃ at a rate of 5℃ per hour, and crystallization continued for 18 h. The mixture was filtered, and the powder was washed with an appropriate amount of 1,4-dioxane. The powder was then vacuum-dried at 45℃ until the moisture content met the requirements. The yield was 92%, and the purity was 99.3%. Figure 4 The powder X-ray diffraction pattern shown is the same as that in Example 1, obtained by measuring Cu-Kα rays.

[0049] Example 5

[0050] At 25℃, prepare 100 mL of a 500 g / L hexapeptide-9 aqueous solution and stir continuously at 100 rpm. Add 1 to 3 times the volume of isopropanol dropwise at a rate of 45 mL / h. After the addition is complete, maintain the temperature and stir for 6 h to grow crystals. Then, reduce the temperature to 10℃ at a rate of 6℃ per hour and continue crystallization for 8 h. After filtration, wash the powder with isopropanol and dry it under vacuum at 45℃ until the moisture content meets the requirements.

[0051] The yields and purities are shown in the table below. Higher yields were achieved when the volume ratio of isopropanol to aqueous solution was between 1.5 and 2. Since the method optimization used an aqueous solution of hexapeptide-9, the purity of the active substance itself was high, and no additional impurities were introduced; therefore, the purity results are not very meaningful.

[0052] Table 3

[0053] Example 6

[0054] At 25℃, prepare 100 mL of a 500 g / L hexapeptide-9 aqueous solution and stir continuously at 100 rpm. Add 2 times the volume of isopropanol dropwise at a rate of 15~90 mL / h. After the addition is complete, maintain the temperature and stir for 6 h to grow crystals. Then, reduce the temperature to 10℃ at a rate of 6℃ per hour and continue crystallization for 8 h. After filtration, wash the powder with isopropanol and dry it under vacuum at 45℃ until the moisture content meets the requirements.

[0055] The yield and purity are shown in the table below. The flow rate should be controlled at ≤45 mL / h. Too fast a flow rate will cause the active substance to turn into oil instead of precipitating crystals.

[0056] Table 4

[0057] Example 7

[0058] At 25℃, prepare 100 mL of a 500 g / L hexapeptide-9 aqueous solution and stir continuously at 100 rpm. Add 2 times the volume of isopropanol dropwise at a rate of 45 mL / h. After the addition is complete, maintain the temperature and stir for 6 h to grow crystals. Then, reduce the temperature to 10℃ at a rate of 6℃ per hour and continue crystallization for 8 h. After filtration, wash the powder with isopropanol and dry it under vacuum at 45℃ until the moisture content meets the requirements.

[0059] The yield and purity are shown in the table below. A cooling rate of 4~6℃ / h is preferred. Too rapid a cooling rate will result in poor crystal precipitation.

[0060] Table 5

[0061] Comparative Example 1

[0062] At 25℃, 100 mL of a 500 g / L hexapeptide-9 aqueous solution was prepared, rapidly frozen at -80℃ under a vacuum of 50 Pa and an ambient temperature of 20℃, and freeze-dried for 24 h to obtain an amorphous powder with a yield of 91% and a purity of 99.5%. Its XRD pattern is shown below. Figure 6 .

[0063] Comparative Example 2

[0064] At 25℃, 100 mL of a 500 g / L hexapeptide-9 aqueous solution was prepared and stirred continuously at 100 rpm. Two volumes of ethanol / n-butanol / n-hexane / dichloromethane / methyl tert-butyl ether / ethyl acetate / butyl acetate / ethyl formate / n-heptane / dimethyl sulfoxide / cyclohexane were added dropwise at a rate of 45 mL / h. After the addition was completed, the temperature was maintained and stirred for 6 h to allow crystals to grow. Then, the temperature was lowered to 10℃ at a rate of 6℃ per hour, and crystallization continued for 8 h. No crystals precipitated, and the solution remained clear.

[0065] Compared with Example 1, except that the antisolvent was changed from isopropanol to other solvents, all other conditions remained the same. No crystals precipitated after the antisolvent was changed. None of the solvents listed in Comparative Example 2 are suitable as antisolvents under these conditions.

[0066] Application Example 1

[0067] The hygroscopicity test method is as follows: Nine new 20 mL glass bottles were selected, dried in an oven at 105℃ until constant weight, and numbered. After the products of Example 1, Example 2 and Comparative Example 1 were dried to constant weight, 1 g of powder was filled into each glass bottle and placed in a constant temperature and humidity chamber (room temperature, 95% humidity). The samples were weighed every 24 hours until the samples reached constant weight. The results are shown in Table 6. The crystal form of the present invention is more stable in a high humidity environment.

[0068] Table 6 Hygroscopicity Results

[0069] In Table 6, three replicate experiments were conducted for each product.

[0070] The temperature stability test method is as follows: Take 4-5g of powder from Example 1, Example 2 and Comparative Example 1 dried to constant weight and fill it into a clean glass bottle. Place the glass bottle in an incubator at 45℃ for 30 days, take it out and test the powder composition. The results are shown in Table 7. This crystal form has good temperature stability.

[0071] Table 7 Temperature stability test results

[0072] In Table 7, three replicate experiments were conducted for each product.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A crystal form of hexapeptide-9, characterized in that, The X-ray powder diffraction pattern shows diffraction peaks at 2θ ± 0.2°, where 2θ is 5.804°, 9.493°, 11.566°, 18.475°, 23.393°, and 25.877°.

2. The crystal form according to claim 1, characterized in that, The 2θ values ​​are 5.804°, 9.493°, 11.566°, 18.475°, 18.786°, 19.012°, 19.442°, 20.278°, 20.84°, 20.916°, 22.034°, 22.932°, 23.393°, 25.237°, 25.877°, 26.25°, 29.44°, 29.93°, 30.282°, 30.805°, 32.556°, 33.712°, 34.183°, and 34.778°.

3. The crystal form according to claim 1, characterized in that, The X-ray powder diffraction patterns are shown in Figures 1, 3, or 4.

4. A method for preparing the crystal form of hexapeptide-9 as described in any one of claims 1 to 3, characterized in that, Includes the following steps: An aqueous solution of hexapeptide-9 was mixed with an antisolvent, and crystallization was performed to obtain the crystal form of hexapeptide-9. The antisolvent is selected from any one of isopropanol, dimethylformamide, and 1,4-dioxane.

5. The preparation method according to claim 4, characterized in that, The antisolvent was added dropwise to the aqueous solution of hexapeptide-9 at a rate of 40-50 mL / h; The mixing temperature is 15~45℃; The concentration of the aqueous solution of hexapeptide-9 is 300~500 g / L, and the pH is 4.0~7.0; The volume ratio of the aqueous solution of hexapeptide-9 to the antisolvent is 1:(1~3). After mixing, crystallize at 15~45℃ for 5~10h, then cool down to 5~14℃ at 4~6℃ / h and crystallize for 10~24h.

6. A crystal form of hexapeptide-9, characterized in that, The X-ray powder diffraction pattern shows diffraction peaks at 2θ ± 0.2°, where 2θ is 10.6658°, 19.097°, 20.215°, and 21.587°.

7. The crystal form according to claim 6, characterized in that, The 2θ values ​​are 10.6658°, 11.218°, 12.3787°, 18.325°, 19.097°, 19.536°, 19.740°, 20.215°, 21.291°, 21.587°, 22.503°, 23.072°, 24.361°, 24.962°, 25.748°, 26.941°, 27.453°, 28.083°, 29.173°, 31.397°, 33.433°, and 41.974°.

8. The crystal form according to claim 6, characterized in that, The X-ray powder diffraction pattern is shown in Figure 2.

9. A method for preparing the crystal form of hexapeptide-9 as described in any one of claims 6 to 8, characterized in that, Includes the following steps: An aqueous solution of hexapeptide-9 was mixed with an antisolvent, and crystallization was performed to obtain the crystal form of hexapeptide-9. The antisolvent is selected from methanol.

10. The application of the crystal form of hexapeptide-9 as described in any one of claims 1 to 3, or the crystal form of hexapeptide-9 prepared by the preparation method described in any one of claims 4 to 5, or the crystal form of hexapeptide-9 as described in any one of claims 6 to 8, or the crystal form of hexapeptide-9 prepared by the preparation method described in any one of claims 9, in the preparation of cosmetics.

Images