An ophthalmic gel formulation and its application

Abstract

This invention discloses an ophthalmic gel formulation. The ophthalmic gel of this invention comprises recombinant human β-nerve growth factor, histidine or citrate, hydroxypropyl methylcellulose, polyvinyl alcohol, and a protective agent, wherein the protective agent comprises L-methionine. The ophthalmic gel of this invention exhibits good storage stability and can be stored for extended periods at 5±3℃.

Description

Technical Field

[0001] This invention relates to an ophthalmic gel formulation and its application. Background Technology

[0002] Nerve growth factor (NGF) is a bioactive factor in the neurotrophic factor family, composed of three subunits: α, β, and γ. NGF is a neurotrophic factor with dual biological functions of neurotrophic and neurite-promoting growth, playing a crucial regulatory role in the development, differentiation, growth, regeneration, and functional expression of the central and peripheral nervous systems. The active region is the β subunit, a dimer formed by two single chains of 118 amino acids linked non-covalently. This is the active subunit through which NGF exerts its neuroregeneration and repair functions. Activation of its receptor is regulated by signaling pathways such as PI3K-AKT, PLCγ-PKC, Ras-MAPK, and Rac / CDC42.

[0003] Currently, Oxervate is the only globally approved rhNGF drug for the treatment of neurotrophic keratitis. However, the active ingredient rhNGF in Oxervate is unstable; its purity and content decrease rapidly under the intended use conditions (such as light exposure, refrigeration, and high temperatures). According to the Oxervate instructions, the product must be stored at -20°C; after being provided to patients, it can be stored at 5±3°C for one week; any unused medication stored at 5±3°C for more than one week or any remaining medication opened for more than 12 hours should not be used. These stringent usage requirements significantly limit patient accessibility and result in high clinical costs. Summary of the Invention

[0004] One object of the present invention is to provide an ophthalmic gel and its application.

[0005] One aspect of the present invention provides an ophthalmic gel comprising: Recombinant human β-nerve growth factor, Histidine or citrate Hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol (PVA), and A protective agent comprising L-methionine.

[0006] In one embodiment, the protective agent further comprises one or more of disaccharides, hexatols, glycerol, and amino acids.

[0007] In one embodiment, the disaccharide is selected from either trehalose or sucrose.

[0008] In one embodiment, the hexose alcohol is selected from either mannitol or sorbitol.

[0009] In one embodiment, the amino acid is selected from either arginine or glycine.

[0010] In one embodiment, the mass ratio of the hydroxypropyl methylcellulose to the polyvinyl alcohol is 5:7.

[0011] In one embodiment, the ophthalmic gel comprises: 10-40 µg / ml recombinant human β-nerve growth factor, 10 mM-25 mM histidine or citrate, 10 mg / ml HPMC, 14 mg / ml PVA, and 1 mg / ml L-methionine.

[0012] In one embodiment, the protecting agent is a disaccharide and an amino acid. In one embodiment, the protecting agent is a hexapeptide and glycerol. In one embodiment, the protecting agent is an amino acid and glycerol. In one embodiment, the protecting agent is a disaccharide and a hexapeptide. In one embodiment, the protecting agent is a disaccharide, a hexapeptide, and an amino acid. In one embodiment, the protecting agent is a disaccharide, glycerol, and an amino acid.

[0013] In one embodiment, the disaccharide content is 20-50 mg / ml. In one embodiment, the amino acid content is 60-150 mM. In one embodiment, the hexatol content is 10-50 mg / ml. In one embodiment, the glycerol content is 5-50 mg / ml.

[0014] In one embodiment, the ophthalmic gel further comprises a surfactant.

[0015] In one embodiment, the surfactant is polysorbate 80, PEG6000, F68, or Tween 80.

[0016] In one embodiment, the surfactant content is 0.1-0.5 mg / ml.

[0017] Another object of the present invention is to provide the use of the ophthalmic gel in the preparation of medicaments for treating optic neuropathy and corneal lesions.

[0018] In one embodiment, the corneal lesion is caused by decreased corneal sensitivity, corneal denervation, or corneal damage.

[0019] In one embodiment, the ophthalmic gel is used to treat neurotrophic keratitis or dry eye.

[0020] The ophthalmic gel of the present invention has good storage stability and can be stored for a long time at 5±3℃. Detailed Implementation

[0021] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0022] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0023] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] This invention does not limit the source of raw materials. Unless otherwise specified, all raw materials and excipients used are pharmaceutical-grade commercially available products, and all solvents used are water for injection unless otherwise specified. Regarding analytical methods, the embodiments of this invention all use the following methods.

[0025] Recombinant human β-nerve growth factor, specifically the recombinant human β-nerve growth factor prepared under patent 201910390559.2; Evaluation of main peak purity and peak area using RP-HPLC (reversed-phase chromatography) The purity was determined according to the Pharmacopoeia of the People's Republic of China (2025 edition, 0512 High Performance Liquid Chromatography), using a C18 alkyl-bonded silica gel column. When used for purity determination, the sample purity was calculated using the area normalization method. For formulations based on HPMC and PVA, a precise amount was weighed, diluted 2-fold with the mobile phase, thoroughly shaken and mixed, centrifuged, and the supernatant was injected for analysis. The chromatogram, peak area of ​​the target protein, and purity were recorded. The peak area and purity results of each group of samples were compared and evaluated.

[0026] I. Ophthalmic Gel Formulation - Histidine Cache System (1) Ophthalmic gel preparation 1-3

[0027] Weigh out recombinant human β-nerve growth factor (rhNGF), dissolve it in water for injection, add excipients and mix well, then add histidine hydrochloric acid to adjust the pH to 6.5.

[0028] The compositions of ophthalmic gel formulations 1-3 are shown in Table 1 below.

[0029] Table 1. Composition of ophthalmic gel formulations

[0030] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 2.

[0031] Table 2. Purity of rhNGF after storage at different temperatures

[0032] The ophthalmic gel formulation 3 showed the least decrease in rhNGF purity, and its stability was superior to that of ophthalmic gel formulations 1 and 2. The use of a combination of HPMC and PVA 1 as the gel matrix had a crucial impact on the stability of the ophthalmic gel formulations. (2) Ophthalmic gel preparation 4-7

[0033] Ophthalmic gel formulation 4-7 was prepared according to the method described above. The composition of ophthalmic gel formulation 4-7 is shown in Table 3 below.

[0034] Table 3. Composition of ophthalmic gel formulations

[0035] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 4.

[0036] Table 4. Purity of rhNGF after storage at different temperatures

[0037] The rhNGF purity of ophthalmic gel formulations 4-7 was greater than 85% after storage at 5±3℃ for 1 month and at 25℃ for 45 days. Using a combination of HPMC and PVA 1 as the gel matrix, the stability of the ophthalmic gel formulations was satisfactory, provided that no other protective agents were present besides L-Met. Under these conditions, the ophthalmic gel formulations remained relatively stable even when other protective agents, such as trehalose, sucrose, and mannitol, were added.

[0038] (3) Ophthalmic gel preparation 8-14 Ophthalmic gel formulation 8-14 was prepared according to the method described above. The composition of ophthalmic gel formulation 8-14 is shown in Table 5 below.

[0039] Table 5. Composition of ophthalmic gel formulations

[0040] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 6.

[0041] Table 6. Purity of rhNGF after storage at different temperatures

[0042] The ophthalmic gel formulation 8-14 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 3 months, greater than 85% after storage at 25℃ for 30 days, and greater than 80% after storage at 37℃ for 10 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulation remained relatively stable with the addition of polysorbate 80 and other protective agents, such as trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, and glycine, in addition to L-Met.

[0043] (4) Ophthalmic gel preparations 15-19 Ophthalmic gel formulation 15-19 was prepared according to the method described above. The composition of ophthalmic gel formulation 15-19 is shown in Table 7 below.

[0044] Table 7. Composition of ophthalmic gel formulations

[0045] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 8.

[0046] Table 8. Purity of rhNGF after storage at different temperatures

[0047] The ophthalmic gel formulations 15-19 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 3 months, greater than 85% after storage at 25℃ for 30 days, and greater than 85% after storage at 37℃ for 5 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable when, in addition to L-Met, two or more of the following protective agents were added: polysorbate 80 and other protective agents such as trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, and glycine.

[0048] (5) Ophthalmic gel preparations 20-22 Ophthalmic gel formulation 20-22 was prepared according to the above method. The composition of ophthalmic gel formulation 20-22 is shown in Table 9 below.

[0049] Table 9. Composition of ophthalmic gel formulations

[0050] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 10.

[0051] Table 10. Purity of rhNGF after storage at different temperatures

[0052] The ophthalmic gel formulations 20-22 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 3 months, greater than 90% after storage at 25℃ for 30 days, and greater than 85% after storage at 37℃ for 5 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable when any three of the following protective agents were added besides L-Met: trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, and glycine.

[0053] II. Ophthalmic Preparations - Citric Acid Caching System (6) Ophthalmic gel preparations 23-30

[0054] The preparation of ophthalmic gel formulations is the same as above. The composition of ophthalmic gel formulations 23-30 is shown in Table 11 below.

[0055] Table 11. Composition of ophthalmic gel formulations

[0056] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 12.

[0057] Table 12. Purity of rhNGF after storage at different temperatures

[0058] The ophthalmic gel formulations 23-30 maintained a rhNGF purity greater than 95% after storage at 5±3℃ for one month and greater than 85% after storage at 25℃ for 45 days. Using a combination of HPMC and PVA 1 as the gel matrix, and assuming no other protective agents besides L-Met, the stability of the ophthalmic gel formulations was satisfactory. Adding other protective agents, such as any one of trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, or glycine, further improved the stability of the ophthalmic gel formulations.

[0059] (7) Ophthalmic gel preparations 31-38 The preparation of ophthalmic gel formulations is the same as above. The composition of ophthalmic gel formulations 31-38 is shown in Tables 13 and 14 below.

[0060] Table 13. Composition of ophthalmic gel formulations

[0061] Table 14. Composition of ophthalmic gel formulations

[0062] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 15.

[0063] Table 15. Purity of rhNGF after storage at different temperatures

[0064] The ophthalmic gel formulations 31-38 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 12 months, greater than 85% after storage at 25℃ for 45 days, and greater than 80% after storage at 37℃ for 7 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable with the addition of other protective agents besides L-Met, such as any one of trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, glycine, etc., as well as surfactants.

[0065] (8) Ophthalmic gel preparations 39-41 The preparation of ophthalmic gel formulations is the same as above. The composition of ophthalmic gel formulations 39-41 is shown in Table 16 below.

[0066] Table 16. Composition of ophthalmic gel formulations

[0067] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 17.

[0068] Table 17. Purity of rhNGF after storage at different temperatures

[0069] The ophthalmic gel formulations 39-41 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 12 months, greater than 85% after storage at 25℃ for 45 days, and greater than 85% after storage at 37℃ for 10 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable with the addition of any two of the following protective agents besides L-Met: trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, glycine, and surfactants.

[0070] (9) Ophthalmic gel preparations 42-46 The preparation of ophthalmic gel formulations is the same as above. The composition of ophthalmic gel formulations 42-46 is shown in Table 18 below.

[0071] Table 18. Composition of ophthalmic gel formulations

[0072] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 19.

[0073] Table 19. Purity of rhNGF after storage at different temperatures

[0074] The ophthalmic gel formulations 42-46 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 15 months, greater than 85% after storage at 25℃ for 45 days, and greater than 85% after storage at 37℃ for 7 days. When using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable when any two or three of the following protective agents were added besides L-Met: trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, and glycine.

[0075] (10) Ophthalmic gel preparations 47-49 The preparation of ophthalmic gel formulations is the same as above. The composition of ophthalmic gel formulations 47-49 is shown in Table 20 below.

[0076] Table 20. Composition of ophthalmic gel formulations

[0077] The purity of rhNGF in different gel formulations after storage at different temperatures was determined by HPLC-FLD method, and the results are shown in Table 21.

[0078] Table 21. Purity of rhNGF after storage at different temperatures

[0079] The ophthalmic gel formulations 47-49 maintained a rhNGF purity greater than 90% after storage at 5±3℃ for 12 months, greater than 85% after storage at 25℃ for 60 days, and greater than 85% after storage at 37℃ for 7 days. Using a combination of HPMC and PVA 1 as the gel matrix, the ophthalmic gel formulations remained relatively stable with the addition of any three of the following protective agents besides L-Met: trehalose, sucrose, mannitol, glycerin, sorbitol, arginine, and glycine, as well as surfactants.

Claims

1. An ophthalmic gel, characterized in that, The ophthalmic gel comprises: Histidine or citrate Recombinant human β-nerve growth factor, Hydroxypropyl methylcellulose and polyvinyl alcohol, and A protective agent comprising L-methionine.

2. The ophthalmic gel according to claim 1, characterized in that, The protective agent also includes any one or more of disaccharides, hexatols, glycerol, and amino acids.

3. The ophthalmic gel according to claim 2, characterized in that, The disaccharide is selected from either trehalose or sucrose.

4. The ophthalmic gel according to claim 2, characterized in that, The hexose alcohol is selected from either mannitol or sorbitol.

5. The ophthalmic gel according to claim 2, characterized in that, The amino acid is selected from either arginine or glycine.

6. The ophthalmic gel according to claim 1 or 2, characterized in that, The mass ratio of the hydroxypropyl methylcellulose to the polyvinyl alcohol is 5:

7.

7. The ophthalmic gel according to claim 1 or 2, characterized in that, The ophthalmic gel also contains surfactants.

8. The ophthalmic gel according to claim 7, characterized in that, The surfactant is polysorbate 80, PEG6000, F68, or Tween 80.

9. Use of the ophthalmic gel according to any one of claims 1-8 in the preparation of a medicament for treating optic neuropathy and corneal lesions.

10. The application according to claim 9, characterized in that, The corneal lesions are caused by decreased corneal sensitivity, corneal denervation, or corneal damage.

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