Composition containing recombinant human nerve growth factor and application and ophthalmic preparation thereof

Abstract

The invention discloses a composition containing a recombinant human nerve growth factor, application of the composition and an ophthalmic preparation. The composition is prepared from the following components in parts by weight: 0.005 to 1 part of recombinant human nerve growth factor, 20 to 70 parts of trehalose, 5 to 30 parts of mannitol and 1 to 13 parts of polyvinyl alcohol. The trehalose, the mannitol and the polyvinyl alcohol are compounded with the recombinant human nerve growth factor, so that the stability of the recombinant human nerve growth factor can be remarkably improved, and the loss of pharmacological active ingredients of the ophthalmic preparation in the preparation, transportation and long-term storage periods is reduced, so that the medication safety and the treatment effect are ensured; and the recombinant human nerve growth factor in the ophthalmic preparation is good in stability, so that the clinical requirements of patients with eye diseases can be better met.

Description

Technical Field

[0001] The invention belongs to the technical field of ophthalmic preparations, and specifically relates to a composition containing recombinant human nerve growth factor and an application thereof and an ophthalmic preparation. Background Art

[0002] The cornea is located at the front 1 / 6 of the eyeball wall. It is a blood-free transparent membrane with abundant sensory nerve endings. Corneal diseases are very harmful and can cause corneal edema, eye pain, vision loss, etc. In severe cases, it can cause corneal perforation or even blindness. There are many causes of corneal diseases, including tear film abnormalities, inflammation, systemic diseases, surgical injuries that reduce corneal sensitivity, corneal denervation or corneal damage. At present, corneal diseases are one of the most common eye pathological conditions.

[0003] Nerve growth factor (NGF) is a cytokine that can promote the growth, development, differentiation, maturation of central and peripheral neurons, and accelerate the repair of nerve damage. Many in vitro and in vivo experimental studies have shown that NGF has a good effect in treating some diseases of the visual system, such as retinitis pigmentosa, optic neuritis, eye contusion, glaucoma, keratitis, dry eyes, etc., which cause optic neuropathy.

[0004] In 2018, the U.S. FDA approved Oxervate, recombinant human nerve growth factor (rhNGF) eye drops, for the treatment of neurotrophic keratitis, which can promote corneal epithelial repair and re-nervate the cornea. In clinical trials, 70% of patients treated with Oxervate had complete corneal healing after 8 weeks. However, the stability of the active ingredient rhNGF in this product is poor. Under the intended conditions of use (such as light, refrigeration, high temperature, etc.), the purity and content of rhNGF decrease rapidly. According to the Oxervate instructions, the product needs to be stored at -20°C; after being provided to the patient, it can be stored at 2-8°C for 1 week; any unused drugs stored at 2-8°C for more than 1 week or remaining drugs opened for more than 12 hours cannot be used. The strict usage requirements greatly limit the accessibility of patients and lead to expensive clinical use costs.

[0005] CN113307850A discloses an antimicrobial peptide eye drop, comprising an antimicrobial peptide, a thickener, an osmotic pressure regulator, a pH buffer, a protective agent, a surfactant and water. The invention aims to solve the problem that the activity of the antimicrobial peptide decreases rapidly after being prepared into a liquid preparation and the activity stability is poor at room temperature.

[0006] CN112826829A discloses an ophthalmic preparation comprising trehalose, mannitol and cellulose ether, which is effective in inducing healing of corneal epithelial defects when the combination of trehalose, mannitol and cellulose ether is topically applied to the eye.

[0007] Therefore, there is an urgent need to develop a composition that can significantly improve the stability of recombinant human nerve growth factor and reduce the cost of ophthalmic preparations containing recombinant human nerve growth factor and the medical burden of patients, which is of great significance for improving the treatment accessibility of patients with corneal-related lesions. Summary of the invention

[0008] In order to solve the above technical problems, the present invention provides a composition containing recombinant human nerve growth factor and its application and ophthalmic preparation. The composition, including a composition containing low-concentration recombinant human nerve growth factor, has better stability and reduces the loss of pharmacologically active ingredients in ophthalmic preparations during manufacturing, transportation and long-term storage. The ophthalmic preparation prepared using the composition has good stability, thereby reducing the cost of use for patients and meeting the clinical needs of eye disease treatment.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A composition containing recombinant human nerve growth factor comprises recombinant human nerve growth factor, trehalose, mannitol and polyvinyl alcohol. The present invention utilizes trehalose, mannitol and polyvinyl alcohol to compound with recombinant human nerve growth factor, which can significantly improve the stability of recombinant human nerve growth factor, especially improve the stability of recombinant human nerve growth factor at low concentration.

[0011] The amount of recombinant human nerve growth factor in the composition of the present invention is 0.005-1 part by weight; for example, 0.54-1 part by weight, 0.18-0.54 part by weight, 0.1-0.18 part by weight, 0.06-0.1 part by weight, 0.04-0.06 part by weight, 0.02-0.04 part by weight, 0.01-0.02 part by weight, 0.005-0.01 part by weight, 0.005-1 part by weight or a combination of any of the above endpoint values ​​or range values, such as: 0.01-1 part by weight, 0.005-0.54 part by weight, 0.005-0.18 part by weight, 0.005-0.06 part by weight, 0.005-0.04 part by weight, 0.005-0.02 part by weight, etc.

[0012] The amount of trehalose in the composition of the present invention is 20-70 parts by weight, for example, 40-70 parts by weight; 45-60 parts by weight.

[0013] The amount of mannitol in the composition of the present invention is 5-30 parts by weight, for example, 5-20 parts by weight; 8-14 parts by weight.

[0014] The amount of polyvinyl alcohol in the composition of the present invention is 1-13 parts by weight, for example, 1-11 parts by weight; 5-9 parts by weight.

[0015] The present invention also provides the use of the composition containing recombinant human nerve growth factor in the preparation of ophthalmic preparations.

[0016] The ophthalmic preparation of the present invention can be used to treat corneal lesions caused by reduced corneal sensitivity, corneal denervation or corneal damage, such as neurotrophic keratitis, trauma / surgery-related corneal damage / defective lesions, dry eyes, etc.

[0017] The present invention also provides an ophthalmic preparation, comprising the above composition containing recombinant human nerve growth factor.

[0018] Compared with the prior art, the ophthalmic preparation of the present invention has better stability, thereby reducing the loss of active ingredients in the ophthalmic preparation during manufacture, transportation and long-term storage.

[0019] The osmotic pressure of the ophthalmic preparation of the present invention is 200-380 mOsmol / kg, preferably 250-350 mOsmol / kg, and more preferably 280-320 mosm / kg.

[0020] The viscosity of the ophthalmic preparation of the present invention is 1-25 mm 2 / s; preferably 2-24mm 2 / s.

[0021] The ophthalmic preparation of the present invention may further comprise a buffer to adjust the pH to meet the requirements. Preferably, the pH of the ophthalmic preparation is 6.5-7.5; more preferably 7.0-7.4. The buffer is selected from at least one of phosphate, citrate and histidine.

[0022] The ophthalmic preparation of the present invention may further comprise an antioxidant compound, specifically, the antioxidant compound is selected from L-methionine and / or cysteine. Preferably, the content of the antioxidant compound is 0.01-0.1 mg / ml.

[0023] The ophthalmic preparation of the present invention may further comprise a high molecular weight polymer, specifically, the high molecular weight polymer is polyethylene glycol, and the molecular weight is 4000-8000. Preferably, the content of the high molecular weight polymer is 5-25 mg / ml.

[0024] The beneficial effects of the present invention are:

[0025] (1) The present invention utilizes trehalose, mannitol and polyvinyl alcohol to compound with recombinant human nerve growth factor, which can significantly improve the stability of recombinant human nerve growth factor, especially the stability of low-concentration recombinant human nerve growth factor.

[0026] (2) The ophthalmic preparation of the present invention can significantly restore corneal perception of model rats, reduce corneal sodium fluorescein staining scores, improve tear secretion, and promote corneal nerve recovery, confirming that the ophthalmic preparation of the present invention has a good effect of repairing corneal epithelium and nerves.

[0027] (3) The ophthalmic preparation of the present invention has good stability and thus has low storage requirements. It can be stored at 10-14°C for at least 7 days, which greatly reduces the cost of use for patients. Compared with public prescriptions, it has more cost advantages and can better meet the clinical needs of patients with eye diseases.

[0028] (4) The ophthalmic preparation (low concentration) of the present invention has good light stability. After 10 days of illumination, the purity of the recombinant human nerve growth factor is greater than 90%, which is higher than the release standard of marketed products. After 15 days of illumination, the purity of the recombinant human nerve growth factor is greater than 85%, which meets the release standard of marketed products. The quality of the ophthalmic preparation of the present invention is better.

[0029] (5) The ophthalmic preparation of the present invention has good stability, thereby reducing the loss of pharmacologically active substances during the manufacture, transportation and long-term storage of the ophthalmic preparation, and ensuring the therapeutic effect and safety of medication. DETAILED DESCRIPTION

[0030] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0031] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0032] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0033] The present invention does not limit the source of raw materials. Unless otherwise specified, the raw materials and excipients used are all commercially available pharmaceutical grade products, and the solvents are all water for injection unless otherwise specified. In terms of analytical methods, the following methods are used in the embodiments of the present invention. Unless otherwise specified, the percentages described in the present invention are all percentages by mass.

[0034] 1. Experimental Materials

[0035] Recombinant human nerve growth factor is a recombinant human β-nerve growth factor prepared by Chongqing Kerun Biopharmaceutical Co., Ltd. Patent 201910390559.2. Commercially available recombinant human nerve growth factor products can also achieve similar technical effects;

[0036] Polyvinyl alcohol was purchased from Jiangsu Alpha Pharmaceutical Group;

[0037] L-methionine was purchased from Hubei Bafeng Pharmaceutical Chemical Co., Ltd.;

[0038] Disodium hydrogen phosphate dodecahydrate was purchased from Sichuan Huayi Pharmaceutical Excipient Manufacturing Company;

[0039] Sodium dihydrogen phosphate monohydrate was purchased from Sichuan Huayi Pharmaceutical Excipient Manufacturing Company;

[0040] Mannitol was purchased from Qingdao Mingyue Seaweed Group Co., Ltd.

[0041] PEG6000, purchased from Hunan Erkang Pharmaceutical Co., Ltd.;

[0042] Trehalose dihydrate was purchased from Japan Co., Ltd.;

[0043] Sodium hyaluronate was purchased from Shaanxi Zhengyi Pharmaceutical Excipients Co., Ltd.

[0044] 2. RP-HPLC (reverse phase chromatography) method to evaluate the purity and content of the main peak

[0045] The determination was carried out in accordance with the Pharmacopoeia of the People's Republic of China (2015 edition, 0521 high performance liquid chromatography method), and the detection was carried out using a reverse phase chromatography column. When this method is used for purity detection, the sample purity is calculated by area normalization. When this method is used for content testing, 20 μg / ml of recombinant human nerve growth factor is used for external standard method quantification, and the mean peak area of ​​the target protein at this concentration is 170. Samples above this concentration are diluted with phosphate buffer; samples below this concentration are concentrated by ultrafiltration and 12000g centrifugation; all are analyzed at 20 μg / ml and the chromatogram is recorded.

[0046] Content evaluation: Relative percentage content (%) = (test sample peak area / 170) × 100%.

[0047] 1. Stability test of the composition containing recombinant human nerve growth factor

[0048] The formulas of the compositions containing recombinant human nerve growth factor of Examples 1-15 are shown in Table 1.

[0049] Table 1

[0050]

[0051] The preparation method of the above-mentioned composition containing recombinant human nerve growth factor is as follows: trehalose, mannitol and polyvinyl alcohol are weighed separately and dissolved in injection water, and then the recombinant human nerve growth factor is added and mixed evenly, phosphate buffer is added to adjust the pH to 7.2, and filtered.

[0052] Under the conditions of a temperature of 12±2°C and an intense light of 5000±500 Lux, RP-HPLC was used to investigate the effect of light exposure on the stability of the composition at different storage times.

[0053] The comparative composition 1 is: 180 μg / ml recombinant human nerve growth factor, 50.4 mg / ml trehalose, 13.1 mg / ml mannitol, 1.1 mg / ml hydroxypropyl methylcellulose (HPMC, 4000 mPa.s), and the preparation method is the same as the above composition containing recombinant human nerve growth factor.

[0054] Comparative composition 2: recombinant human nerve growth factor 180 μg / ml, trehalose 50.4 mg / ml, mannitol 13.1 mg / ml, sodium hyaluronate 0.1 mg / ml. The preparation method is the same as the above composition containing recombinant human nerve growth factor.

[0055] The results are shown in Tables 2 and 3.

[0056] Table 2

[0057]

[0058] As can be seen from Table 2, after the compositions containing recombinant human nerve growth factor of Examples 1-15 of the present invention were exposed to light for 7 days at 12±2°C, the purity of the recombinant human nerve growth factor in the compositions was greater than 90%, the purity of the recombinant human nerve growth factor in the comparative composition 1 was greater than 90%, and the purity of the recombinant human nerve growth factor in the comparative composition 2 was less than 90%.

[0059] Table 3

[0060]

[0061]

[0062] As shown in Table 3, after 7 days of illumination at 12±2°C, the relative percentage of recombinant human nerve growth factor in the compositions containing recombinant human nerve growth factor in Examples 1-15 of the present invention is within the target concentration range of 80-120%; while after 3 days of illumination, the relative percentage of recombinant human nerve growth factor in the comparative composition 1 is less than 80%; after 1 day of illumination, the relative percentage of recombinant human nerve growth factor in the comparative composition 2 is already 61%. The above results show that the composition containing recombinant human nerve growth factor of the present invention has good light stability. The combination of trehalose, mannitol and polyvinyl alcohol with recombinant human nerve growth factor can improve the light stability of recombinant human nerve growth factor.

[0063] It can be seen from Table 2 and Table 3 that the compositions containing recombinant human nerve growth factor in Examples 1-15 of the present invention are exposed to light for 7 days at 12±2°C, and the purity and relative percentage content of the recombinant human nerve growth factor in the compositions meet the requirements of purity and target concentration range; while the comparative composition 1 is exposed to light for 7 days at 12±2°C, although the purity of its recombinant human nerve growth factor meets the requirements, the relative percentage content of the recombinant human nerve growth factor is less than 80%, which cannot meet the requirements of the target concentration range. After 7 days of light exposure at 12±2°C, the purity and relative percentage content of the recombinant human nerve growth factor in the comparative composition 2 do not meet the requirements of purity and target concentration range. Therefore, compared with the comparative composition 1 and the comparative composition 2, the composition containing recombinant human nerve growth factor of the present invention has better photostability, and the composition containing less than 100μg / ml recombinant human nerve growth factor still has better photostability.

[0064] In order to investigate the effects of different amounts of trehalose, mannitol and polyvinyl alcohol combined with recombinant human nerve growth factor on the composition containing recombinant human nerve growth factor, the following comparative examples 1-3 containing recombinant human nerve growth factor were prepared.

[0065] The formulas of the compositions containing recombinant human nerve growth factor of Comparative Examples 1-3 are shown in Table 4.

[0066] Table 4

[0067]

[0068] The preparation method of the composition is: weigh the other components except the recombinant human nerve growth factor respectively, dissolve them in injection water, then add the recombinant human nerve growth factor and mix them evenly, add phosphate buffer to adjust the pH to 7.2, filter, and then the mixture is ready.

[0069] Referring to the aforementioned stability test method, the stability of the compositions of Comparative Examples 1-3 was tested. The results are shown in Tables 5 and 6.

[0070] Table 5

[0071]

[0072] As shown in Table 5, the purity of the recombinant human nerve growth factor of the composition of Comparative Example 1 is lower than 89.5% after being illuminated at 12±2°C for 7 days. The purity of the recombinant human nerve growth factor of the compositions containing recombinant human nerve growth factor of Comparative Examples 2 and 3 is still greater than 90% after being illuminated at 12±2°C for 7 days.

[0073] Table 6

[0074]

[0075] It can be seen from Table 6 that after 7 days of illumination at 12±2°C, the relative percentage content of the recombinant human nerve growth factor in the composition containing recombinant human nerve growth factor in Comparative Example 1 is already at the lower limit of the target concentration range; the relative percentage content of the recombinant human nerve growth factor in Comparative Examples 2 and 3 is both lower than 80%.

[0076] It can be seen from Table 5 and Table 6 that the compositions containing recombinant human nerve growth factor in Comparative Examples 1-3 cannot simultaneously meet the requirements for the purity and target concentration range of the recombinant human nerve growth factor in the light stability test.

[0077] In summary, the composition of the present invention has good photostability, and even the composition containing low concentration of recombinant human nerve growth factor has good photostability, thereby reducing the loss of pharmacologically active ingredients in the ophthalmic preparation during manufacturing, transportation and long-term storage.

[0078] 2. Performance evaluation of ophthalmic preparations

[0079] Taking the compositions of Example 4, Example 12 and Example 15 as examples, the stability and efficacy of ophthalmic preparations containing low concentrations (100 μg / ml, 10 μg / ml and 5 μg / ml) of recombinant human nerve growth factor were investigated.

[0080] Ophthalmic preparation 1, a composition containing the recombinant human nerve growth factor of Example 4, marked as prescription 1; the specific formula is as follows: recombinant human nerve growth factor 100 μg / ml, trehalose 50 mg / ml, mannitol 14 mg / ml, polyvinyl alcohol 9 mg / ml, PEG6000 10 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / ml and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0081] Ophthalmic preparation 2, a composition containing the recombinant human nerve growth factor of Example 12, is marked as Prescription 2; the specific formula is as follows: recombinant human nerve growth factor 10 μg / ml, trehalose 70 mg / ml, mannitol 20 mg / ml, polyvinyl alcohol 11 mg / ml, PEG6000 10 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / ml and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0082] Ophthalmic preparation 3, a composition containing the recombinant human nerve growth factor of Example 15, marked as prescription 3; the specific formula is as follows: recombinant human nerve growth factor 5 μg / ml, trehalose 55 mg / ml, mannitol 12 mg / ml, polyvinyl alcohol 7 mg / ml, PEG6000 10 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / ml and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0083] A comparative ophthalmic preparation, comprising the aforementioned comparative composition 1, has a specific formula as follows: recombinant human nerve growth factor 180 μg / ml, trehalose 50.4 mg / ml, mannitol 13.1 mg / ml, HPMC (4000 mPas) 1.1 mg / ml, PEG6000 10.7 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / ml and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0084] 2.1 Long-term stability

[0085] The above ophthalmic preparations (2000 tubes each, 2 mL in size) were placed at -20°C for a long period of time for 9 months. The samples were evaluated by RP-HPLC. The results are shown in Table 7.

[0086] Table 7

[0087]

[0088] As shown in Table 7, the purity of the recombinant human nerve growth factor in the ophthalmic preparation containing a low concentration of recombinant human nerve growth factor of the present invention remains stable when placed at -20°C for 9 months, and is superior to that of the comparative ophthalmic preparation, thus meeting the requirement of long-term storage.

[0089] 2.2 Acceleration stability

[0090] The above ophthalmic preparations (2000 tubes each, 2 mL in size) were subjected to accelerated stability testing at 2-8°C, and the samples were evaluated by RP-HPLC. The results are shown in Table 8.

[0091] Table 8

[0092]

[0093] As can be seen from Table 8, the purity of the recombinant human nerve growth factor of the ophthalmic preparation containing a low concentration of recombinant human nerve growth factor of the present invention remains at a stable level when stored at 2-8°C for 4 months, and the purity of the recombinant human nerve growth factor is greater than 90%; while the purity of the recombinant human nerve growth factor of the comparative ophthalmic preparation is less than 90% when stored at 2-8°C for 4 months.

[0094] 2.3 Photostability

[0095] The above ophthalmic preparations (2000 tubes each, specification 2 ml) were stored at 12±2°C and under strong light (5000±500 Lux) for 15 days. The purity of the samples was evaluated by RP-HPLC. The results are shown in Table 9.

[0096] Table 9

[0097]

[0098] As can be seen from Table 9, the ophthalmic preparation containing a low concentration of recombinant human nerve growth factor of the present invention has good light stability. After 10 days of illumination, the purity of the recombinant human nerve growth factor is greater than 90%, and after 15 days of illumination, the purity of the recombinant human nerve growth factor is greater than 85%, which still meets the release standard of marketed products; while the comparative ophthalmic preparation has a recombinant human nerve growth factor purity of less than 90% after 10 days of illumination, and a purity of the recombinant human nerve growth factor of less than 85% after 15 days of illumination, which does not meet the release standard of marketed products.

[0099] Table 10

[0100]

[0101] As shown in Table 10, the ophthalmic preparation containing a low concentration of recombinant human nerve growth factor of the present invention has a relative percentage content of the recombinant human nerve growth factor within the target concentration range after 15 days of illumination; while the relative percentage content of the recombinant human nerve growth factor of the comparative ophthalmic preparation is lower than 80% after 3 days of illumination.

[0102] In summary, the ophthalmic preparation containing low concentration of recombinant human nerve growth factor of the present invention has good photostability.

[0103] 2.4 Evaluation of the effect of neurotrophic keratitis

[0104] (I) Experimental Animals

[0105] SD rat pups (3 days old), weighing 7-10 g.

[0106] (II) Test methods

[0107] Newborn mice were randomly divided into groups according to their weight, and capsaicin (8 mg / ml) was injected subcutaneously at the back of the neck once to establish the corneal denervation animal model. After modeling, both the model group and the normal animal group underwent ophthalmological examinations. The model was successful if there was a significant difference in corneal perceptual sensitivity and corneal fluorescein sodium staining. The animals with successful modeling were randomly divided into a model control group, an original preparation treatment group, and a new preparation treatment group; 16 normal mice were taken as a normal control group.

[0108] In this example, the effective daily dose of the composition for humans is calculated at 0.1 μg / kg (calculated as recombinant human nerve growth factor), an adult weighs 60 kg, and the dose conversion factor between mice and humans is 10.0, so the equivalent daily dose for mice is 1 μg / kg (calculated as recombinant human nerve growth factor).

[0109] At 8:00 every day, all animals except the normal control group were given medication, with a daily dosage of 10 μl / eye. The model control group was given injection water, the original preparation treatment group was given the aforementioned comparative ophthalmic preparation, and the new preparation treatment group was given the ophthalmic preparation of the aforementioned prescription 1. The specific formula of the ophthalmic preparation is as follows:

[0110] Comparative ophthalmic preparation: recombinant human nerve growth factor 180 μg / ml, trehalose 50.4 mg / ml, mannitol 13.1 mg / ml, HPMC (4000 mPas) 1.1 mg / ml, PEG6000 10.7 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / mL and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0111] Prescription 1: recombinant human nerve growth factor 100 μg / ml, trehalose 50 mg / ml, mannitol 14 mg / ml, polyvinyl alcohol 9 mg / ml, PEG6000 10 mg / ml, L-methionine 0.01 mg / ml, disodium hydrogen phosphate dodecahydrate 7.24 mg / mL and sodium dihydrogen phosphate monohydrate 1.08 mg / ml.

[0112] After successful modeling, ophthalmological examination was performed and recorded as Day 1 (abbreviated as D1), the 4th day of administration was recorded as Day 5 (abbreviated as D5), and the 8th day of administration was recorded as Day 9 (abbreviated as D9). On Day 1, Day 5 and Day 9 (20:00 on the same day), the corneal nerve perception of the animals was measured with a corneal sensory sensitivity meter; after sodium fluorescein staining, the corneal epithelial defect was observed and evaluated by slit lamp; on Day 9, the animal cornea was taken and gold chloride staining was performed to evaluate the recovery of the corneal nerve.

[0113] Judgment criteria: corneal sodium fluorescein staining score was used to evaluate the corneal epithelial defect of the animals, corneal sensory sensitivity meter was used to measure corneal nerve perception, and gold chloride corneal nerve counting was used to quantify the recovery of corneal nerves.

[0114] The results are shown in Tables 11-13 below.

[0115] Table 11 Corneal perception sensitivity

[0116]

[0117] As shown in Table 11, compared with the normal control group, the D1 examination showed that the corneal sensitivity of the model control group, the original preparation treatment group and the new preparation treatment group was significantly different ( a P≤0.001), the model was successfully established.

[0118] After 4 days of administration, the corneal sensitivity of the original preparation treatment group and the new preparation treatment group was significantly different from that of the model control group ( b P≤0.05). This indicates that after 4 consecutive days of administration, the comparative ophthalmic preparation and the ophthalmic preparation of prescription 1 have the effect of improving corneal sensitivity.

[0119] After 8 days of administration, the corneal sensitivity of the original preparation treatment group and the new preparation treatment group was significantly different from that of the model control group ( c P≤0.001). This indicates that after 8 days of continuous administration, the comparison ophthalmic preparation and the ophthalmic preparation of prescription 1 can further improve corneal sensitivity.

[0120] Table 12 Corneal epithelial defect repair

[0121]

[0122] As shown in Table 12, compared with the normal control group, the D1 examination showed that the fluorescent staining scores of the model control group, the original preparation treatment group and the new preparation treatment group showed extremely significant differences ( a P≤0.001), the model was successfully established.

[0123] After 4 days of administration, the fluorescent staining scores of the original preparation treatment group and the new preparation treatment group were significantly different from those of the model control group ( b P≤0.05). This indicates that after 4 consecutive days of administration, the comparative ophthalmic preparation and the ophthalmic preparation of prescription 1 have the efficacy of repairing corneal epithelial defects.

[0124] After 8 days of administration, the fluorescent staining scores of the original preparation treatment group and the new preparation treatment group showed a very significant difference compared with the model control group ( c P≤0.001). This indicates that after 8 days of continuous administration, the comparative ophthalmic preparation and the ophthalmic preparation of prescription 1 can further repair corneal epithelial defects.

[0125] Table 13 Corneal nerve recovery

[0126] Group Test animals (only) Corneal nerve length (mean±SD) Normal control group 16 1240.99±171.27 Model control group 16 <![CDATA[862.45±59.46 a ]]> Original preparation treatment group 16 <![CDATA[1140.60±78.41 c ]]> New preparation treatment group 16 <![CDATA[1130.62±79.87 c ]]>

[0127] As shown in Table 13, compared with the normal control group, the D9 examination showed that the length of the corneal nerve in the model control group was significantly different ( a P≤0.001), the model was successfully established.

[0128] After 8 days of administration, the corneal nerve lengths in the original preparation treatment group and the new preparation treatment group were significantly different from those in the model control group ( c P≤0.001). This indicates that after continuous administration for 8 days, the comparative ophthalmic preparation and the ophthalmic preparation of prescription 1 have the effect of increasing the length of the corneal nerve of the model mice.

[0129] In summary, after continuous administration of the ophthalmic preparation of Prescription 1 for 4 and 8 days, it can significantly improve the corneal sensory sensitivity of the model mice, significantly reduce the corneal sodium fluorescein staining score of the model mice, and significantly increase the corneal nerve length of the model mice.

Claims

1. A composition containing recombinant human nerve growth factor, characterized in that: The invention comprises the following components in parts by weight: 0.005-1 part of recombinant human nerve growth factor, 20-70 parts of trehalose, 5-30 parts of mannitol and 1-13 parts of polyvinyl alcohol.

2. The composition according to claim 1, characterized in that The invention comprises the following components in parts by weight: 0.005-0.1 parts of recombinant human nerve growth factor, 40-70 parts of trehalose, 5-20 parts of mannitol and 1-11 parts of polyvinyl alcohol.

3. The composition according to claim 2, characterized in that The invention comprises the following components in parts by weight: 0.005-0.06 parts of recombinant human nerve growth factor, 45-60 parts of trehalose, 8-14 parts of mannitol and 5-9 parts of polyvinyl alcohol.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of ophthalmic preparations.

5. The use according to claim 4, characterized in that: The ophthalmic preparation is used for treating corneal lesions.

6. The use according to claim 5, characterized in that: The corneal lesions are caused by decreased corneal sensitivity, corneal denervation or corneal damage.

7. An ophthalmic preparation, characterized in that: The invention comprises the composition according to any one of claims 1 to 3.

8. The ophthalmic preparation according to claim 7, characterized in that The ophthalmic preparation has a pH of 6.5-7.5, an osmotic pressure of 200-380 mOsmol / kg, and a viscosity of 1-25 mm 2 / s.

9. The ophthalmic preparation according to claim 7, characterized in that The invention also comprises an antioxidant compound, wherein the antioxidant compound comprises L-methionine and / or cysteine; and the content of the antioxidant compound is 0.01-0.1 mg / ml.

10. The ophthalmic preparation according to claim 7, characterized in that It also includes a high molecular polymer, which is polyethylene glycol with a molecular weight of 4000-8000 and a content of 5-25 mg / ml.

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