High-stability hEGF sustained-release gel external preparation and preparation method

Abstract

The application discloses a high-stability hEGF sustained-release gel external preparation and a preparation method, and belongs to the technical field of sustained-release gel preparations, and comprises the following steps: preparing a poloxamer 407 solution, preparing a sodium carboxymethyl cellulose solution; adding the sodium carboxymethyl cellulose solution into the poloxamer 407 solution to obtain a mixed solution A, preparing a borneol solution, adding the borneol solution into the mixed solution A to obtain a mixed solution B; adding human epidermal growth factor into the mixed solution B to obtain a mixed solution C; and filtering and sterilizing to obtain the hEGF sustained-release gel preparation. The low-temperature gel drug loading combination process is adopted in combination with a stable pH system, so that the biological activity of the human epidermal growth factor can be effectively maintained, the preparation can normally play its core biological functions of regulating the proliferation, differentiation and migration of related target cells, and the basic demand for drug efficiency in actual application is met.

Description

Technical Field

[0001] This invention relates to the field of sustained-release gel formulation technology, specifically to a highly stable hEGF sustained-release gel topical formulation and its preparation method. Background Technology

[0002] Topical human epidermal growth factor (hEGF) is a polypeptide cytokine containing 53 amino acid residues. Its core biological function lies in regulating the proliferation, differentiation, and migration of target cells such as epithelial cells and fibroblasts. As the application of this factor in wound repair, burn treatment, ophthalmology, and medical aesthetics deepens, single formulations can no longer meet the diverse clinical needs and market application scenarios.

[0003] The current mainstream topical human epidermal growth factor products in clinical practice are mainly administered by reconstituted lyophilized powder and soaking gauze. This dosage form has the disadvantage of a single delivery carrier, resulting in insufficient clinical efficacy for administration to joints or other sites where gauze is inconvenient. The effectiveness is limited by the method of administration.

[0004] Topical human epidermal growth factor sustained-release gel represents a significant technological breakthrough. Its core advantages lie in its significantly improved formulation stability, which effectively maintains the bioactivity of hEGF during storage and use; excellent biocompatibility, reducing the risk of local tissue irritation and adapting to different wound environments.

[0005] Based on this, the present invention designs a highly stable hEGF sustained-release gel topical formulation and its preparation method. Summary of the Invention

[0006] In view of the above-mentioned shortcomings of the existing technology, the present invention provides a highly stable hEGF sustained-release gel topical formulation and its preparation method.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A highly stable hEGF sustained-release gel for external use and its preparation method, comprising the following steps:

[0009] S1: Add 180-210g of poloxamer 407 to water and stir to obtain a poloxamer 407 solution;

[0010] S2: Add 48-52g of sodium carboxymethyl cellulose to water and stir to obtain a sodium carboxymethyl cellulose solution;

[0011] S3: Add sodium carboxymethyl cellulose solution to poloxamer 407 solution and stir to obtain mixture A;

[0012] S4: Add 1-3g of borneol to water and stir to obtain a borneol solution;

[0013] First, cool the mixture to 15-20℃, add the borneol solution to mixture A, add water to make up to 800mL, and continue stirring. During stirring, add 0.1-1mol / L sodium hydroxide solution to adjust the pH to 6.5-7.0 to obtain mixture B.

[0014] S5: Thaw 49-52mg of human epidermal growth factor, store it at low temperature after thawing, dilute it with water and add it to mixture B, add water to make up the total volume of 1000mL, stir to obtain mixture C;

[0015] S6: Filter and sterilize to obtain hEGF sustained-release gel formulation;

[0016] S7: Filling.

[0017] Furthermore, S1 specifically involves adding 180-210g of poloxamer 407 to 240-300mL of water for injection at a rate of 5-15g / min, and stirring at 150-250rpm for 3-6h to obtain a poloxamer 407 solution.

[0018] Furthermore, S2 specifically involves: taking 48-52g of sodium carboxymethyl cellulose and adding it to 160-220mL of water for injection at 80-90℃ at a rate of 8-20g / min, stirring at 300-400rpm for 3-6h, and cooling to 15-20℃ at 2-4℃ / min to obtain a sodium carboxymethyl cellulose solution.

[0019] Furthermore, S3 specifically involves adding a sodium carboxymethyl cellulose solution to a poloxamer 407 solution at a rate of 100-200 mL / min, and stirring at 150-250 rpm for 1-2 hours at 3-8°C to obtain a mixture A.

[0020] Furthermore, S4 specifically involves: taking 1-3g of borneol and adding it to 80-120mL of water for injection at 80-90℃ at a rate of 0.2-0.5g / min, and stirring at 200-300rpm for 1-2 hours to obtain a borneol solution;

[0021] Cool the mixture to 15-20℃ at a rate of 2-4℃ / min, reduce the stirring speed to 150-250 rpm, add the borneol solution to mixture A at a rate of 80-150 mL / h, add water for injection to bring the volume to 800 mL, and continue stirring at a speed of 150-250 rpm for 1-3 h. During stirring, add 0.1-1 mol / L sodium hydroxide solution at a rate of 5-15 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

[0022] Furthermore, S5 specifically involves: thawing 49-52 mg of human epidermal growth factor at 2-8℃ for 5-15 minutes, then storing it in a refrigerator at 2-8℃ for 15-25 minutes while stirring at 100-150 rpm; diluting the human epidermal growth factor with 5-10 mL of water for injection at 2-8℃ and adding it to mixture B at a rate of 5-10 mL / h; adding water for injection at 2-8℃ to bring the total volume to 1000 mL; and stirring at 100-150 rpm for 30-60 minutes to obtain mixture C.

[0023] Furthermore, S6 specifically involves aseptically filtering the mixture C using a filter with a pore size of 0.22-0.30 μm. During filtration, sterile nitrogen gas at 0.08-0.12 MPa is introduced for low-pressure filtration, with a filtration rate of 50-100 mL / min, to obtain a sterile drug solution. The filtered sterile drug solution is then collected in a sterile storage tank at 18-20℃ and sterilized with saturated steam at 121℃ for 15-30 min to obtain the hEGF sustained-release gel formulation.

[0024] Furthermore, S7 specifically refers to: filling, controlling the filling rate at 10-30 mL / min, controlling the total filling time for each batch at 6-8 h, and immediately aseptically sealing the formulation container after filling, with the sealing time controlled at 3-5 s.

[0025] An hEGF sustained-release gel formulation prepared according to the preparation method described above.

[0026] Compared with the prior art, the beneficial effects of this invention are as follows:

[0027] 1. This invention employs a low-temperature gel-loaded drug delivery process, combined with a stable pH system, to effectively maintain the biological activity of human epidermal growth factor (hEGF), ensuring that the formulation can normally perform its core biological functions such as regulating the proliferation, differentiation, and migration of related target cells, thus meeting the basic requirements for drug efficacy in practical applications.

[0028] 2. The formulation of this invention relies on its own temperature-sensitive properties. It is in a liquid state at room temperature, which makes it easy to apply evenly to the treatment site and reduces the difficulty of operation. After entering the application environment, it can quickly transform into a gel form at body temperature, prolonging the residence time of the drug at the treatment site and providing continuous local drug action support for the relevant repair process, avoiding the problem of short-lasting effect due to rapid drug loss.

[0029] 3. This invention significantly improves the strength and adhesion properties of the gel by combining poloxamer 407 with sodium carboxymethyl cellulose, enabling the gel to adhere more stably to the treatment site during action and preventing it from falling off due to slight external contact or changes in body position. At the same time, the gel structure can form a physical barrier, reducing the interference of external factors on the treatment area, creating a stable local environment for the drug to exert its effects, and further optimizing the local effect of the drug. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Example 1: This example discloses a highly stable hEGF sustained-release gel topical formulation and its preparation method, including the following steps:

[0032] S1: Take 180g of poloxamer 407 and add it to 240mL of water for injection at 2℃ at a rate of 5g / min. Stir at 150rpm for 3h to obtain a poloxamer 407 solution.

[0033] S2: Take 48g of sodium carboxymethyl cellulose and add it to 160mL of water for injection at 80℃ at a rate of 8g / min. Stir at 300rpm for 3h and cool down to 15℃ at 2℃ / min to obtain sodium carboxymethyl cellulose solution.

[0034] S3: Add poloxamer 407 solution to sodium carboxymethyl cellulose solution at a rate of 100 mL / min, and stir at 150 rpm for 1 h at 3 °C to obtain mixture A;

[0035] S4: Take 1g of borneol and add it to 80mL of water for injection at 80℃ at a rate of 0.2g / min. Stir at 200rpm for 1h to obtain a borneol solution.

[0036] The mixture was cooled to 15℃ at a rate of 2℃ / min and the stirring speed was reduced to 150 rpm. The borneol solution was added to the mixture A at a rate of 80 mL / h. Then, water for injection was added to bring the volume to 800 mL. The mixture was stirred at 150 rpm for 1 h. During the stirring process, 0.1 mol / L sodium hydroxide solution was added at a rate of 5 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

[0037] S5: Thaw 49 mg of human epidermal growth factor (hEGF) at 2℃ for 5 min. After thawing, keep it in a refrigerator at 2℃ for low temperature storage. Stir at 100 rpm for 15 min. Dilute the human epidermal growth factor with 5 mL of water for injection at 2℃ and add it to the mixture B at a rate of 5 mL / h. Then add water for injection at 2℃ to a total volume of 1000 mL and stir at 100 rpm for 30 min to obtain the mixture C.

[0038] S6: The mixture C was aseptically filtered using a filter (polyethersulfone material) with a pore size of 0.22μm. During filtration, sterile nitrogen gas of 0.08MPa was introduced for low-pressure filtration at a filtration rate of 50mL / min to obtain a sterile drug solution. The filtered sterile drug solution was collected in a sterile storage tank at 18℃ and sterilized with saturated steam at 121℃ for 15min to obtain the hEGF sustained-release gel formulation.

[0039] S7: Use a pre-sterilized (121℃ saturated steam sterilization for 15 min) aseptic filling machine for filling; before filling, rinse the filling head, pipes and other parts of the filling machine that come into contact with the liquid medicine with sterile saline, the rinsing volume is 3-5 times the pipe volume; control the filling rate at 10 mL / min, and control the total filling time of each batch to 6 h; after filling, immediately aseptically seal the preparation container, the sealing time is controlled to 3-5 s.

[0040] Example 2: This example discloses a highly stable hEGF sustained-release gel topical formulation and its preparation method, including the following steps:

[0041] S1: Take 210g of poloxamer 407 and add it to 300mL of water for injection at 8℃ at a rate of 15g / min. Stir at 250rpm for 6h to obtain a poloxamer 407 solution.

[0042] S2: Take 52g of sodium carboxymethyl cellulose and add it to 220mL of water for injection at 90℃ at a rate of 20g / min. Stir at 400rpm for 6h and cool down to 20℃ at 4℃ / min to obtain sodium carboxymethyl cellulose solution.

[0043] S3: Add poloxamer 407 solution to sodium carboxymethyl cellulose solution at a rate of 200 mL / min, and stir at 250 rpm for 2 h at 8 °C to obtain mixture A;

[0044] S4: Take 3g of borneol and add it to 120mL of water for injection at 90℃ at a rate of 0.5g / min. Stir at 300rpm for 2h to obtain a borneol solution.

[0045] The mixture was cooled to 20℃ at a rate of 4℃ / min and the stirring speed was reduced to 250 rpm. The borneol solution was added to the mixture A at a rate of 150 mL / h. Then, water for injection was added to bring the volume to 800 mL. The mixture was stirred at 250 rpm for 3 h. During the stirring process, 1 mol / L sodium hydroxide solution was added at a rate of 15 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

[0046] S5: Thaw 52mg of human epidermal growth factor (hEGF) at 8℃ for 15min. After thawing, keep it in the refrigerator at 8℃ for low temperature storage. Stir at 150rpm for 25min. Dilute the human epidermal growth factor with 10mL of water for injection at 8℃ and add it to the mixture B at a rate of 10mL / h. Then add water for injection at 8℃ to the total volume of 1000mL and stir at 150rpm for 60min to obtain the mixture C.

[0047] S6: The mixture C was aseptically filtered using a filter (polyethersulfone material) with a pore size of 0.30μm. During filtration, sterile nitrogen gas of 0.12MPa was introduced for low-pressure filtration at a filtration rate of 100mL / min to obtain a sterile drug solution. The filtered sterile drug solution was collected in a sterile storage tank at 20℃ and sterilized with saturated steam at 121℃ for 30min to obtain the hEGF sustained-release gel formulation.

[0048] S7: Use a pre-sterilized (121℃ saturated steam sterilization for 15-30 min) aseptic filling machine for filling; before filling, rinse the filling head, pipes and other parts of the filling machine that come into contact with the liquid medicine with sterile saline, the rinsing volume is 3-5 times the pipe volume; control the filling rate at 30 mL / min, and control the total filling time of each batch to 8 hours; after filling, immediately aseptically seal the preparation container, the sealing time is controlled to 3-5 seconds.

[0049] Example 3: This example discloses a highly stable hEGF sustained-release gel topical formulation and its preparation method, including the following steps:

[0050] S1: Take 200g of poloxamer 407 and add it to 260mL of water for injection at 5℃ at a rate of 10g / min. Stir at 210rpm for 4h to obtain a poloxamer 407 solution.

[0051] S2: Take 50g of sodium carboxymethyl cellulose and add it to 180mL of water for injection at 86℃ at a rate of 15g / min. Stir at 360rpm for 4h and cool down to 18℃ at 3℃ / min to obtain sodium carboxymethyl cellulose solution.

[0052] S3: Add poloxamer 407 solution to sodium carboxymethyl cellulose solution at a rate of 150 mL / min, and stir at 170 rpm for 1.5 h at 6 °C to obtain mixture A;

[0053] S4: Take 2g of borneol and add it to 100mL of water for injection at 86℃ at a rate of 0.4g / min. Stir at 260rpm for 1.5h to obtain a borneol solution.

[0054] The mixture was cooled to 18℃ at a rate of 3℃ / min and the stirring speed was reduced to 220 rpm. The borneol solution was added to the mixture A at a rate of 120 mL / h. Then, water for injection was added to bring the volume to 800 mL. The mixture was stirred at 220 rpm for 2 h. During the stirring process, 0.6 mol / L sodium hydroxide solution was added at a rate of 12 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

[0055] S5: Thaw 50 mg of human epidermal growth factor (hEGF) at 5°C for 13 min. After thawing, keep it in a 5°C refrigerator for low-temperature storage. Stir at 110 rpm for 21 min. Dilute the human epidermal growth factor with 8 mL of water for injection at 5°C and add it to mixture B at a rate of 6 mL / h. Then add water for injection at 5°C to a total volume of 1000 mL and stir at 130 rpm for 45 min to obtain mixture C.

[0056] S6: The mixture C was aseptically filtered using a filter (polyethersulfone material) with a pore size of 0.26μm. During filtration, sterile nitrogen gas of 0.10MPa was introduced for low-pressure filtration, and the filtration rate was 70mL / min to obtain a sterile drug solution. The filtered sterile drug solution was collected in a sterile storage tank at 20℃ and sterilized with saturated steam at 121℃ for 25min to obtain the hEGF sustained-release gel formulation.

[0057] S7: Use a pre-sterilized (121℃ saturated steam sterilization for 24 min) aseptic filling machine for filling; before filling, rinse the filling head, pipes and other parts of the filling machine that come into contact with the drug solution with sterile physiological saline, the rinsing volume is 3-5 times the pipe volume; during filling, control the filling rate at 25 mL / min, and control the total filling time for each batch to 7 hours (to avoid the aseptic state of the drug solution being affected by long-term operation); after filling, immediately aseptically seal the preparation container, the sealing time is controlled to 3-5 seconds.

[0058] Example 4: This example discloses a highly stable hEGF sustained-release gel topical formulation and its preparation method, including the following steps:

[0059] S1: Take 190g of poloxamer 407 and add it to 240-300mL of water for injection at 4℃ at a rate of 13g / min. Stir at 230rpm for 5h to obtain a poloxamer 407 solution.

[0060] S2: Take 51g of sodium carboxymethyl cellulose and add it to 200mL of water for injection at 88℃ at a rate of 10g / min. Stir at 320rpm for 5h and cool down to 20℃ at 4℃ / min to obtain sodium carboxymethyl cellulose solution.

[0061] S3: Add poloxamer 407 solution to sodium carboxymethyl cellulose solution at a rate of 110 mL / min, and stir at 210 rpm for 1.5 h at 4 °C to obtain mixture A;

[0062] S4: Take 1g of borneol and add it to 110mL of water for injection at 84℃ at a rate of 0.4g / min. Stir at 300rpm for 2h to obtain a borneol solution.

[0063] The mixture was cooled to 17℃ at a rate of 4℃ / min and the stirring speed was reduced to 180 rpm. The borneol solution was added to the mixture A at a rate of 100 mL / h. Then, water for injection was added to bring the volume to 800 mL. The mixture was stirred at 180 rpm for 3 h. During the stirring process, 0.8 mol / L sodium hydroxide solution was added at a rate of 8 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

[0064] S5: Thaw 51 mg of human epidermal growth factor (hEGF) at 6°C for 8 min. After thawing, keep it in a refrigerator at 6°C for 20 min and stir at 130 rpm. Dilute the human epidermal growth factor with 7 mL of water for injection at 6°C and add it to the mixture B at a rate of 7 mL / h. Then add water for injection at 6°C to a total volume of 1000 mL and stir at 110 rpm for 50 min to obtain the mixture C.

[0065] S6: The mixture C was aseptically filtered using a filter (polyethersulfone material) with a pore size of 0.30μm. During filtration, sterile nitrogen gas of 0.12MPa was introduced for low-pressure filtration, and the filtration rate was 80mL / min to obtain a sterile drug solution. The filtered sterile drug solution was collected in a sterile storage tank at 18℃ and sterilized with saturated steam at 121℃ for 20min to obtain the hEGF sustained-release gel formulation.

[0066] S7: Use a pre-sterilized (121℃ saturated steam sterilization for 15-30 min) aseptic filling machine for filling; before filling, rinse the filling head, pipes and other parts of the filling machine that come into contact with the drug solution with sterile physiological saline, the rinsing volume is 3-5 times the pipe volume; during filling, control the filling rate at 30 mL / min, and control the total filling time for each batch to 8 hours (to avoid the aseptic state of the drug solution being affected by long-term operation); after filling, immediately aseptically seal the preparation container, the sealing time is controlled to 3-5 seconds.

[0067] Comparative Example 1: The difference between this comparative example and Example 3 is that the mass of borneol in S4 is 8g.

[0068] Comparative Example 2: The difference between this comparative example and Example 3 is that the mass of the poloxamer 407 solution in S1 is 280g.

[0069] Comparative Example 3: The difference between this comparative example and Example 3 is that the mixture A is a poloxamer 407 solution and does not contain sodium carboxymethyl cellulose solution.

[0070] Comparative Example 4: This comparative example differs from Example 3 in that the mass of poloxamer 407 solution in S1 is 280g, the mass of borneol in S4 is 8g, and the mixture A does not contain sodium carboxymethyl cellulose solution.

[0071] Experimental Example 1: Detection of the retention rate of topical human epidermal growth factor (hEGF) activity;

[0072] Cells: Balb / c 3T3 fibroblasts were selected;

[0073] Culture medium: DMEM high glucose medium (with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin added, stored at 4℃, and preheated to 37℃ before use);

[0074] Standard: hEGF standard (activity known, concentration 100 μg / mL, stored at -80℃, diluted with culture medium to a gradient of 0, 0.5, 2, 10, 20 ng / mL);

[0075] Test samples: hEGF sustained-release gel formulations prepared in the embodiments and comparative examples of this invention (taken out at 2-8℃ and equilibrated at room temperature for 30 min).

[0076] Weigh 1.0g of the gel to be tested, add 9mL of sterile PBS, vortex for 3min until the gel is dispersed, centrifuge at 3000rpm for 8min, take the supernatant (i.e. the original sample solution), dilute the original sample solution 100 times with culture medium (referred to as the test solution), and store in an ice bath;

[0077] Remove the Balb / c 3T3 cell cryovials and thaw them rapidly in a 37°C water bath. Transfer the thawed cell suspension to a 15mL sterile centrifuge tube, add 5mL of DMEM medium containing 10% FBS, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend the cells in 5mL of fresh medium, and transfer to a culture flask (25cm). 2 The cells were placed in a CO2 incubator (37℃, 5% CO2) and cultured for 24 hours. The culture medium was changed after each incubation. When the cells reached the logarithmic growth phase, they were passaged. During passage, the culture medium was aspirated from the culture flask, and the cells were washed twice with sterile PBS. 1 mL of 0.25% trypsin-EDTA solution was added, and the cells were incubated at 37℃ for 2-3 minutes until cell detachment. 5 mL of culture medium was added to stop digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM medium containing 10% FBS. Cells were counted using a counting chamber, and the cell concentration was adjusted to 5 × 10⁶ cells / mL. 4 pcs / mL, for later use;

[0078] Add 100 μL of adjusted cell suspension (approximately 5 × 10⁶ cells per well) to each well of a 96-well cell culture plate. 3 Add 100 μL of sterile culture medium to the edge wells (columns 1 and 12), and the remaining wells are experimental wells. Place the 96-well plate in a CO2 incubator and incubate at 37°C and 5% CO2 for 24 h.

[0079] The hEGF standard was diluted to the following concentration gradient using DMEM medium containing 2% FBS:

[0080] Seven concentrations were tested: 0 ng / mL (blank control), 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL.

[0081] Discard the original culture medium in the 96-well plate (retain the edge wells), add standard solutions of different concentrations to the experimental wells, set up 3 replicates for each concentration, 100 μL per well, and mark the area of ​​standard wells as the standard group.

[0082] Add the test sample solution to the remaining experimental wells of the 96-well plate, with 3 replicates per sample, 100 μL per well (denoted as the sample group). Set up a matrix control group: take a blank gel without hEGF (prepared according to Example 3, only without hEGF), process and dilute it as described above, and add it to the corresponding well, 100 μL per well, 3 replicates. Set up a blank control group: add 100 μL of DMEM medium containing 2% FBS to the corresponding well, 3 replicates. Place the 96-well plate back in the CO2 incubator and incubate at 37°C and 5% CO2 for 48 h.

[0083] After incubation for 48 hours, remove the 96-well plate, aspirate the culture medium from each well in a clean bench, add 100 μL of medium containing MTT (final MTT concentration 0.5 mg / mL, diluted in advance with DMEM containing 2% FBS) to each well, and return it to the CO2 incubator for further incubation at 37°C and 5% CO2 for 4 hours.

[0084] After incubation for 4 hours, remove the 96-well plate, aspirate the MTT solution from each well, add 150 μL of sterile DMSO to each well, place it on a shaker (100 rpm) and shake for 10 minutes. Immediately place the 96-well plate into a microplate reader, use 490 nm as the detection wavelength and 630 nm as the reference wavelength, measure the absorbance (OD value) of each well, and record the OD data of all wells.

[0085] Calculate the average OD value for each group;

[0086] Standard group: The average OD value of three replicates for the same concentration is recorded as the OD standard;

[0087] Sample group: The average OD value of three replicates of the same sample is taken as OD sample;

[0088] Matrix control group: The average OD value of the three replicates was taken as OD matrix;

[0089] Blank control group: The average OD value of the three replicates is recorded as OD blank;

[0090] Correct OD value:

[0091] Standard group calibrated OD value = OD standard - OD blank;

[0092] Corrected OD value for sample group = OD sample - OD basis;

[0093] Plotting a standard curve: Plot the hEGF standard concentration (ng / mL) on the x-axis and the corresponding corrected OD value on the y-axis. Using linear regression analysis, the standard curve equation is obtained as: y = ax + b (where a is the slope and b is the intercept). The correlation coefficient R0 is required. 2 ≥0.98;

[0094] Calculate the concentration of active hEGF in the sample: Substitute the corrected OD value of the sample group into the standard curve equation to calculate the concentration of active hEGF in the test sample solution, denoted as Csample (ng / mL); Calculate the concentration of active hEGF in the original sample solution according to the dilution factor (denoted as n) described above:

[0095] Cstock solution = Csample × n (ng / mL), then convert to μg / g (1g sample corresponds to 10mL stock solution, i.e., Cstock solution (ng / mL) × 10mL / 1000 = μg / g).

[0096] Calculate the theoretical hEGF concentration (Ctheoretical): Based on the amount of hEGF added in the preparation process, for example, in Example 3, 50 mg hEGF was added to 1000 mL of gel, and the gel density was calculated as 1 g / mL. The theoretical concentration Ctheoretical = 50 mg / 1000 g = 50 μg / g.

[0097] Calculate the activity retention rate:

[0098] Activity retention rate (%) = (actual active hEGF concentration in the sample / Ctherapeutic) × 100%.

[0099] For the same batch of samples, at least three parallel tests should be performed, and the average of the three activity retention rates should be taken as the final result, which should be retained to one decimal place.

[0100] Experiment Example 2: Temperature Sensitivity Test;

[0101] For each set of examples and comparative examples, take 3 gel samples to be tested, weigh 2.0g of each sample accurately (error ≤ 0.02g), and add them to 3 10mL sterile transparent centrifuge tubes (labeled as sample tubes 1, 2, and 3, for parallel experiments). Place the 3 sample tubes in a constant temperature environment of 25±1℃.

[0102] Remove the three sample tubes, which have been equilibrated to 25±1℃, from the constant temperature environment. Set the temperature of the constant temperature water bath to 37±0.5℃. After the water temperature stabilizes (fluctuation ≤0.2℃), add an appropriate amount of sterile saline to the water bath (the water level should be 1cm higher than the sample liquid level in the sample tubes to ensure uniform heating of the samples). Place the three sample tubes (sample tubes 1, 2, and 3) vertically on the support of the constant temperature water bath, so that the liquid level in the sample tubes is flush with the water bath liquid level. Insert a thermocouple temperature sensor into the middle of the sample in sample tube 1 (rinse the sensor with sterile saline beforehand to avoid contaminating the sample). Connect the data logger to monitor the internal temperature of the sample in real time, ensuring that the sample temperature can rise to 37±0.5℃.

[0103] When the internal temperature of sample tube 1 stabilizes at 37±0.5℃, immediately start the stopwatch (marked as the start point of the transition timing). Every 3 seconds, remove sample tube 1, quickly invert it 180°, and observe whether the sample flows. If the sample still flows along the tube wall, immediately put the sample tube back into the water bath and continue timing. If the sample does not flow within 1 minute of inversion (no liquid residue on the tube wall and the sample maintains a fixed shape), immediately stop the stopwatch and record the time (marked as the transition time T). Measure the transition times of sample tubes 2 and 3 in the same way, and take the average of 3 measurements (if the deviation of a single measurement from the average value is >20%, the sample tube needs to be measured again to eliminate operational errors).

[0104] The three sample tubes that have formed gels were left in a constant temperature water bath at 37±0.5℃ for 1 hour (simulating the continuous body temperature environment of the wound). Every 15 minutes, the sample tubes were taken out and inverted for observation. If the gel always maintained a fixed shape and there was no flow, collapse or liquid exudation after 1 minute of inversion, it was determined that the gel stability at body temperature was qualified. If the gel collapsed, flowed or exuded liquid, it was determined that the stability was unqualified and it could not continue to play a role in the wound.

[0105] The results are shown in the table below:

[0106] project Activity retention rate (%) Transition time T(s) stability Example 1 96.1 12 No flow, no collapse, no liquid seepage Example 2 96.5 12 No flow, no collapse, no liquid seepage Example 3 96.4 12 No flow, no collapse, no liquid seepage Example 4 96.2 12 No flow, no collapse, no liquid seepage Comparative Example 1 96.3 12 Slight flow and collapse, no liquid seepage Comparative Example 2 95.8 15 No flow, slight collapse, no liquid seepage Comparative Example 3 92.5 12 No flow, no collapse, but a small amount of liquid seepage. Comparative Example 4 91.7 15 Slightly flowing and collapsing, with a small amount of liquid seeping out.

[0107] As shown in the table above, the hEGF sustained-release gel formulation prepared in the embodiments of the present invention exhibits good consistency and reliability in the three core technical indicators of activity retention, thermosensitive transition characteristics, and structural stability at body temperature. This indicates that the raw material dosage, mixing process parameters, and component combination scheme set in the embodiments can effectively ensure the overall performance stability of the formulation.

[0108] In contrast, when the amount of a single key raw material deviates from the design range of the example during the preparation process (such as excessive use of borneol or poloxamer 407), the structural stability of the formulation at body temperature will decrease to varying degrees, and in some cases, the thermosensitive transition rate will be slowed down. When the core component (such as sodium carboxymethyl cellulose) is missing, not only will the activity retention capacity of the formulation be significantly reduced, but liquid leakage problems will also occur at body temperature. If both the deviation in the amount of raw material and the absence of the core component exist simultaneously, the activity retention capacity, thermosensitive transition characteristics and structural stability of the formulation will be negatively affected at the same time, and the performance degradation will be more obvious.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a highly stable hEGF sustained-release gel for external use, characterized in that, Includes the following steps: S1: Add 180-210g of poloxamer 407 to water and stir to obtain a poloxamer 407 solution; S2: Add 48-52g of sodium carboxymethyl cellulose to water and stir to obtain a sodium carboxymethyl cellulose solution; S3: Add sodium carboxymethyl cellulose solution to poloxamer 407 solution and stir to obtain mixture A; S4: Add 1-3g of borneol to water and stir to obtain a borneol solution; First, cool the mixture to 15-20℃, add the borneol solution to mixture A, add water to make up to 800mL, and continue stirring. During stirring, add 0.1-1mol / L sodium hydroxide solution to adjust the pH to 6.5-7.0 to obtain mixture B. S5: Thaw 49-52mg of human epidermal growth factor, store it at low temperature after thawing, dilute it with water and add it to mixture B, add water to make up the total volume of 1000mL, stir to obtain mixture C; S6: Filter and sterilize to obtain hEGF sustained-release gel formulation; S7: Filling.

2. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S1 is specifically as follows: Take 180-210g of poloxamer 407 and add it to 240-300mL of water for injection at 2-8℃ at a rate of 5-15g / min. Stir at 150-250rpm for 3-6h to obtain a poloxamer 407 solution.

3. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S2 is specifically as follows: Take 48-52g of sodium carboxymethyl cellulose and add it to 160-220mL of water for injection at 80-90℃ at a rate of 8-20g / min. Stir at 300-400rpm for 3-6h and cool down to 15-20℃ at 2-4℃ / min to obtain a sodium carboxymethyl cellulose solution.

4. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S3 specifically involves adding a sodium carboxymethyl cellulose solution to a poloxamer 407 solution at a rate of 100-200 mL / min, and stirring at 150-250 rpm for 1-2 hours at 3-8°C to obtain mixture A.

5. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S4 is specifically as follows: Take 1-3g of borneol and add it to 80-120mL of water for injection at 80-90℃ at a rate of 0.2-0.5g / min. Stir at 200-300rpm for 1-2h to obtain a borneol solution. Cool the mixture to 15-20℃ at a rate of 2-4℃ / min, reduce the stirring speed to 150-250 rpm, add the borneol solution to mixture A at a rate of 80-150 mL / h, add water for injection to bring the volume to 800 mL, and continue stirring at a speed of 150-250 rpm for 1-3 h. During stirring, add 0.1-1 mol / L sodium hydroxide solution at a rate of 5-15 mL / h to adjust the pH value to 6.5-7.0, thus obtaining mixture B.

6. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, Specifically, S5 involves: thawing 49-52 mg of human epidermal growth factor at 2-8℃ for 5-15 minutes, then storing it in a refrigerator at 2-8℃ for 15-25 minutes while stirring at 100-150 rpm. The human epidermal growth factor is then diluted with 5-10 mL of water for injection at 2-8℃ and added to mixture B at a rate of 5-10 mL / h. Water for injection at 2-8℃ is then added to bring the total volume to 1000 mL, and the mixture is stirred at 100-150 rpm for 30-60 minutes to obtain mixture C.

7. The method for preparing a highly stable hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S6 specifically involves aseptically filtering the mixture C using a filter with a pore size of 0.22-0.30 μm. During filtration, sterile nitrogen gas at 0.08-0.12 MPa is introduced for low-pressure filtration, with a filtration rate of 50-100 mL / min, to obtain a sterile drug solution. The filtered sterile drug solution is then collected in a sterile storage tank at 18-20℃ and sterilized with saturated steam at 121℃ for 15-30 min to obtain the hEGF sustained-release gel formulation.

8. The method for preparing a high-stability hEGF sustained-release gel topical formulation according to claim 1, characterized in that, S7 specifically refers to filling, controlling the filling rate at 10-30 mL / min, controlling the total filling time for each batch at 6-8 hours, and immediately aseptically sealing the formulation container after filling, with the sealing time controlled at 3-5 seconds.

9. An hEGF sustained-release gel formulation prepared by the preparation method according to any one of claims 1-8.