Stable epidermal growth factor composition and application thereof
By combining EGF with buffer salts and stabilizers to form a stable EGF composition, the problem of degradation of EGF in aqueous formulations is solved, and EGF maintains activity and effectiveness in long-term storage, which is suitable for the treatment of wound healing.
Patent Information
- Application Number
- CN202311678509.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-08
- Publication Date
- 2025-06-10
AI Technical Summary
In the prior art, epidermal growth factor (EGF) is prone to degradation in the presence of water, resulting in a short storage time of aqueous formulations and is unable to be effectively used for long-term wound healing.
A stable EGF composition is formed by combining EGF with a buffer salt (such as phosphate) and a stabilizer (such as sucrose). The composition can be stored at room temperature for two years and remains active after being left at 37°C in a light for 60 days.
The EGF composition maintains the stability and activity of EGF in long-term storage, can effectively promote the healing of wound wounds, and is suitable for the treatment of various surgical wounds, physical trauma, acute and chronic ulcers, burns and scalds and other wounds.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to a stable epidermal growth factor (EGF) composition and its application in treating wound healing. Background Art
[0002] Epidermal growth factor (EGF) consists of 53 amino acids and has three pairs of disulfide bonds in the molecule. It is a polypeptide that promotes cell proliferation, differentiation, and migration. EGF binds to receptors on the cell surface to carry out a series of reactions, thereby stimulating cell proliferation, differentiation, and migration, and participating in a series of physiological processes and embryonic development, tissue regeneration, and wound healing, etc. At present, human epidermal growth factor (hEGF) has been widely used in medical and other fields, and has been used in the treatment of wound healing of various surgical wounds, wounds caused by various ulcers, and ophthalmology, etc.
[0003] However, EGF is prone to degradation and loss of activity in the presence of moisture, which makes aqueous preparations containing EGF unable to be stored for a long time. Therefore, there is an urgent need to develop a composition containing EGF that can be effectively used for wound healing and has good stability. Summary of the Invention
[0004] The primary object of the present invention is to overcome the deficiencies of the prior art and provide a stable epidermal growth factor (EGF) composition. The composition has a simple formulation, and EGF can still maintain effective activity after being placed at room temperature for two years.
[0005] Another object of the present invention is to provide the application of the stable EGF composition in the preparation of drugs for treating various wound healings.
[0006] The object of the present invention is achieved by the following technical solutions: A stable epidermal growth factor composition, comprising epidermal growth factor (EGF), buffer salt, and stabilizer; wherein, the buffer salt is phosphate or citrate or acetate, and the stabilizer is sucrose.
[0007] As one of the implementation schemes, the buffer salt is phosphate, selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, or potassium dihydrogen phosphate, preferably disodium hydrogen phosphate.
[0008] As one of the implementation schemes, the composition further comprises a preservative, and the preservative is at least one of sodium methyl paraben, sodium propyl paraben, ethyl paraben, butyl paraben, phenol, triclosan, m-cresol, phenoxyethanol, 1,2-hexanediol, preferably methyl paraben and propyl paraben.
[0009] As one of the embodiments, the pH of the composition is 6.0 - 8.5, preferably 6.5 - 8.0. By way of example, it includes but is not limited to 6.5, 7.0, 7.5, 8.0.
[0010] As one of the embodiments, the composition is in terms of weight - volume percentage (w / v),
[0011] the content of EGF is 0.001 - 0.015%, preferably 0.005 - 0.015%;
[0012] the content of phosphate is 0.01 - 0.5%, preferably 0.1%;
[0013] the content of sucrose is 1 - 8%, preferably 2 - 5%.
[0014] As one of the embodiments, the composition is in terms of weight - volume percentage (w / v),
[0015] the content of methyl paraben is 0.02 - 0.3%, preferably 0.18%;
[0016] the content of propyl paraben is 0.01 - 0.1%, preferably 0.02%.
[0017] As one of the embodiments, the composition further contains a pH regulator and water. The pH regulator includes but is not limited to dilute hydrochloric acid or sodium hydroxide solution.
[0018] As one of the preferred embodiments, the composition consists of the following components in weight - volume percentage (w / v): 0.005 - 0.015% EGF, 0.1% disodium hydrogen phosphate, 2 - 5% sucrose, appropriate amount of water, pH 6.5 - 8.0.
[0019] As one of the preferred embodiments, the composition consists of the following components in weight - volume percentage (w / v): 0.005 - 0.015% EGF, 0.1% disodium hydrogen phosphate, 2 - 5% sucrose, 0.18% methyl paraben, 0.02% propyl paraben, appropriate amount of water, pH 6.5 - 8.0.
[0020] The application of the composition in the preparation of a drug for treating wound healing such as surgical wounds, physical trauma, acute and chronic ulcers, burns and scalds.
[0021] The composition of the present invention can be made into various external dosage forms, including but not limited to sprays, ointments, gels or eye drops.
[0022] A method for preparing the composition of the present invention comprises the following steps: Optionally weigh the preservative in the prescribed amount, add water and stir until dissolved and clarified; add the buffer salt and sucrose in the prescribed amount and stir until dissolved; add the EGF stock solution or lyophilized powder in the prescribed amount, stir until clarified, and adjust the pH to 6.5 - 8.0 with a pH regulator, and make up the volume to 100 ml with purified water.
[0023] The technical effects of the present invention are as follows:
[0024] The present invention is an unexpected result discovered by the inventor through a large number of experiments on various excipients in the early stage. The simultaneous presence of a buffer salt (such as disodium hydrogen phosphate) and a stabilizer (such as sucrose) can well solve the problem that the EGF aqueous preparation is easily degraded and loses its activity during long-term storage. Disodium hydrogen phosphate and sucrose, as excipients, have a synergistic effect on improving the stability of EGF.
[0025] After the composition of the present invention is placed in the dark at 37°C for 60 days, there is no precipitation or separation, the appearance is clear, and the stability of EGF is good; after being placed at 37°C for 90 days, the EGF content remains at about 90%, and it can be stored at room temperature for two years and still has effective activity.
[0026] The composition formula of the present invention is simple and can effectively promote the healing of wound surfaces. Detailed implementation manners
[0027] The following will describe the implementation schemes of the present invention in detail in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.
[0028] Those skilled in the art should understand that the reagents used in the following examples are all obtained by purchasing from the market unless otherwise specified.
[0029] The amino acid sequence of epidermal growth factor EGF described in the present invention is NSDSECPLSH DGYCLHDGVCMYIEALDKYA CNCVVGYIGE RCQYRDLKWW ELR. The EGF lyophilized powder can be obtained by carrier construction, fermentation purification, ultrafiltration and then vacuum freeze-drying. The specific preparation method refers to Pichia Fermentation Process Guidelines, Expression, purification, and characterization of the native intracellular domain of human epidermal growth factor receptors 1 and 2 in Escherichia coli.
[0030] Example 1
[0031] Prepare the EGF composition according to the ratio shown in Table 1:
[0032] Table 1. Formulations of Compositions 1 - 4
[0033]
[0034] Preparation method of Composition 1: Add 4.60 μL of EGF stock solution with a concentration of 3258.67 μg / ml to the preparation tank, add an appropriate amount of purified water and stir evenly, adjust the pH to 6.5 with 10% dilute hydrochloric acid or 10% sodium hydroxide solution, make up the volume to 100 ml with purified water, and confirm the pH is 6.5 after stirring, then a preparation with a final EGF concentration of 150 μg / ml is obtained.
[0035] Preparation method of Composition 2: Weigh 0.1 g of disodium hydrogen phosphate, add water and stir to dissolve, add 7.72 μL of EGF stock solution with a concentration of 1942.63 μg / ml, stir until clear, then adjust the pH to 6.5 with 10% dilute hydrochloric acid or 10% sodium hydroxide solution, make up the volume to 100 ml with purified water, and confirm the pH is 6.5 after stirring, then a preparation with a final EGF concentration of 150 μg / ml is obtained.
[0036] Preparation method of Composition 3: Weigh 0.1 g of disodium hydrogen phosphate, add water and stir to dissolve, add 9.26 μL of EGF stock solution with a concentration of 539.83 μg / ml, stir until clear, then adjust the pH to 6.5 with 10% dilute hydrochloric acid or 10% sodium hydroxide solution, make up the volume to 100 ml with purified water, and confirm the pH is 6.5 after stirring, then a preparation with a final EGF concentration of 50 μg / ml is obtained.
[0037] Preparation method of Composition 4: Weigh 0.1 g of disodium hydrogen phosphate and 5 g of sucrose, add water and stir to dissolve, add 9.26 μL of EGF stock solution with a concentration of 539.83 μg / ml, stir until clear, then adjust the pH to 6.5 with 10% dilute hydrochloric acid or 10% sodium hydroxide solution, make up the volume to 100 ml with purified water, and confirm the pH is 6.5 after stirring, then a preparation with a final EGF concentration of 50 μg / ml is obtained.
[0038] Place the prepared Compositions 1 and 2 at 40°C for accelerated experiments to examine the relative content of EGF. A total of 10 days are placed, and samples are taken at 0 day, 5 days, and 10 days respectively. Use octadecylsilane-bonded silica gel as the filler, 0.1% TFA-aqueous solution as mobile phase A, 0.1% TFA-acetonitrile solution as mobile phase B, the flow rate is 1 ml / min, the column temperature is 30°C, the detection wavelength is 280 nm, take 10 μl of the sample and inject it into the liquid chromatograph for detection. The data are shown in Table 2 below.
[0039] Table 2. Relative Contents of EGF in Compositions 1 and 2
[0040] 0 days 20 days 60 days Composition 1 94.80％ 73.45％ 69.11％ Composition 2 94.47％ 90.59％ 87.81％
[0041] The prepared Compositions 3 and 4 were respectively placed at 37°C for accelerated experiments to investigate the relative contents of EGF. They were placed for a total of 30 days, and samples were taken at 0 day, 10 days, 20 days, and 30 days. Octadecylsilane-bonded silica gel was used as the filler, 0.1% TFA-aqueous solution was used as mobile phase A, 0.1% TFA-acetonitrile solution was used as mobile phase B, the flow rate was 1 ml / min, the column temperature was 30°C, the detection wavelength was 280 nm, 10 μl of the sample was injected into the liquid chromatograph for detection, and the data are shown in Table 3 below.
[0042] Table 3. Relative Contents of EGF in Compositions 3 and 4
[0043] 0 days 10 days 20 days 30 days Composition 3 97.27％ 96.30％ 88.96％ 85.09％ Composition 4 98.01％ 97.61％ 96.94％ 96.75％
[0044] The experimental results in Table 2 and Table 3 show that when disodium hydrogen phosphate and sucrose are simultaneously added to the formulation, the two can synergistically improve the stability of EGF.
[0045] Example 2
[0046] Prepare the EGF composition according to the ratios shown in Table 4:
[0047] Table 4. Formulations of Compositions 5 - 9
[0048]
[0049] Preparation method: Weigh the methyl paraben and propyl paraben in the prescription amount, add an appropriate volume of 65°C hot water, stir and dissolve until clear, add the disodium hydrogen phosphate and sucrose in the prescription amount, stir and dissolve, after restoring to room temperature, add the EGF lyophilized powder in the prescription amount, stir until clear, and adjust the pH to 6.5 - 8.0 with 10% dilute hydrochloric acid or 10% sodium hydroxide solution, make up the volume to 100 ml with purified water, stir until clear, and confirm that the pH is 6.5 - 8.0.
[0050] The prepared Compositions 5 - 9 were respectively placed at 37°C for accelerated experiments to investigate the relative contents of EGF. They were placed for a total of 60 days, and samples were taken at 0 day, 20 days, and 60 days. Octadecylsilane-bonded silica gel was used as the filler, 0.1% TFA-aqueous solution was used as mobile phase A, 0.1% TFA-acetonitrile solution was used as mobile phase B, the flow rate was 1 ml / min, the column temperature was 30°C, the detection wavelength was 280 nm, 10 μl of the sample was injected into the liquid chromatograph for detection, and the data are shown in Table 5 below.
[0051] Table 5. Appearance of Compositions 5 - 9 and Relative Contents of EGF under Accelerated Experiments
[0052] 0 days 20 days 60 days Property Composition 5 100％ 96.93％ 90.52％ Clear Composition 6 100％ 97.75％ 94.66％ Clear Composition 7 100％ 99.97％ 97.49％ Clear Composition 8 100％ 99.04％ 91.95％ Clear Composition 9 100％ 97.83％ 89.43％ Clear
[0053] The test data show that after the composition solution containing 2 - 5% (w / v) sucrose and with a pH of 6.5 - 8.0 is placed in the dark at 37°C for 60 days, there is no precipitation or separation, the appearance is clear, and the EGF has good stability.
[0054] Example 3 Shelf - life Test
[0055] The prescriptions of Composition 6 were respectively prepared in parallel for three batches of samples to conduct the shelf - life test of the preparation prescription. According to the change of the EGF content over time under different storage conditions of the samples, its stability was evaluated to predict the shelf - life of the product.
[0056] Table 6. Stability Results of Three - batch Prescription Samples
[0057]
[0058] Referring to the "Disinfection Technical Specification", when the samples are stored at 37°C for 90 days and the degradation rate of the active ingredient content ≤ 10%, the storage shelf - life can be set as 2 years. According to the above results, after the three batches of samples are accelerated at 37°C for 90 days, the EGF contents are 89.92%, 90.02%, and 90.11% of the initial content, and the average degradation rate of the active ingredient content ≤ 10%, indicating that the samples have good stability.
[0059] Example 4 Efficacy Test of Animal Wound Model
[0060] 1. Selection of Animal Model
[0061] Three animals with skin wounds (2 cats and 1 dog) were selected in a qualified animal hospital (Aibejia Pet Hospital in the Economic Development Zone). One of the cats had two wounds, and there were a total of 4 wounds on the 3 animals, which were respectively marked as groups A, B, C, and D.
[0062] 2. Administration Method
[0063] The wounds in groups A, B, C, and D of the experiment were respectively treated with the preparation prescription of Composition 6 in Example 2 (where the EGF concentrations were 25 μg / ml, 50 μg / ml, 100 μg / ml, and 150 μg / ml) by spraying three times a day in the morning, noon, and evening, 2 - 3 sprays each time, and the healing situation was observed continuously for 3 - 14 days.
[0064] 3. Wound Healing Index
[0065] According to clinical treatment, it is divided into: cured, markedly effective, effective, and ineffective.
[0066] Recovery: After medication, the wound has completely healed, including no inflammatory reaction, no infection, no inflammation, no exudate from the wound, no swelling or redness, no hyperplasia, and no scar, with a neat wound edge, then it is considered a recovery.
[0067] Marked effect: After medication, the wound has significantly healed, including no inflammatory reaction, no infection, no inflammation, no hyperplasia, and no scar, with a neat wound edge, then it is considered a marked effect.
[0068] Effective: After medication, the wound has healed. The healing speed is not fast but faster than natural healing, including no inflammatory reaction, no infection, and no inflammation, then it is considered effective.
[0069] Ineffective: After medication, there is no difference in the healing speed of the wound compared to natural healing. The wound heals slowly and may have swelling, pain, exudate on the wound surface, and a risk of bacterial infection, then it is considered ineffective.
[0070] 4. Results
[0071] EGF Concentration Animal Experiment Results Evaluation Results 25 ug / ml Pharmacodynamic Effect Not Obvious Invalid 50 ug / ml Pharmacodynamic Effect Not Obvious Effective 100 ug / ml Pharmacodynamic Effect Obvious, Healing Accelerated Cured 150 ug / ml Pharmacodynamic Effect Obvious, Healing Accelerated Cured
[0072] Experiments show that within the range of EGF concentration from 50 μg / ml to 150 μg / ml, it has an effective healing effect on various surgical wound surfaces of pets. Within the range of 100 μg / ml to 150 μg / ml, it has an obvious promoting healing effect on various surgical wound surfaces of pets.
[0073] It can be understood that the above embodiments are merely exemplary embodiments adopted to illustrate the principle of the present invention. However, the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principle and essence of the present invention, and these modifications and improvements are also regarded as the protection scope of the present invention.
Claims
1. A stable epidermal growth factor composition, It is characterized in that It comprises epidermal growth factor (EGF), buffer salt and stabilizer; wherein the buffer salt is phosphate or citrate or acetate, and the stabilizer is sucrose.
2. The composition according to claim 1, It is characterized in that The buffer salt is a phosphate, selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate or potassium dihydrogen phosphate, preferably disodium hydrogen phosphate.
3. The composition according to claim 1, It is characterized in that The invention also comprises a preservative, which is at least one of sodium methylparaben, sodium propylparaben, ethylparaben, butylparaben, phenol, triclosan, m-cresol, phenoxyethanol and 1,2-hexanediol, preferably methylparaben and propylparaben.
4. The composition according to claim 1, It is characterized in that The pH is 6.0-8.5, preferably 6.5-8.
0.
5. The composition according to claim 1, It is characterized in that Calculated by weight volume percentage (w / v), The content of EGF is 0.001-0.015%, preferably 0.005-0.015%; The content of the buffer salt is 0.01-0.5%, preferably 0.1%; The content of sucrose is 1-8%, preferably 2-5%.
6. The composition according to claim 3, It is characterized in that Calculated by weight volume percentage (w / v), The content of methyl paraben is 0.02-0.3%, preferably 0.18%; The content of propyl paraben is 0.01-0.1%, preferably 0.02%.
7. The composition according to claim 5, It is characterized in that The invention comprises the following components in weight volume percentage (w / v): 0.005-0.015% EGF, 0.1% disodium hydrogen phosphate, 2-5% sucrose, appropriate amount of water, and pH 6.5-8.
0.
8. The composition according to claim 6, It is characterized in that The invention comprises the following components in weight volume percentage (w / v): 0.005-0.015% EGF, 0.1% disodium hydrogen phosphate, 2-5% sucrose, 0.18% methyl parahydroxybenzoate, 0.02% propyl parahydroxybenzoate, appropriate amount of water, and pH 6.5-8.
0.
9. Use of the composition according to any one of claims 1 to 8 in the preparation of a medicament for treating wound healing such as surgical wounds, physical trauma, acute and chronic ulcers, burns and scalds.
10. A composition according to any one of claims 1 to 8, It is characterized in that The dosage form is spray, ointment, gel or eye drops.