Recombinant human platelet-derived growth factor eye drops

Abstract

The invention provides a liquid composition containing recombinant human platelet-derived growth factors. The composition comprises the following components: polyvinyl alcohol, hydroxypropyl methyl cellulose, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, mannitol and the like. The invention also provides a preparation method of the composition and application of the composition in preparation of corneal injury treatment medicines. The eye drops prepared from the composition disclosed by the invention have good stability and safety, and have a good effect in repairing corneal injury.

Description

Technical Field

[0001] The present invention belongs to the field of biological products, and in particular, relates to recombinant human platelet-derived growth factor eye drops, a preparation process thereof, and an application thereof in the auxiliary treatment of corneal damage. Background Art

[0002] Platelet derived growth factor (PDGF) is a basic protein stored in platelet alpha granules. It is secreted by various types of cells including platelets, endothelial cells, epithelial cells, glial cells and inflammatory cells. It can stimulate fibroblasts, glial cells, smooth muscle cells and other cells that are stagnant in the G0 / G1 phase to enter the division and proliferation cycle. It is an effective mitogen. PDGFs have four monomers, A, B, C, and D, which are connected by disulfide bonds to form homo- or hetero-dimers (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-AB). After PDGF binds to its receptor PDGFR, it promotes the formation of two receptor molecules into dimers and activates the phosphorylation of tyrosine residues in the intracellular domain, thereby transmitting signals into the cell and regulating cell division, proliferation and other life activities through a cascade amplification waterfall effect.

[0003] The main biological characteristics of PDGF are: ① Chemotaxis: stimulates the chemotactic migration of fibroblasts, smooth muscle cells, neutrophils, and monocytes, which is extremely important for the body's damage repair; ② Mitogenicity: promotes DNA synthesis, cell lysis, and proliferation of various cells; ③ Increases the synthesis and accumulation of extracellular matrix (ECM): promotes the synthesis of various ECM components such as collagen fibers, integrins, fibronectin (FN), laminin (LN), mucopolysaccharides, etc., inhibits the degradation of ECM, and participates in the occurrence and development of tissue and organ fibrosis and sclerosis; ④ Vascular active effect: can induce contraction of different types of blood vessels.

[0004] The cornea is an important refractive medium of the eye. When the damage is serious, such as stromal layer damage or more serious corneal laceration, the repair time may be as long as 1-3 months. This long-term passive repair is prone to corneal infection and ulcers, and at the same time forms scars of varying degrees, or changes in corneal curvature, which seriously affect vision. Platelet-derived growth factor, as an effective mitogen, can promote the proliferation and division of various cells, chemotaxis of fibroblasts and immune cells, and accelerate the repair of damaged tissues and organs. These characteristics of platelet-derived growth factor give it great application potential in the repair of corneal damage in the eye, and it is expected to become a good drug for the treatment of corneal damage.

[0005] The purpose of the present invention is to provide a composition containing human platelet-derived growth factor, and further provide the use of the composition in preparing a drug for treating corneal injury. Summary of the invention

[0006] Based on the above purpose, the present invention first provides a liquid composition containing recombinant human platelet-derived growth factor, characterized in that the composition contains: Recombinant human platelet-derived growth factor 800-7000 IU / ml Thickener 0.1-2% (g / ml) Protein protective agent 0.05-3% (g / ml) Phosphate buffer 10-30 mM Osmotic pressure regulator 0.25-0.75% (g / ml) The pH of the liquid composition is 6.8-7.4, Wherein, the thickener is selected from one or more of polyvinyl alcohol, sodium hyaluronate, hydroxypropyl methylcellulose, and methylcellulose; the protein protectant is selected from one or more of glycine, glutamic acid, lysine, histidine, arginine, sucrose, trehalose, polyethylene glycol 400, and mannitol; and the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, or sorbitol.

[0007] In a preferred embodiment, the amount of recombinant human platelet-derived growth factor in the composition is 1000-6000 IU / ml.

[0008] In a preferred embodiment, the thickener is polyvinyl alcohol, and the amount of polyvinyl alcohol used is 0.5%-1.5%.

[0009] In a preferred embodiment, the thickener is hydroxypropyl methylcellulose, and the amount of hydroxypropyl methylcellulose is 0.1%-0.5%.

[0010] In a preferred embodiment, the protein protective agent is mannitol, and the dosage of mannitol is 0.05%-3%.

[0011] In a preferred embodiment, the osmotic pressure regulator is sodium chloride, wherein the amount of sodium chloride is 0.25%-0.75%.

[0012] In a more preferred embodiment, the amounts of the components in the composition are: Recombinant human platelet-derived growth factor 3000 IU / ml Polyvinyl alcohol 1% (g / ml) Hydroxypropyl methylcellulose 0.1% (g / ml) Mannitol 1% (g / ml) Sodium chloride 0.56% (g / ml) Phosphate buffer is 20 mM, pH 6.8-7.4.

[0013] Among them, the 20mM phosphate buffer system can be prepared by 0.52% (g / ml) disodium hydrogen phosphate dodecahydrate and 0.08% (g / ml) sodium dihydrogen phosphate monohydrate.

[0014] Secondly, the present invention provides a method for preparing the above-mentioned liquid composition, the method comprising the following steps: (1) Disperse the thickener polyvinyl alcohol in water for injection, sterilize by autoclaving at 121°C, and set aside after cooling; (2) Dissolve recombinant human platelet-derived growth factor, hydroxypropyl methylcellulose, phosphate buffer, osmotic pressure regulator, and protein protectant in water for injection, and filter aseptically using a 0.22 μm filter membrane; (3) Under sterile conditions, mix the solution obtained in step (1) and the solution obtained in step (2) evenly, make up to volume with sterile water for injection, and immediately aliquot for use.

[0015] In a specific embodiment, the preparation method of the above-mentioned recombinant human platelet-derived growth factor eye drops comprises the following steps: (1) Disperse 0.1-2% polyvinyl alcohol evenly in an appropriate amount of water for injection, sterilize by autoclave at 121°C, and set aside after cooling; (2) Dissolve 800-7000 IU / ml of recombinant human platelet-derived growth factor, 0.1%-0.3% of hydroxypropyl methylcellulose, 0.05-3% of protein protectant, 10-30 mM of phosphate buffer, and 0.25-0.75% of osmotic pressure regulator in an appropriate amount of water for injection, and sterile filter with a 0.22 um filter membrane; (3) Under sterile conditions, mix the solution obtained in step (1) and the solution obtained in step (2) uniformly, make up to volume with sterile water for injection, and then immediately package the mixture to obtain the product.

[0016] Finally, the present invention provides the use of the above liquid composition in preparing a drug for treating corneal damage.

[0017] In a preferred embodiment, the corneal injury includes chemical burns and mechanical injuries.

[0018] In a preferred embodiment, the liquid composition is prepared as eye drops.

[0019] Compared with the prior art, the present invention has the following advantages: 1. This product is fast and effective, and can repair a variety of corneal injuries, mechanical injuries, mild to moderate chemical burns, corneal surgery and poor postoperative healing, mild to moderate dry eye syndrome, etc., and is supplemented with excipients to produce a synergistic effect between the excipients and recombinant human platelet-derived growth factor, thereby increasing the biological activity and stability of recombinant human platelet-derived growth factor in eye drops.

[0020] 2. This product has good safety, low irritation, and does not contain preservatives by designing the formula and selecting excipients for recombinant human platelet-derived growth factor, thus avoiding the damage of preservatives to the eyes such as corneal ulcers. The production process is simple, the industrialization prospects are broad, and the application potential is huge.

[0021] 3. The present invention selects an eye drop formula suitable for human platelet-derived growth factor, which solves the eye discomfort and vision obstruction caused by eye gels and ointments, improves the residence time of platelet-derived growth factor in the cornea, and increases its bioavailability. In addition, the eye drop prescription also improves the stability of PDGF, not only does not affect the biological activity of PDGF, but can improve its biological activity, forming an enhancement effect, so that a lower dose of PDGF can achieve a better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Observation of corneal epithelial healing after administration of recombinant human platelet-derived growth factor eye drops in a rabbit alkali burn model; Figure 2 . Dynamic graph of the development of corneal damage and repair ratio after administration of recombinant human platelet-derived growth factor eye drops in the rabbit alkali burn model. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0024] According to the principle of quality by design, the present invention screens the key excipients in the prescription of recombinant human platelet-derived growth factor eye drops, including thickeners, protein protectants, etc., and evaluates and screens eye drops of different prescriptions according to the key quality parameters of growth factor eye drops in the pharmacopoeia, such as pH, osmotic pressure, biological activity, etc.

[0025] Example 1. Design and screening of prescriptions In view of the physicochemical properties of recombinant human platelet-derived growth factor, the present invention screened some candidate types of pharmaceutical-grade eye drop thickeners approved by the State Food and Drug Administration, protein protective agents, and buffer concentrations for screening. According to the quality standard of the pH of growth factor eye drops in the pharmacopoeia (pH 6.5-7.5), it is just in the buffer range of phosphate buffer. At the same time, according to the characteristics of recombinant human platelet-derived growth factor, the preferred buffer system of the present invention is phosphate buffer, so the selection of the buffer system is not included in the prescription screening experiment in the present invention.

[0026] Table 1. Thickener type and concentration screening formula table

[0027] The nine prescriptions in Table 1 have the same components, such as buffer system and osmotic pressure regulator, except for the different thickeners (see Table 2). No protein protective agent was added in the screening of thickeners, thus forming nine different eye drop screening prescriptions. Based on key quality parameters such as protein content (PDGF), pH, sterility, appearance, and biological activity, the type and concentration of thickeners were screened through high temperature accelerated experiments.

[0028] Table 2. Results of accelerated test at high temperature (40°C) for thickener type and concentration screening formula

[0029] Note: F3H is the formula of F3 with 0.02% g / ml of heparin sodium added, and F7H is the formula of F7 with 0.02% g / ml of heparin sodium added.

[0030] The determination method of recombinant human platelet-derived growth factor is enzyme-linked immunosorbent assay (ELISA), and the detection method of the biological activity of eye drops is reporter gene method, and the method refers to patent CN 109912710 A.

[0031] The experimental results show that the biological activity of recombinant human platelet-derived growth factor eye drops prepared using sodium hyaluronate as a thickener (F2: 1430 IU / ml) is less than half of the biological activity of recombinant human platelet-derived growth factor eye drops prepared using polyvinyl alcohol, hydroxypropyl methylcellulose and methylcellulose as thickeners (F3: 3100 IU / ml, F4: 2810 IU / ml, F5: 2800 IU / ml, respectively), and this decrease in activity is not improved by changes in the concentration of sodium hyaluronate (the biological activity of 0.3% sodium hyaluronate eye drops is 1410 IU / ml). Therefore, sodium hyaluronate is abandoned as a thickener for this product. Polyvinyl alcohol, hydroxypropyl methylcellulose, methylcellulose and three thickeners have a certain synergistic effect with recombinant human platelet-derived growth factor, which can improve the biological activity of the growth factor. The biological activity of the human platelet-derived growth factor eye drops without thickener is 1580IU / ml, while the biological activity of the recombinant human platelet-derived growth factor eye drops after adding thickeners polyvinyl alcohol, hydroxypropyl methylcellulose or methylcellulose is about 3000IU / ml, and the activity increases by about 1 times; among them, polyvinyl alcohol has the best effect and the best stability, with the highest initial biological activity (3100IU / ml), higher than hydroxypropyl methylcellulose (2810IU / ml) and methylcellulose (2800IU / ml). After one month of acceleration, polyvinyl alcohol as a thickener still has the highest biological activity (2636IU / ml). Hydroxypropyl methylcellulose and methylcellulose are second in terms of thickening effect and stability (2388 IU / ml and 2364 IU / ml, respectively), and low concentrations are more suitable as thickeners for recombinant platelet-derived growth factor eye drops. At the same time, the material cost is lower, more economical, and more stable. It is worth noting that in the prescription screening experiment, attempts were made to add sodium heparin (0.02% g / ml) to the prescriptions with polyvinyl alcohol as the thickener, such as F3 and F7, in the hope of producing a synergistic effect with recombinant human platelet-derived growth factor and increasing the biological activity and stability of the growth factor. However, the experimental results showed that this effect did not occur. The biological activity of the eye drops after adding sodium heparin was not much different from that of the eye drops without sodium heparin (both around 3100 IU / ml), and 0.02% sodium heparin also caused the growth factor to reversibly precipitate and produce precipitation.

[0032] Three potential candidate thickeners for recombinant human platelet-derived growth factor eye drops were found through single thickener screening experiments, and further experiments were designed to investigate whether the mixed use of two thickeners can further increase the biological activity and stability of recombinant human platelet-derived growth factor and improve the bioavailability of the growth factor. Due to the principle of excipient cost and prescription simplification, the eye drop prescription design and screening experiments using a mixture of more than two thickeners were not further conducted in the present invention.

[0033] Table 3. Thickener combination formula

[0034] Except for the thickener, other excipients in the above-screened prescriptions, such as buffer system and osmotic pressure regulator, are the same. Each prescription is screened based on key quality parameters such as protein content, eye drop biological activity, pH, sterility, appearance, etc. The stability of each prescription is evaluated through high temperature accelerated experiments, and finally the prescriptions with high biological activity and good stability are screened out.

[0035] Table 4. Results of accelerated test of thickener combination formula at high temperature (40°C)

[0036] The results of the screening experiment showed that when hydroxypropyl methylcellulose and methylcellulose were combined with polyvinyl alcohol as thickeners for recombinant platelet-derived growth factor eye drops, the effect of 0.1% concentration was better than that of 0.3%. After one month of accelerated testing, the biological activity of 0.1% concentration (3248 IU / ml (F17), 2904 IU / ml (F10)) was higher than that of 0.3% concentration (2480 IU / ml (F12), 2460 IU / ml (F13)). Therefore, the hydroxypropyl methylcellulose and methylcellulose used in the subsequent dual thickener prescriptions were all at a concentration of 0.1%.

[0037] After completing the screening test of the thickener using a single factor variable method, the protein protective agent of the recombinant platelet-derived growth factor eye drops was also screened using a single factor variable method. Based on the physicochemical properties of recombinant human platelet-derived growth factor, the candidate protein protective agents selected in the present invention include but are not limited to lysine, histidine, glycine, glutamic acid, arginine, sucrose, trehalose, polyethylene glycol 400, mannitol, and dextran. According to the physicochemical properties of recombinant human platelet-derived growth factor and the results of previous stability studies, the content of the protein protective agent in each prescription in the screening experiment of different protein protective agents was 0.5%, and the optimal content was screened later.

[0038] Table 5. Protein protective agent type and concentration screening formula

[0039] In the above different eye drop prescriptions, the buffer system, osmotic pressure regulator and thickener are also the same. According to the previous thickener screening test results, 1% polyvinyl alcohol is selected as the thickener here. All prescriptions are different only in the protein protectant. Similarly, each prescription is screened according to the key quality parameters of the eye drops such as protein content, appearance, pH, sterility, and biological activity of the eye drops. Each prescription is evaluated by the stability of each prescription after the high temperature accelerated test to screen the best candidate prescription for the protein protectant.

[0040] Table 6. Results of high temperature (40°C) accelerated test for protein protectant type and concentration screening formula

[0041] The experimental results show that when arginine, mannitol and polyethylene glycol are used as protein protectants, recombinant human platelet-derived growth factor eye drops have good stability and biological activity. After three months of accelerated testing, the biological activities are (2772IU / ml (F23), 2772IU / ml (F27), 2706IU / ml (F26)) higher than other test groups. Based on this experimental result, the optimal concentration of each protection is further screened.

[0042] Table 7. Protein protective agent combination formula

[0043] The thickener, buffer system, and osmotic pressure regulator are the same in the above sixteen prescriptions. Only the type and content of the protein protective agent are different. When setting the content of the protein protective agent, because the eye drops need to maintain isotonicity with the human body, the content of the protein protective agent cannot be increased indefinitely. Considering various factors, the upper limit of the content of the protein protective agent in this experiment is set to 3%. The candidate eye drop prescriptions are evaluated and screened through key quality parameters and stability, and the optimal protective agent and its content are screened out.

[0044] Table 8. Results of high temperature (40°C) accelerated test of protein protective agent combination formula

[0045] The results of the screening experiment showed that when the protein protective agent was 1% mannitol, the physical and chemical parameters, biological activity and stability of the recombinant human platelet-derived growth factor eye drops were the best. After three months of the accelerated test, the biological activity of the eye drops was 2982 IU / ml (F39), which was higher than that of other experimental groups.

[0046] Following the same idea and experimental method, the buffer system concentration and the type and content of osmotic pressure regulators in recombinant human platelet-derived growth factor eye drops were screened. At the same time, referring to relevant literature, the optimal buffer system concentration of recombinant human platelet-derived growth factor eye drops was finally selected as 20mM, and the osmotic pressure regulator was 0.56% sodium chloride.

[0047] According to the above screening experiments, the best thickener, buffer system, osmotic pressure regulator, and protein protective agent are screened to form different candidate formulations of recombinant human platelet-derived growth factor eye drops, and the best eye drops formulation is screened through the following implementation case.

[0048] Preparation Example 1: Prescription F39 Recombinant human platelet-derived growth factor 1200000IU Polyvinyl alcohol 4g Mannitol 4g Sodium dihydrogen phosphate monohydrate 0.32g Disodium hydrogen phosphate dodecahydrate 2.08g Sodium chloride 2.24g Add water for injection to 400ml Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121°C for 30 minutes, and set aside after cooling; dissolve recombinant human platelet-derived growth factor, mannitol, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically with a 0.22um filter membrane; under sterile conditions, mix the solutions obtained in the above two steps evenly, make up to volume with sterile water for injection, and immediately package to obtain the product.

[0049] Preparation Example 2: Prescription F7 with Mannitol Protein Protectant Recombinant human platelet-derived growth factor 1200000IU Polyvinyl alcohol 5.6g Mannitol 4g Sodium dihydrogen phosphate monohydrate 0.32g Disodium hydrogen phosphate dodecahydrate 2.08g Sodium chloride 2.24g Add water for injection to 400ml Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121°C for 30 minutes, and set aside after cooling; dissolve recombinant human platelet-derived growth factor, mannitol, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically with a 0.22um filter membrane; under sterile conditions, mix the solutions obtained in the above two steps evenly, make up to volume with sterile water for injection, and immediately package to obtain the product.

[0050] Preparation Example 3: Prescription F10 with addition of protein protective agent Recombinant human platelet-derived growth factor 1200000IU Polyvinyl alcohol 4g Hydroxypropyl methylcellulose 0.4g Mannitol 4g Sodium dihydrogen phosphate monohydrate 0.32g Disodium hydrogen phosphate dodecahydrate 2.08g Sodium chloride 2.24g Add water for injection to 400ml Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121°C for 30 minutes, and set aside after cooling; dissolve recombinant human platelet-derived growth factor, hydroxypropyl methylcellulose, mannitol, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically with a 0.22um filter membrane; mix the solutions obtained in the above two steps evenly under sterile conditions, make up to volume with sterile water for injection and immediately package to obtain the product.

[0051] Preparation Example 4: Prescription F11 with addition of protein protective agent Recombinant human platelet-derived growth factor 1200000IU Polyvinyl alcohol 4g Methylcellulose 0.4g Mannitol 4g Sodium dihydrogen phosphate monohydrate 0.32g Disodium hydrogen phosphate dodecahydrate 2.08g Sodium chloride 2.24g Add water for injection to 400ml Specific preparation method: Disperse polyvinyl alcohol in an appropriate amount of water for injection, sterilize at 121 degrees for 30 minutes, and set aside after cooling; dissolve recombinant human platelet-derived growth factor, methylcellulose, mannitol, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dodecahydrate and sodium chloride in an appropriate amount of water for injection, and filter aseptically with a 0.22um filter membrane; under sterile conditions, mix the solutions obtained in the above two steps evenly, make up to volume with sterile water for injection and immediately package to obtain the product.

[0052] Application Example 1: Experiment on the repair of rabbit corneal alkali burn model promoted by recombinant human platelet-derived growth factor eye drops Experimental animals: Healthy New Zealand white rabbits, weighing 2-3 kg, both male and female, no abnormalities were found in the anterior segment of the eye by slit lamp examination. 60 animals were randomly divided into 6 groups, 10 in each group.

[0053] Rabbit corneal alkali burn model: The external eye examination of the normal animal was performed, and the left eye was selected as the experimental eye. General anesthesia was performed by intraperitoneal injection of chloral hydrate, and the right eye was washed twice with normal saline, and 50ul of dicaine eye drops were delivered to the eye twice for eye anesthesia. A filter paper disc with a diameter of 8mm was soaked with 1M sodium hydroxide, and the excess sodium hydroxide in the filter paper disc was absorbed with dry filter paper. Then, the rabbit's eyelid was opened with an eyelid opener, and the excess moisture on the ocular surface was wiped off with a cotton swab. The filter paper disc was carefully placed in the center of the rabbit's right cornea for 40s, and then the ocular surface and conjunctival sac were fully rinsed with excess normal saline for 1min, thereby completing the experimental chemical burn on the rabbit's ocular surface.

[0054] Test samples: The negative control was a model control group without any drug administration, the test group was a recombinant human platelet-derived growth factor eye drop with four different prescriptions in Preparation Examples 1, 2, 3, and 4, and the positive control group was Beifushu (recombinant bovine basic growth factor eye drop 4200 IU / ml). 1-2 drops were administered each time, 4 times a day, for two consecutive weeks.

[0055] Evaluation index: On the 1st, 3rd, 5th, 7th, 10th and 14th days of treatment, the injured cornea of ​​the rabbit was stained with sodium fluorescein, and then the cornea was photographed with a slit lamp fluorescence microscope. The corneal epithelial healing rate of each group at different times was analyzed by a computer image analysis system. The higher the corneal epithelial healing area and corneal epithelial healing rate, the better the treatment effect.

[0056] The test results are shown in Table 9: Table 9: Corneal epithelial healing area of ​​each group at different times (%, X±S)

[0057] The experimental results show that when the recombinant human platelet-derived growth factor is used in the formulation of Example 3 (formulation F10 with protein protective agent added), the repair effect is the best in the rabbit corneal alkali burn model. The damaged cornea has completely recovered after 7 days of treatment, which is significantly shorter than other experimental groups (10-14 days). Compared with the negative control, positive control and other experimental groups, the formulation of Example 3 has a shorter repair time, and the epithelial repair of the damaged cornea is completed in about seven days, without sodium fluorescein staining, and the effect is significantly better than other groups.

[0058] After the prescription screening was completed, the screening of the content of recombinant human platelet-derived growth factor in the recombinant human platelet-derived growth factor eye drops was continued on the same rabbit alkali burn model. The screening experiment set up a negative control group, a solvent control group, a low concentration group of recombinant platelet-derived growth factor, a medium concentration group of recombinant platelet-derived growth factor, a high concentration group of recombinant platelet-derived growth factor, and a positive control group, wherein the negative control group was a model control group without any drug administration; the solvent control group was an eye drop prescription without recombinant platelet-derived growth factor, and the auxiliary material composition was the same as that in Preparation Example 3; the content of low-concentration recombinant platelet-derived growth factor was 800IU / ml (other components were the same as those in Preparation Example 3); the content of medium-concentration recombinant platelet-derived growth factor was 3000IU / ml (Preparation Example 3), and the content of high-concentration recombinant platelet-derived growth factor was 7000IU / ml (other components were the same as those in Preparation Example 3), and the positive control group was Beifushu (recombinant bovine basic growth factor eye drops 4200IU / ml). The administration method and frequency were the same as those in the above prescription screening experiment. The epithelial healing rate of the damaged cornea was observed by fluorescein sodium staining at different time points using a slit lamp microscope. The test results are shown in Table 10. Table 10: Corneal epithelial healing area in each group at different times (%, X±S)

[0059] Statistical results showed that the corneal epithelial healing area in the group of the present invention (medium concentration group) was the highest. On the 7th day of medication, the healing area was close to 100%, which was significantly better than other experimental groups and the positive control group.

[0060] Figure 1 The figure is an observation diagram of the healing of the corneal epithelium of the experimental rabbit, wherein the upper part is the cornea at the beginning of medication (day 0), and the lower part is the cornea on the 7th day of medication. The results show that: seven days after medication, there is basically no sodium fluorescein staining in the damaged cornea of ​​the present invention group (medium concentration group), and the cornea is completely repaired, while the negative control group, low concentration group, high concentration group and positive control group all have sodium fluorescein staining, and the damaged cornea is not completely repaired.

[0061] Figure 2 This is a dynamic diagram of the proportion of corneal damage repair after drug administration to rabbit corneal alkali burns. The results show that the eye drops of the present invention (medium concentration group) show the advantages of rapid onset and good therapeutic effect. Compared with the positive control group and other test groups, it only takes a shorter time to completely repair the damaged cornea.

[0062] The experimental results showed that when the content of recombinant platelet-derived growth factor in the eye drops was 3000IU / ml (medium concentration group), the repair effect of damaged cornea was best, the corneal epithelial repair time was shorter, and it was significantly better than the negative control group, solvent control group, positive control group and other experimental groups.

[0063] Application Experiment 2: Stability Study High temperature and high humidity test The eye drops prepared in Example 3 were placed in a colorless plastic bottle, sealed with a cap, and placed in an accelerated test at 40°C and a relative humidity of 75%. The content, appearance, pH, osmotic pressure, sterility, and biological activity of the eye drops were tested at 0, 1, 2, and 3 months. The results are shown in the following table: Table 11: High temperature and high humidity test results

[0064] The test results show that the eye drops of the present invention have stable quality in the high temperature and high humidity test.

[0065] 2. Low temperature long-term test The eye drops prepared in Example 3 were bottled in colorless plastic bottles, sealed and placed at 4°C for testing. The content, appearance, pH, osmotic pressure, sterility and biological activity of the eye drops were tested at 0, 1, 3, 6 and 12 months. The results are shown in the following table: Table 12: Low temperature test results

[0066] The test results show that the eye drops of the present invention have stable quality in a low-temperature long-term test.

[0067] 3. Light Acceleration Test The eye drops prepared in Example 3 were placed in a colorless plastic bottle, sealed and placed under 3000Lx light for testing. The content, appearance, pH, osmotic pressure, sterility and biological activity of the eye drops were tested at 0, 1, 2 and 3 months. The results are shown in the following table: Table 13. Light accelerated test

[0068] The test results show that the eye drops of the present invention have stable quality in the light accelerated test.

[0069] Application Experiment Example 3: Irritation Test The eye drops prepared by the eye drops prescription in Preparation Example 3 and blank excipients were selected to observe the stimulation reaction of the conjunctiva, cornea and iris after the animal eyes were exposed to the test samples. Grouping of administration: 9 healthy rabbits with no damage in eye examination (no turbidity in the cornea, no conjunctival congestion, edema and secretions, round pupils, equal size on both sides, good light reflection) were taken and randomly divided into 3 groups according to body weight, 3 rabbits / group, the left and right eyes of the first group were given normal saline, the left eye of the second group was given normal saline as a self-control, and the right eye was given blank excipient eye drops. The left eye of the third group was also given normal saline as a self-control, and the right eye was given the eye drops prepared in Example 4. Method of administration: The liquid medicine was dripped into the conjunctival sac of the rabbit eye, the nasolacrimal duct was compressed, and the eyes were passively closed for about 5 to 10 seconds. 4 times / d, 1-2 drops (about 50ul) each time, continuous eye drops for 14 days, before each administration and 1, 2, 4, 24, 48 and 72h after the last administration, observe the cornea, iris and conjunctiva directly with naked eyes or with a magnifying glass to see if they are irritating, and score the irritation, stain with 2% sodium fluorescein when appropriate, and observe whether each part is colored. The results are scored for irritation reaction according to Table 14, and the irritation reaction scores of the cornea, iris and conjunctiva of each rabbit at each observation time are added together to obtain the total score, and the total score of a group is divided by the number of animals to obtain the final average score, which is compared with the left eye. Then, the eye irritation of the sample is judged according to the evaluation criteria in Table 15. When the irritation intensity of the cornea, iris and conjunctiva is inconsistent, evaluations should be made separately. After all observations are completed, the rabbits are killed, both eyes and upper and lower eyelids are cut, and abnormal changes such as redness and swelling are observed with naked eyes, and then fixed in 10% formaldehyde solution, embedded in paraffin, sliced, HE stained, and histopathological examination is performed. The irritation test results are shown in Table 16.

[0070] Table 14: Eye irritation response score standard

[0071] Table 15: Eye irritation evaluation criteria

[0072] Table 16: Results of irritation test of recombinant human platelet-derived growth factor eye drops

[0073] The results of the animal eye irritation test show that the eye drops prepared in Preparation Example 3 are non-irritating to the blank excipients and normal saline.

[0074] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements can be made without departing from the spirit and scope of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A liquid composition containing recombinant human platelet-derived growth factor, It is characterized in that The composition contains: Recombinant human platelet-derived growth factor 800-7000 IU / ml Thickener 0.1-2% (g / ml) Protein protective agent 0.05-3% (g / ml) Phosphate buffer 10-30 mM Osmotic pressure regulator 0.25-0.75% (g / ml) The pH of the liquid composition is 6.8-7.4, Wherein, the thickener is selected from one or more of polyvinyl alcohol, sodium hyaluronate, hydroxypropyl methylcellulose, and methylcellulose; the protein protectant is selected from one or more of glycine, glutamic acid, lysine, histidine, arginine, sucrose, trehalose, polyethylene glycol 400, and mannitol; and the osmotic pressure regulator is selected from one or more of sodium chloride, glucose, or sorbitol.

2. The liquid composition according to claim 1, It is characterized in that The dosage of the recombinant human platelet-derived growth factor in the composition is 1000-6000 IU / ml.

3. The liquid composition according to claim 1, It is characterized in that The thickener is polyvinyl alcohol, and the dosage of polyvinyl alcohol is 0.5%-1.5%.

4. The liquid composition according to claim 1, It is characterized in that The thickener is hydroxypropyl methylcellulose, and the dosage of hydroxypropyl methylcellulose is 0.1%-0.5%.

5. The liquid composition according to claim 1, It is characterized in that The protein protective agent is mannitol, and the dosage of mannitol is 0.05%-3%.

6. The liquid composition according to claim 1, It is characterized in that The osmotic pressure regulator is sodium chloride, and the dosage of the sodium chloride is 0.25%-0.75%.

7. The liquid composition according to claim 1, It is characterized in that The dosage of each component in the composition is respectively: Recombinant human platelet-derived growth factor 3000 IU / ml Polyvinyl alcohol 1% (g / ml) Hydroxypropyl methylcellulose 0.1% (g / ml) Mannitol 1% (g / ml) Sodium chloride 0.56% (g / ml) Phosphate buffer is 20 mM, pH 6.8-7.

4.

8. A method for preparing the liquid composition according to any one of claims 1 to 7, It is characterized in that The method comprises the following steps: (1) Disperse polyvinyl alcohol in water for injection, sterilize by autoclave at 121°C, and set aside after cooling; (2) Dissolve recombinant human platelet-derived growth factor, hydroxypropyl methylcellulose, phosphate buffer, osmotic pressure regulator, and protein protectant in water for injection, and filter aseptically using a 0.22 μm filter membrane; (3) Under sterile conditions, mix the solution obtained in step (1) and the solution obtained in step (2) evenly, make up to volume with sterile water for injection, and immediately aliquot for use.

9. Use of the liquid composition according to any one of claims 1 to 7 in the preparation of a drug for treating corneal damage.

10. The use according to claim 9, It is characterized in that The corneal injuries include chemical burns and mechanical injuries.

11. The use according to claim 10, It is characterized in that The liquid composition is prepared as eye drops.

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