A nerve growth factor composition and use thereof

Abstract

The application belongs to the field of biological preparations and provides a nerve growth factor (NGF) composition. The NGF composition contains mannitol, the content of NGF in the composition is greatly increased, and the storage space is greatly saved when the NGF is stored as a protein stock solution. The liquid preparation of the application has good stability, does not need to be stored by freezing, has high safety, has a wide applicable range of NGF concentration, and can be used for eye drop administration to treat ocular fundus diseases such as glaucoma, and ocular surface diseases such as dry eye disease and neurotrophic keratitis.

Description

Technical Field

[0001] This invention belongs to the field of biological agents and relates to compositions containing nerve growth factor and the application of such compositions. Background Technology

[0002] Nerve growth factor (NGF) possesses dual biological functions of neuronal nutrition and neurite growth promotion. It plays a crucial regulatory role in the development, differentiation, growth, regeneration, and functional expression of central and peripheral neurons, and has been studied and reported in Alzheimer's disease, diabetic peripheral neuropathy, arthritis, pain, and ophthalmic diseases. In 2017 and 2018, Domperidone's NGF eye drops (Eosavi) were launched in the EU and US as an orphan drug for neurotrophic keratitis, and subsequently expanded to include clinical trials for indications such as glaucoma, although significant efficacy has not yet been shown in glaucoma. Since then, research on the application of NGF in ophthalmic diseases has become a hot topic, with several domestic products under development focusing on ocular surface diseases such as neurotrophic keratitis or dry eye disease, while research on fundus diseases is relatively limited.

[0003] Currently, ocular surface diseases are typically treated with topical eye drops, while fundus diseases tend to be treated with injections or other methods that directly reach the fundus. However, for chronic fundus diseases requiring long-term medication, repeated intraocular injections increase the risk of ocular inflammation and bleeding, and vitreous injections may lead to vitreous opacities. Therefore, if eye drops can effectively treat fundus diseases, this method of administration is more readily accepted by clinicians and patients due to its safety and convenience. Given the important applications of NGF in ocular diseases, screening an eye drop formulation that ensures NGF efficacy is of great clinical research value. Furthermore, if the same formulation can be used for both ocular surface and fundus diseases, the medicinal value of NGF can be maximized.

[0004] Most current NGF formulations are lyophilized powders, and even solutions often use albumin as a stabilizer or are directly frozen for long-term storage. According to its instructions, the Italian company Tonpai's NGF eye drops require long-term storage at -20°C, and storage at 2-8°C for no more than one week, indicating stringent storage conditions. Patent CN 1163265C provides a solution using HSA protein as a carrier, raising uncertain safety concerns. Furthermore, due to the strong hydrophobicity of nerve growth factor protein, its solubility in ordinary solutions is low, and precipitation occurs at slightly higher concentrations. Therefore, the concentration of the NGF protein stock solution cannot be high. However, low-concentration stock solutions require larger storage volumes during production, especially since biological protein stock solutions are typically stored at -80°C. Large volumes of stock solutions would require more and larger ultra-low temperature freezers, causing numerous inconveniences in stock solution storage. Therefore, there is an urgent need to develop an NGF formulation that can be stored long-term at 2-8°C, is protein-free, and has good stability. Summary of the Invention

[0005] The purpose of this invention is to provide a nerve growth factor composition with a stable protein content to solve the problems existing in the prior art.

[0006] In a first aspect, the present invention provides a nerve growth factor composition comprising mannitol, wherein the mannitol content is 20-50 mg / mL.

[0007] In a preferred embodiment, the mannitol content is 30-44 mg / mL.

[0008] In some embodiments, the NGF content is 0.02~20 mg / mL; preferably, the NGF content is 0.02~18.8 mg / mL; more preferably, the NGF content is 0.02~2 mg / mL.

[0009] In some embodiments, the nerve growth factor composition further comprises a buffer solution, the buffer solution being PB or Tris-HCl buffer solution, the buffer solution concentration being 10 mM, and the pH being 6.0 to 6.5.

[0010] Preferably, the buffer solution is a PB solution with a concentration of 10 mM and a pH of 6.0 to 6.5.

[0011] In some embodiments, the nerve growth factor composition comprises a 10 mM PB solution at pH 6.0–6.5, 40 mg / mL mannitol, and 0.02–20 mg / mL NGF.

[0012] In some embodiments, the nerve growth factor composition further comprises methionine at a concentration of 0.01 to 0.2 mg / mL; preferably, the concentration of methionine is 0.05 mg / mL.

[0013] In some embodiments, the nerve growth factor composition further comprises a surfactant selected from PEG6000 or PEG8000 at a concentration of 1 to 10 mg / mL.

[0014] In some embodiments, the nerve growth factor composition may further contain PS80 at a concentration of 0.1 to 0.6 mg / mL.

[0015] In some preferred embodiments, the concentration of PS80 in the nerve growth factor composition is 0.2 mg / mL.

[0016] In a preferred embodiment, the nerve growth factor composition comprises: 10 mmol / L PB, pH 6.0~6.5, 0.02~2 mg / mL NGF, 44 mg / mL mannitol, 10 mg / mL PEG8000, and 0.05 mg / mL methionine.

[0017] In some preferred embodiments, the concentration of NGF in the nerve growth factor composition is 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, or 2 mg / mL.

[0018] In some embodiments, the nerve growth factor composition is an eye drop.

[0019] In some embodiments, the nerve growth factor composition does not contain human serum albumin or a lyophilization protectant.

[0020] In a second aspect, the invention provides the use of the nerve growth factor composition in the preparation of medicaments for treating ocular surface diseases such as dry eye or neurotrophic keratitis, and fundus diseases such as optic nerve damage in glaucoma.

[0021] In a third aspect, the present invention provides the use of mannitol in the preparation of nerve growth factor compositions, wherein the use is to increase the NGF content; preferably, the NGF content can reach 20 mg / mL.

[0022] In a fourth aspect, the present invention provides a nerve growth factor composition for treating ocular surface diseases such as dry eye or neurotrophic keratitis, and fundus diseases such as optic nerve damage in glaucoma.

[0023] In a fifth aspect, a method is provided for treating ocular surface diseases such as dry eye or neurotrophic keratitis, and fundus diseases such as optic nerve damage in glaucoma, comprising administering the nerve growth factor composition of the present invention to a subject in need. The method includes administration by eye drops.

[0024] In this invention, the amino acid sequence of the NGF is shown in SEQ ID NO: 1.

[0025] The nerve growth factor composition of the present invention has good stability, can effectively inhibit the increase of fragments and aggregates, and can support blow-fill-seal (BFS) filling; it does not contain human serum albumin or other protein components, has a simple composition, and good safety; the NGF concentration can reach 20 mg / mL, and it can be stably stored for at least 6 months at 2~8℃, and can be stored as a protein stock solution; the composition does not require lyophilization to prepare, nor freezing to store, and is convenient to carry and use. Attached Figure Description

[0026] Figure 1 : SEC-HPLC small molecule fragment results in the formulation component screening of Example 3;

[0027] Figure 2 The IOD values ​​of the corneal fluorescein sodium-stained areas in animals with dry eye disease, compared with the solvent control group, P <0.05, P <0.01; Figure 3 The corneal fluorescein sodium staining score of animals with neurotrophic keratitis compared with the solvent control group. P <0.0001; Figure 4 RGC count (cells / mm) on retinal slices of glaucoma animals 2 Mean ± SD), where, compared with the solvent control group, P <0.05, P <0.01, P <0.001. Detailed Implementation

[0028] The present invention will be described below through specific embodiments, but the content of the present invention is not limited thereto.

[0029] In the following embodiments, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the art and can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art can perform the methods and obtain the corresponding results without any doubt based on the description of the embodiments.

[0030] In the following examples, the nerve growth factor composition is prepared into a formulation for use. Therefore, "nerve growth factor composition" and "NGF formulation" have the same meaning and can be used interchangeably.

[0031] Example 1: Experiment on the effect of mannitol on NGF solubility Recombinant human nerve growth factor (NGF) protein (SEQ ID NO: 1) was expressed and prepared using commercially licensed CHO-K1 cells. The study found that NGF has low solubility in common phosphate-buffered saline (PBS). Specifically, during chromatographic purification, when the protein concentration collected with PBS solution (i.e., 10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (PB), approximately 9 mg / mL NaCl, pH 7.2) was as high as about 1 mg / mL, the collected protein exhibited a distinct milky appearance and precipitated within a short time, becoming irreversibly insoluble upon dilution. If PBS was added immediately while the milky state was still present, diluting to approximately 0.5 mg / mL, the solution regained its clarity. PB buffer is salt-free and has low conductivity, making it even worse than PBS for the highly hydrophobic NGF. In PB, an NGF concentration of approximately 0.6 mg / mL already exhibited an opalescent appearance, and the solution was not completely transparent.

[0032] Based on the above observations, the applicant explored methods to improve the solubility of NGF. To prepare PBS or PB-mannitol solutions, PB solutions containing approximately 9 mg / mL NaCl or 40 mg / mL mannitol were added to NGF solutions in PB solvent. These solutions were then concentrated using ultrafiltration centrifuge tubes to increase the NGF protein concentration. The results showed that even with extended centrifugation times (20-30 min or more) after ultrafiltration, the protein concentration in PBS-solvent samples was difficult to increase to 1 mg / mL; instead, protein yield decreased significantly due to turbidity and precipitation. However, with the addition of mannitol to PB, the NGF protein concentration was easily increased to over 2 mg / mL using ultrafiltration centrifuge tubes. Extending the ultrafiltration centrifugation time to over 10 min (depending on the initial sample concentration) increased the protein concentration to approximately 20 mg / mL, and the concentrated sample remained clear throughout. Therefore, NGF protein can be effectively concentrated using PB solutions with added mannitol as the stock solution, avoiding the problems of low protein concentration and large stock solution storage volume associated with PBS solvents.

[0033] Table 1: Ultrafiltration Concentration Analysis of Small-Scale NGF Samples

[0034] Note: Yield = (protein concentration after concentration × volume) / (protein concentration before concentration × volume) Example 2: Different buffer systems and pH screening Based on the commonly used pH of eye drops, the pH range of the formulation solution was set to 6.0–7.5, and both PB and Tris buffers were tested. The NGF protein stock solution was prepared using the same method (desalted and replaced with 10 mM PB and 10 mM Tris-HCl, respectively), and other excipients were added according to the design in Table 2 to prepare a formulation with a final NGF concentration of 0.2 mg / mL. The prepared formulation was manually injected into low-density polyethylene (LDPE) eye drop packaging using a disposable syringe, sealed with sealing film, and placed in a stability incubator at 40°C for 4 weeks. Two samples were taken weekly for testing. The testing items were based on the Chinese Pharmacopoeia, and key testing items included visible foreign matter, SEC-HPLC purity, RP-HPLC purity, protein content, and in vitro biological activity, as shown in Table 3.

[0035] Table 2: Buffer and pH Screening Formulation Design

[0036] Table 3: Analytical Methods for Formulation Detection (Chinese Pharmacopoeia)

[0037] (1) Visible foreign matter: Regardless of whether it was the Tris-HCl or PB buffer system, FI, F-II, and FV (pH 6.0 and 6.5) were clear solutions after preparation and remained clear during the 4-week acceleration process. However, F-III, F-IV, F-VI, and F-VII (pH 7.0 and 7.5) became turbid immediately after preparation, indicating that the samples were unstable at pH ≥ 7.0, and therefore no further tests were performed.

[0038] (2) SEC-HPLC purity: During the 4-week accelerated SEC-HPLC process at 40℃, the purity of FI, F-II, and FV all decreased, but the decrease was small, within 4%. The trend of change was consistent across groups, with no significant differences. However, the retention time of all samples in the 4th week shifted, and the results were not representative and were not recorded. The results are shown in Table 4.

[0039] (3) RP-HPLC: Overall, the RP purity of FI and F-II showed a consistent decreasing trend, while the purity of FV decreased slightly slower than that of FI and F-II, indicating that the Tris-HCl system was slightly more stable than the phosphoric acid system at pH 6.5. However, the optimal buffer range for Tris-HCl is pH 7.0–9.0, so the phosphoric acid buffer system was ultimately chosen as the buffer system. The decrease in RP-HPLC purity was mainly due to the increase in oxidation peaks, with the oxidation peaks of FI and F-II increasing slightly faster than that of FV. The denaturation peaks of all groups increased. The results are shown in Table 4.

[0040] Table 4: Purity Analysis by RP-HPLC

[0041] (4) Protein content and biological activity: During the 40℃ acceleration process, the protein content of each group remained relatively stable, ranging from 0.16 to 0.22 mg / mL. Biological activity: The biological activity of each group did not change much during the 3 weeks of 40℃ acceleration, but decreased in the 4th week. There was no significant difference between the groups. The biological activity data are shown in Table 5.

[0042] Table 5: Biological activity of the target protein

[0043] In summary, FI, F-II, and FV exhibit good stability, indicating that the sample demonstrates good stability within the pH range of 6.0–6.5. Both Tris-HCl and PB can be used as buffer solutions, but considering that Tris-HCl's buffering capacity is inferior to that of the phosphate system within this pH range, 10 mmol / L PB was chosen as the buffer solution for this product.

[0044] Example 3: Formulation stability screening Based on the screening results above, 10 mM PB was used as the buffer solution at pH 6.3. Other components in the formulation were further screened. The formulation of this example was prepared according to the contents shown in Table 6, with an NGF protein content of 0.2 mg / mL. After packaging and sealing as described in Example 2, it was placed in a stability incubator at 40°C for 4 weeks, with two samples taken each week for testing.

[0045] Table 6: Formulation Design for Screening Pharmaceutical Components (Units for each component are mg / mL)

[0046] The test results are as follows: (1) Visible foreign matter: The sample of formulation F1 in week 1 was not detected due to a placement error (the formulation itself does not affect subsequent experiments), while other formulations remained clear throughout.

[0047] (2) Osmotic pressure: Osmotic pressure was tested on the zero-point samples to confirm whether the osmotic pressure of each formulation met the requirements. The expected optimal osmotic pressure was around 300 mOsm / kg. The test results showed that, except for F8 which had a slightly higher osmotic pressure, the initial osmotic pressure of the other samples was around 300 mOsm / kg. The results are shown in Table 7.

[0048] Table 7: Osmotic pressure test results (mOsm / kg)

[0049] (3) Protein content: After four weeks of high-temperature treatment, the protein content of each preparation did not change significantly.

[0050] (4) SEC-HPLC: The purity of SEC in groups F6 and F7 decreased at a slightly faster rate than in other groups, mainly due to the growth of the peak after acceleration. Compared with the F1 control group, this indicates that glycerol and propylene glycol did not have a better effect on product stability than PEG.

[0051] Compared to F1, F8, and F9, F9 did not contain PS80, yet its SEC purity was not lower than that of F9 containing PS80; in fact, it was slightly higher. This indicates that the addition of PS80 has no significant effect on formulation stability.

[0052] Specifically, during the 4-week storage of the F9 formulation at 40°C, small molecule fragments were not detected by SEC-HPLC for the first 3 weeks, and only a very small amount (0.04%) was detected at 40°C for 4 weeks. (See attached image.) Figure 1 Other prescriptions showed varying degrees of small molecule fragments in tests conducted within 1-3 weeks.

[0053] The effects of methionine concentration on formulation stability were investigated in four groups (F1-F4). No significant differences were found in SEC purity among the four groups, with all groups showing a decrease rate of less than 5%, and the differences between groups were small. Therefore, the formulation exhibits good SEC-HPLC purity stability within the methionine concentration range of 0.01–0.2 mg / mL (Table 8).

[0054] Table 8: Purity Analysis by SEC-HPLC

[0055] (5) RP-HPLC: Compared with F1, F8 and F9, there were no significant differences in the trends of the target protein, oxidation peak and denaturation peak in RP, indicating that the addition of PS80 had no significant effect on the purity of the formulation by RP-HPLC.

[0056] The effects of methionine concentration on formulation stability were investigated in four groups (F1-F4). No significant differences were observed among the four groups, but a trend emerged where the oxidation peak increased more slowly and the target protein decreased more slowly with increasing methionine concentration. Overall, the results indicate that the RP-UPLC purity of the formulation did not differ significantly within the methionine concentration range of 0.01–0.2 mg / mL. The results are shown in Table 9.

[0057] Table 9: Purity Analysis of RP-UPLC

[0058] Analysis of all test results: (1) Methionine concentration: F1~F4 contain different concentrations of methionine. The appearance and protein content showed no significant changes during the accelerated protein detection process. The purity of SEC-HPLC and RP-HPLC decreased, but there was no significant difference. It is recommended that the concentration of methionine be 0.01~0.2 mg / mL.

[0059] (2) Stabilizers: Formulations F1 (PEG8000), F6 (glycerol), and F7 (propylene glycol) showed no significant difference in protein content and RP-HPLC purity. Formulations F6 and F7, which added glycerol and propylene glycol, showed lower SEC-HPLC purity, with significantly higher peak values ​​for small molecule fragments compared to F1. (See [reference]). Figure 1 Therefore, glycerol and propylene glycol are detrimental to protein stability.

[0060] (3) Surfactant PS80: For formulations F1, F8, and F9, the changes in the target protein RP, oxidation peak, and denaturation peak were not significantly different when 0.2 mg / mL, 0.1 mg / mL, and no PS80 was added, respectively. Combined with other detection items, the content of these three formulations and the SEC-HPLC detection results were also relatively consistent. Therefore, PS80 does not help the stability of the formulations, and PS80 is not added.

[0061] Formulations F1 and F5 differ only in PEG: F1 uses PEG8000 and F5 uses PEG6000. During the acceleration process, the protein content, SEC-HPLC, and RP-HPLC analyses of these two formulations were identical, with no significant differences.

[0062] Therefore, the optimal formulation ultimately selected included: 44 mg / mL mannitol, 10 mg / mL PEG8000 or PEG6000, and 0.01–0.2 mg / mL methionine. During 4 weeks of storage at 40°C, the content of small molecule fragments in this formulation remained consistently low according to SEC-HPLC.

[0063] Example 4: Validation of the suitability of different concentration formulations and BFS suitability Based on the previous development results of the 0.2 mg / mL protein formulation, one formulation with good stability was selected: 44 mg / mL mannitol, 10 mg / mL PEG8000, 0.05 mg / mL methionine, 10 mmol / L PB, pH 6.3. Protein solutions of different concentrations (20, 50, 100, 200, and 400 μg / mL) were prepared and tested at 40°C to determine if the formulation was suitable for other protein concentrations.

[0064] Consistent with previous findings, no significant changes were observed in the content of visible foreign matter, methionine, protein content, and specific activity of formulations at different protein concentrations during a 4-week high-temperature treatment at 40°C. Table 10 shows that the RP-UPLC purity, oxidation peak, and denaturation peak trends were consistent across the four concentration formulations. These results indicate that this formula is suitable for NGF protein formulations with concentrations ranging from 20 to 400 μg / mL.

[0065] Table 10: RP-HPLC analysis of the stability of different formulations at 40℃

[0066] Furthermore, considering the ease of use of eye drops, the formulation samples were filled into single-dose eye drops using a blow-fill-seal (BFS) system. This filling system uses a blow-molding process to form low-density polyethylene granules at 180°C, followed by water cooling and shaping, then filling with the sample, and finally sealing with residual heat. Quality testing of the BFS-filled formulation samples confirmed that BFS had no significant impact on product quality; therefore, this formulation is suitable for BFS filling.

[0067] Example 5 Stability Study From the optimal formulation selected through screening, 10 mmol / L PB, 44 mg / mL mannitol, 10 mg / mL PEG 8000, and 0.05 mg / mL methionine were used to prepare high-concentration and low-concentration formulations, namely 0.2 mg / mL and 2 mg / mL, and their stability was analyzed. Both concentrations of the samples were stored at 2–8°C for 6 months under long-term storage conditions, and samples were taken for quality testing to analyze their stability. The results are shown in Table 11.

[0068] Table 11: Stability Analysis of Formulation Samples

[0069] As shown in Table 11, after 6 months of storage at 2-8℃, the RP-HPLC data for the 0.2 and 2.0 mg / mL formulations showed no significant changes in key quality attributes such as purity, protein concentration, and biological activity. This indicates that the selected formulations are of good quality and can support long-term storage of high and low concentrations of NGF protein at 2-8℃.

[0070] Example 6: Pharmacodynamic study of a formulation sample for treating ocular surface disease, dry eye. A dry eye disease animal model was established. Using the selected optimal formulation (10 mmol / L PB, 44 mg / mL mannitol, 10 mg / mL PEG 8000, 0.05 mg / mL methionine), a formulation of 0.05 mg / mL NGF was prepared as the test sample to evaluate the safety and efficacy of the formulation in treating ocular diseases.

[0071] A dry eye disease animal model was established by administering benzalkonium chloride solution to the right eye of SPF-grade mice (Thacker, Abhishek Sahoo, et al. Benzalkonium chloride-induced dry eye disease animal models: Current understanding and potential for translational research[J]. Indian Journal of Ophthalmology, 2023, 71: 1256-62. DOI: 10.4103 / IJO.IJO_2791_22). The successfully modeled mice were randomly divided into two groups: a solvent control group (containing no NGF, but other components were the same as the NGF preparation) and a test substance administration group (containing the NGF preparation). Mice in each group received 5 μL / eye of either solvent or NGF preparation, three times daily for two weeks. The day before administration was designated as D0, and the day of the first administration was designated as D1. Evaluation indicators included: general clinical observation, body weight, tear secretion, and corneal fluorescein staining test (James S. Wolffsohn, et al. TFOS DEWS II Diagnostic Methodology report[J]. The Ocular Surface.2017, 15: 539-574. http: / / dx.doi.org / 10.1016 / j.jtos. 2017.05.001).

[0072] During the experiment, no abnormal changes related to drug administration were observed in the general clinical observations or body weight indicators of the animals in each group. Compared with the solvent control group, the tear secretion of the animals in the test product administration group was significantly increased, and the IOD value of the corneal fluorescein staining area on days 5-14 was significantly decreased in the test product administration group. Figure 2 .

[0073] Conclusion: Under the experimental conditions, a mouse model of dry eye disease was successfully established using benzalkonium chloride solution eye drops. No adverse reactions were observed in the screened formulation, indicating good safety. Furthermore, NGF administration effectively increased tear secretion and repaired corneal functional epithelial damage in the mouse model, demonstrating that continuous administration of NGF for two weeks had a significant therapeutic effect on the animal model of dry eye disease.

[0074] Example 7: Pharmacodynamic study of a formulation sample for treating neurotrophic keratitis, an ocular surface disease. The same formulation as in Example 6 was used to evaluate its efficacy in treating neurotrophic keratitis. A neurotrophic keratitis animal model was established by subcutaneous injection of capsaicin solution into pups produced from SPF-grade rats (Cai Yongmin. Efficacy evaluation of recombinant human nerve growth factor in a rat model of neurotrophic keratitis [D]. Tianjin Medical University, 2021.). Based on the corneal fluorescein staining score, the successfully modeled animals were randomly divided into two groups: a solvent control group and a test product treatment group. Each group of animals received 10 μL / eye of either solvent or NGF formulation via eye drops, three times daily for two consecutive weeks. The day before administration was designated as D1. Evaluation indicators included: general clinical observation, body weight, corneal fluorescein staining score, and corneal nerve length measurement.

[0075] During the experiment, no abnormal changes related to drug administration were observed in the general clinical observations or body weight indicators of any group of animals. Compared with the solvent control group, the corneal nerve length was significantly increased in the test product administration group, while the corneal fluorescein sodium staining scores on D8 and D15 were significantly decreased. (See attached figures). Figure 3 .

[0076] Conclusion: Under the conditions of this experiment, a rat model of neurotrophic keratitis was successfully established by subcutaneous injection of capsaicin. NGF administration effectively repaired functional corneal epithelial damage and increased corneal nerve length in the model rats, indicating that continuous administration of NGF for two weeks has a significant therapeutic effect on the neurotrophic keratitis animal model. This experiment provides a safety and efficacy reference for clinical research.

[0077] Example 8: Pharmacodynamic study of a formulation sample for the treatment of glaucoma, a fundus disease. A 0.2 mg / mL NGF formulation was prepared using the same formulation as in Example 6, and a glaucoma animal model was established to evaluate the safety and efficacy of the NGF formulation in treating fundus diseases.

[0078] A chronic ocular hypertension glaucoma model was established by injecting Tenon's capsule fibroblasts into the right anterior chamber of SPF-grade SD rats (Ayumi Nakagawa, Osamu Sakai, et al. Development and characterization of a new rat ocular hypertension model induced by intracameral injection of conjunctival fbroblasts[j]. Scientific Reports. 2019, 9: 6593. https: / / doi.org / 10.1038 / s41598-019-43048-2). Based on the intraocular pressure in the right eye, the successfully modeled rats were randomly divided into two groups: a solvent control group and a test substance administration group; a non-modeled group (injected with the same volume of PBS in the right anterior chamber) served as a control. The non-modeled group received no treatment, while the solvent control group and the test substance administration group received 10 μL / eye of solvent or NGF preparation twice daily for 8 weeks. Evaluation indicators included: general clinical observation, body weight, intraocular pressure, and retinal ganglion cell (RGC) count.

[0079] During the experiment: Compared with the non-membrane group, no abnormal changes related to drug administration were observed in general clinical observation or body weight indicators in any of the drug-treated groups. Intraocular pressure in the right eye of rats in the non-membrane group remained at a normal level; intraocular pressure in the right eye of rats in both the solvent control group and the test drug group remained significantly elevated, with no statistically significant difference between the two groups. After 8 weeks of drug administration, compared with the solvent control group, the number of near, middle, and distal RGCs in the retina of the test drug group was significantly increased (…). P <0.05), see data histogram. Figure 4 .

[0080] Conclusion: Under the conditions of this experiment, a rat model of chronic ocular hypertension glaucoma was successfully established by intra-anterior chamber injection of Tenon's capsule fibroblasts. No adverse reactions were observed in the selected formulation, indicating good safety. Furthermore, after 8 weeks of administration, the NGF formulation effectively increased the number of retinal ganglion cells in the model rats, demonstrating that continuous administration of the NGF formulation for 8 weeks has a significant therapeutic effect on the animal model of chronic ocular hypertension glaucoma.

[0081] Sequence information SEQ ID NO: 1 SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKG DEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA。

Claims

1. A nerve growth factor composition comprising mannitol, wherein the mannitol content is 20-50 mg / mL; preferably, the mannitol content is 30-44 mg / mL.

2. The nerve growth factor composition according to claim 1, wherein, The NGF content is 0.02~20 mg / mL; preferably, the NGF content is 0.02~18.8 mg / mL; more preferably, the NGF content is 0.02~2 mg / mL.

3. The nerve growth factor composition according to claim 1 or 2, wherein, The nerve growth factor composition further comprises a buffer solution, wherein the buffer solution is PB or Tris-HCl buffer solution, the buffer solution concentration is 10 mM, and the pH is 6.0~6.5; Preferably, the nerve growth factor composition comprises a 10 mM PB solution with a pH of 6.0-6.5, 40 mg / mL mannitol, and 0.05-20 mg / mL NGF.

4. The nerve growth factor composition according to any one of claims 1 to 3, wherein, The nerve growth factor composition further comprises methionine at a concentration of 0.01-0.2 mg / mL; preferably, the concentration of methionine is 0.05 mg / mL.

5. The nerve growth factor composition according to any one of claims 1 to 4, wherein, The nerve growth factor composition further comprises a surfactant selected from PEG6000 or PEG8000, at a concentration of 1-10 mg / mL.

6. The nerve growth factor composition according to any one of claims 1 to 5, wherein, The nerve growth factor composition also contains PS80 at a concentration of 0.1 to 0.6 mg / mL.

7. The nerve growth factor composition according to any one of claims 1 to 6, wherein, The nerve growth factor composition comprises: 10 mmol / L PB, pH 6.0-6.5, 0.02-2 mg / mL NGF, 44 mg / mL mannitol, 10 mg / mL PEG8000, and 0.05 mg / mL methionine; preferably, the NGF concentration is 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, or 2 mg / mL.

8. The nerve growth factor composition according to any one of claims 1 to 7, wherein, The composition does not contain human serum albumin or lyophilization protectant.

9. The use of the nerve growth factor composition according to any one of claims 1 to 8 in the preparation of a medicament for treating dry eye disease, neurotrophic keratitis, or optic nerve damage in glaucoma.

10. The application of mannitol in the preparation of nerve growth factor compositions, wherein, The NGF content can reach 20 mg / mL.

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