Acidic fibroblast growth factor ophthalmic solution

Abstract

This is a recombinant human acidic fibroblast growth factor preparation, and in particular, an acidic fibroblast growth factor eye drop formulation. The eye drop formulation uses a combination of histidine, polysorbate 80, and sodium heparin as a protein protectant, eliminating the need to use human serum albumin as a protein protectant. This not only reduces costs but also effectively improves product stability, maintaining high biological activity for up to 9 months under conditions of (25±2)°C and a relative humidity of (60±5)%.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and in particular to a stable acidic fibroblast growth factor preparation. [Background technology]

[0002] Fibroblast growth factors (FGFs) are multifunctional and powerful cell factors that play an important role in promoting fibroblast metabolism and collagen formation. They can promote the proliferation of skin tissue, regulate the division, proliferation, and differentiation of epithelial, endothelial, and stromal cells by binding to specific receptors on the cell surface, promote cell metabolism, enhance oxidative activity, promote the rapid proliferation of cells associated with skin damage, regulate the synthesis, secretion, and degradation of intercellular matrix, promote the regeneration of keratinocytes, accelerate the repair of the stratum corneum and basement membrane of the skin, promote the proliferation of human skin cells, and enhance the protein synthesis and cell metabolism of skin cells, delay the aging of skin cells, and promote the repair and proliferation of epidermal cells.

[0003] Fibroblast growth factor ophthalmic solution promotes repair and regeneration in cells derived from mesoderm and ectoderm. Animal testing demonstrated that this product promotes the regeneration of alkali-burned corneal epithelium in rabbits, as well as the repair of the corneal basement membrane and endothelium. Commercially available products include acidic fibroblast growth factor (aFGF or FGF-1) lyophilized powder and alkaline fibroblast growth factor (bFGF or FGF-2) ophthalmic solution. Lyophilized aFGF powder must be stored and transported at 2-8°C, protected from light, and has a shelf life of approximately 24 months. Upon use, the solvent provided in the packaging is poured into a bottle containing rhaFGF lyophilized powder. bFGF ophthalmic solution can be used directly, but must be stored at 2-8°C, has a shelf life of approximately 24 months, and must be used promptly once stored at room temperature. Therefore, research into stabilizers for aFGF and the development of stable aFGF drug compositions are important for improving patient compliance and convenience. There is a strong need in the art to provide a liquid formulation that can maintain the stability of acidic fibroblast growth factor over an extended period of time. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention provides a stable acidic fibroblast growth factor preparation, its preparation method, and applications. By studying each component of the preparation, particularly by selecting and combining different stabilizers and comparing the mixing ratios of each stabilizer, and conducting repeated experiments, a stable preparation containing acidic fibroblast growth factor is obtained.

[0005] The present invention provides a human acidic fibroblast growth factor ophthalmic solution comprising a protein protecting agent, which is a combination selected from histidine, polysorbate 80, and sodium heparin, a moisturizer, a buffer salt, and an osmolality adjusting agent.

[0006] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, which contains 0.01 to 0.2 g of histidine, 0.01 to 0.2 g of polysorbate 80, and 0.005 to 0.1 g of heparin sodium per 100 ml of the ophthalmic solution, and preferably contains 0.1 to 0.2 g of histidine, 0.03 to 0.1 g of polysorbate 80, and 0.01 to 0.05 g of heparin sodium per 100 ml of the ophthalmic solution.

[0007] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution containing 1 to 3 mg of human acidic fibroblast growth factor, preferably 2 mg of human acidic fibroblast growth factor, per 100 ml of the ophthalmic solution.

[0008] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, wherein the humectant is one or more selected from the group consisting of sodium hyaluronate, chondroitin sulfate, polyvinyl alcohol, polyethylene glycol, glycerol, hydroxypropylmethylcellulose, dextran, and carboxymethylcellulose, and the ophthalmic solution contains 0.05 to 1.5 g of the humectant per 100 ml, preferably 0.1 g of the humectant per 100 ml.

[0009] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, wherein the buffer salt is selected from a phosphate buffer system, a histidine buffer system, an acetate buffer system, a citrate buffer system, a Tris buffer system, and a HEPES buffer system, and the buffer salt preferably has a pH of 6.0 to 7.0 and a concentration of 0.01 to 0.05 mol / L, preferably 0.02 mol / L.

[0010] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, wherein the osmolality regulator is one or more selected from the group consisting of sodium chloride, boric acid, and glucose, and the ophthalmic solution preferably contains 0.5 to 0.9 g of the osmolality regulator per 100 ml of the solution.

[0011] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution further comprising a bacteriostatic agent.

[0012] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, wherein the bacteriostatic agent is one or more selected from the group consisting of ethyl hydroxybenzoate, benzalkonium chloride, benzalkonium bromide, polyquaternary ammonium salts, phenylmercuric nitrate, thimerosal, benzalkonium chloride, trichloro-tert-butyl alcohol, parabens, and sorbitol, and the ophthalmic solution preferably contains 0.01 to 0.1 g of the bacteriostatic agent per 100 ml.

[0013] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, which further contains one or more antioxidants selected from sodium thiosulfate, sodium sulfite, vitamin E, sodium ascorbate, and methionine, preferably in an amount of 0.01 to 0.1 g per 100 ml of the ophthalmic solution.

[0014] The present invention provides a recombinant human acidic fibroblast growth factor ophthalmic solution, which further comprises a thickener, preferably one or more selected from polyvinyl alcohol, carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, sodium polypropyleneate, and polyvinylpyrrolidone, and preferably contains 0.5 to 2 g of the thickener per 100 ml of the ophthalmic solution.

[0015] The present invention has the following beneficial effects.

[0016] The ophthalmic solution of the present invention uses a combination of histidine, polysorbate 80, and sodium heparin as a protein protectant, which effectively improves the stability of the product and maintains high biological activity even after 9 months under conditions of (25±2)°C and relative humidity (60±5)%.

[0017] The composite protein protection agent used in the present invention does not require the use of expensive human serum albumin, and effectively reduces production costs while ensuring product quality.

[0018] Through screening, the present invention helps to find multiple preferred combinations of bacteriostatic agents and antioxidants to synergistically improve the stability of the product. DETAILED DESCRIPTION OF THE INVENTION

[0019] In order to better understand the present invention, the following examples are given to illustrate, but not to limit the protection scope of the present invention.

[0020] Example 1: This example provides recombinant human acidic fibroblast growth factor preparation I. Its ingredients and preparation method are as follows:

[0021] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Polysorbate 80 0.05g Heparin sodium 0.02g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0022] A 100ml ophthalmic solution was prepared. Specifically, the moisturizer sodium hyaluronate, the protein protectant histidine, polysorbate 80, sodium heparin, the thickener polyvinyl alcohol, the preservative trichlorotertiary butyl alcohol, and the antioxidant methionine were mixed in the above ratios in 80ml phosphate buffer (0.02mol / L), 0.9g sodium chloride was added to completely dissolve, and then human acidic fibroblast growth factor was added. After uniform mixing, a 0.02mol / L phosphate buffer solution at pH 6.5 was added to the desired volume, and the mixture was sterilized by filtering through a 0.22µm micropore filter. The filtrate was dispensed into PET bottles to prepare the finished ophthalmic solution of the present invention.

[0023] Example 2: The preparation method of this example is the same as that of Example 1, and the specific ingredients are as follows:

[0024] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Polysorbate 80 0.05g Heparin sodium 0.05g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Sodium thiosulfate 0.05g Polyvinyl alcohol 1g

[0025] Example 3: The preparation method of this example is the same as that of Example 1, and the specific ingredients are as follows:

[0026] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Polysorbate 80 0.05g Heparin sodium 0.05g Sodium chloride 0.45g Methyl parahydroxybenzoate 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0027] Example 4: The preparation method of this example is the same as that of Example 1, and the specific ingredients are as follows:

[0028] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Polysorbate 80 0.05g Heparin sodium 0.05g Sodium chloride 0.45g Sodium benzoate 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0029] Comparative Example 1: The preparation method of this comparative example was the same as that of Example 1, and the specific ingredients were as follows:

[0030] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Polysorbate 80 0.05g Heparin sodium 0.05g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0031] Comparative Example 2: The preparation method of this comparative example was the same as that of Example 1, and the specific ingredients were as follows:

[0032] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Heparin sodium 0.05g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0033] Comparative Example 3: The preparation method of this comparative example was the same as that of Example 1, and the specific ingredients were as follows:

[0034] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Polysorbate 80 0.05g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Methionine 0.05g Polyvinyl alcohol 1g

[0035] Comparative Example 4: The preparation method of this comparative example was the same as that of Example 1, and the specific ingredients were as follows:

[0036] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Heparin sodium 0.05g Human serum albumin 0.1g Sodium chloride 0.45g Methionine 0.05g Polyvinyl alcohol 1g

[0037] Comparative Example 5: The preparation method of this comparative example was the same as that of Example 1, and the specific ingredients were as follows:

[0038] Human acidic fibroblast growth factor 2mg Sodium hyaluronate 0.1g Histidine 0.15g Human serum albumin 0.1g Sodium chloride 0.45g Trichlorotertiary butyl alcohol 0.05g Polyvinyl alcohol 1g

[0039] Example of effectiveness: Accelerated stability testing The eye drops prepared in Examples 1 to 4 and Comparative Examples 1 to 4 were stored for 9 months under conditions of (25±2)° C. and relative humidity (60±5)%, and then the activity test was carried out as follows.

[0040] (1) Preparation of sample solution: Recombinant human acidic fibroblast growth factor sample was reconstituted under sterile conditions according to the manufacturer's instructions and diluted to a concentration of 40 IU per ml. The solution was serially diluted four-fold in a 96-well cell culture plate to create eight dilutions, each of which was placed in duplicate wells.

[0041] (2) Preparation of test substance solution: The dilution and treatment methods are the same as for the standard sample.

[0042] (3) The above sample was immediately subjected to the measurement in the following step (4).

[0043] (4) Activity measurement: Balb / c3T3 cells were cultured in complete culture medium at 37°C and 5% CO2 until the cell concentration reached 1.0 x 10 5 ~5.0×10 5 The culture medium in the culture bottle was discarded, and the cells were collected by digestion and diluted to 5.0 × 10 cells / ml in complete culture medium. 4 ~1.3×10 5 A cell suspension was prepared at 100 μL / ml and inoculated into a 96-well cell culture plate, with each well filled at 100 μL. The plate was then cultured at 37°C and 5% CO2 for 24 hours. The suspension was then replaced with maintenance medium and cultured at 37°C and 5% CO2 for 28–32 hours. The maintenance medium was discarded from the plate, and the standard solution and test substance were added, with each well filled at 100 μL. The plate was then cultured at 37°C and 5% CO2 for 64–72 hours. After incubation, 20 μL of MTT solution was added to each well, and the plate was cultured at 37°C and 5% CO2 for 5–6 hours. After mixing, 100 μL of cell lysate was added to each well, mixed evenly, and then placed in a microplate reader. The absorbance was measured at 570 nm with a reference wavelength of 630 nm, and the results were recorded.

[0044] Calculation of results: The test data was processed using a computer program or linear regression algorithm, and the results were calculated using the following formula: Biological activity of test substance (IU / ml) = Pr × Ds × Es / (Dr × Er) Relative activity (%) = Biological activity of test substance (after accelerated testing) / Biological activity of test substance (stability 0 points)

[0045] In the formula, Pr is the biological activity of the preparation in IU / ml, Ds is the pre-dilution factor of the test substance, Dr is the pre-dilution factor of the preparation, Es is the dilution factor of the test substance equivalent to the half-effective dose of the preparation, and Er is the half-effective dilution factor of the preparation. The relative activity (%) measured after the accelerated test is shown in Table 1.

[0046] [Table 1]

[0047] The above data indicate that the combination of histidine, polysorbate 80, and sodium heparin is a protein-protecting agent combination that helps improve the stability of human acidic fibroblast growth factor under accelerated test conditions. Regarding the effects of antioxidants and preservatives on stability, the combination of trichlorotertiary butyl alcohol and methionine in Example 1 is the optimal embodiment, while the effects of other preservatives and antioxidants are somewhat poor.

[0048] Based on the above, it cannot be recognized that the specific implementation of the present invention is limited to these descriptions, and various simple simulations or substitutions that a person skilled in the art can make without departing from the concept of the present invention should all be considered to belong to the protection scope of the present invention, which is determined by the scope of the appended claims.

Claims

1. A recombinant human acidic fibroblast growth factor ophthalmic solution comprising a protein protecting agent, a moisturizing agent, a buffer salt, and an osmotic pressure adjusting agent, wherein the protein protecting agent is a combination selected from the group consisting of histidine, polysorbate 80, and sodium heparin.

2. 2. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, comprising 0.01 to 0.2 g of histidine, 0.01 to 0.2 g of polysorbate 80, and 0.005 to 0.1 g of heparin sodium per 100 ml of the ophthalmic solution, preferably 0.1 to 0.2 g of histidine, 0.03 to 0.1 g of polysorbate 80, and 0.01 to 0.05 g of heparin sodium.

3. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, characterized in that it contains 1 to 3 mg of human acidic fibroblast growth factor, preferably 2 mg of human acidic fibroblast growth factor, per 100 ml of the ophthalmic solution.

4. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, wherein the humectant is one or more selected from the group consisting of sodium hyaluronate, chondroitin sulfate, polyvinyl alcohol, polyethylene glycol, glycerol, hydroxypropyl methylcellulose, dextran, and carboxymethylcellulose, and the humectant is contained in an amount of 0.01 to 1.5 g per 100 ml of the solution, preferably 0.1 g per 100 ml of the solution.

5. 2. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, wherein the buffer salt is selected from the group consisting of a phosphate buffer system, a histidine buffer system, an acetate buffer system, a citrate buffer system, a Tris buffer system, and a HEPES buffer system, and the buffer salt preferably has a pH of 6.0 to 7.0 and a concentration of 0.01 to 0.05 mol / L, preferably 0.02 mol / L.

6. 2. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, wherein the osmotic pressure regulator is one or more selected from the group consisting of sodium chloride, boric acid, and glucose, and the ophthalmic solution preferably contains 0.3 to 0.9 g of the osmotic pressure regulator per 100 ml of the solution.

7. The recombinant human acidic fibroblast growth factor ophthalmic solution of claim 1, further comprising a bacteriostatic agent.

8. 8. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 7, wherein the bacteriostatic agent is one or more selected from the group consisting of ethyl hydroxybenzoate, benzalkonium chloride, benzalkonium bromide, polyquaternary ammonium salts, phenylmercuric nitrate, thimerosal, benzalkonium chloride, trichloro-tert-butyl alcohol, parabens, and sorbitol, and the amount of the bacteriostatic agent is preferably 0.01 to 0.1 g per 100 ml of the solution.

9. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, further comprising an antioxidant.

10. 10. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 9, wherein the antioxidant is one or more selected from the group consisting of sodium thiosulfate, sodium sulfite, vitamin E, sodium ascorbate, and methionine, and the antioxidant is preferably contained in an amount of 0.01 to 0.1 g per 100 ml of the ophthalmic solution.

11. 2. The recombinant human acidic fibroblast growth factor ophthalmic solution according to claim 1, further comprising a thickener, preferably one or more selected from the group consisting of polyvinyl alcohol, carboxymethyl cellulose, methyl cellulose, sodium carboxymethyl cellulose, sodium polypropyleneate, and polyvinylpyrrolidone, and preferably containing 0.5 to 2 g of the thickener per 100 ml of the ophthalmic solution.

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