CN119258198A - Composition containing recombinant human nerve growth factor and its application and ophthalmic preparation - Google Patents
Composition containing recombinant human nerve growth factor and its application and ophthalmic preparation Download PDFInfo
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- CN119258198A CN119258198A CN202310835893.0A CN202310835893A CN119258198A CN 119258198 A CN119258198 A CN 119258198A CN 202310835893 A CN202310835893 A CN 202310835893A CN 119258198 A CN119258198 A CN 119258198A
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- growth factor
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- human nerve
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Zoology (AREA)
- Ophthalmology & Optometry (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Inorganic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a composition containing recombinant human nerve growth factor, application thereof and an ophthalmic preparation. The composition comprises, 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. The invention utilizes the combination of trehalose, mannitol and polyvinyl alcohol with the recombinant human nerve growth factor, can obviously improve the stability of the recombinant human nerve growth factor, reduces the loss of pharmacological active ingredients of the ophthalmic preparation during the manufacturing, transportation and long-term storage, ensures the medication safety and the treatment effect, has good stability of the recombinant human nerve growth factor in the ophthalmic preparation, and can better meet the clinical demands of patients with eye diseases.
Description
Technical Field
The invention belongs to the technical field of ophthalmic preparations, and in particular relates to a composition containing recombinant human nerve growth factor, application thereof and an ophthalmic preparation.
Background
The cornea is positioned at 1/6 of the front end of the eyeball wall, is a vascular-free transparent membrane and contains abundant sensory nerve endings. Keratopathy is harmful, and can cause corneal edema, eye pain, vision deterioration and the like, and serious cases can cause cornea perforation and even blindness. Cornea lesions may be caused by a variety of causes, abnormal tear film, inflammation, systemic disease, reduced corneal sensitivity due to surgical damage, denervation of the cornea, or damage to the cornea. Currently, keratopathy is one of the most common ocular pathological conditions.
Nerve growth factor (nerve growth factor, NGF) is a cytokine that promotes the growth, development, differentiation, maturation, and repair of central and peripheral neurons after nerve damage. Many in vitro and in vivo experimental studies show that NGF has better curative effects on treating some diseases of the visual system, such as optic neuropathy caused by retinitis pigmentosa, optic neuritis, ocular contusion, glaucoma, keratitis, dry eye and the like.
Oxervate, recombinant human nerve growth factor (recombinant human NGF, rhNGF) eye drops, approved by the us FDA in 2018, are used for the treatment of neurotrophic keratitis, which can promote corneal epithelial repair and re-neuronize the cornea. In clinical trials, patients receiving Oxervate treatment reached 70% of the total cornea healing after 8 weeks. However, the active ingredient rhNGF of the product has poor stability, and the purity and content of the rhNGF are rapidly reduced under the conditions (such as illumination, refrigeration, high temperature and the like) to be used. According to Oxervate specifications, the product is stored at-20 ℃, can be stored at 2-8 ℃ for 1 week after being provided to a patient, and any unused drug stored at 2-8 ℃ for more than 1 week or left for more than 12 hours can no longer be used. Stringent usage requirements greatly limit patient accessibility and result in costly clinical usage.
CN113307850a discloses an antibacterial peptide eye drop comprising antibacterial peptide, thickener, osmotic pressure regulator, pH buffer, protectant, surfactant and water. The invention aims to solve the problems of high activity reduction speed and poor activity stability at room temperature of the antibacterial peptide after being prepared into a liquid preparation.
CN112826829a discloses an ophthalmic formulation comprising trehalose, mannitol and cellulose ether, which is effective in inducing healing of corneal epithelial defects when a combination surface of trehalose, mannitol and cellulose ether is applied to the eye.
Therefore, there is a need to develop a composition that can significantly improve the stability of recombinant human nerve growth factor, and reduce the cost of using an ophthalmic formulation containing recombinant human nerve growth factor and the medical burden of patients, which has an important significance in improving the accessibility of treatment for patients with cornea-related lesions.
Disclosure of Invention
In order to solve the technical problems, the invention provides a composition containing recombinant human nerve growth factor, application thereof and an ophthalmic preparation. The compositions, including compositions containing low concentrations of recombinant human nerve growth factor, have improved stability and reduced loss of pharmacologically active ingredients during manufacture, transport and long-term storage of ophthalmic formulations. The ophthalmic preparation prepared by the composition has good stability, thereby reducing the use cost of patients and meeting the clinical requirements of eye disease treatment.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a composition containing recombinant human nerve growth factor comprises recombinant human nerve growth factor, trehalose, mannitol and polyvinyl alcohol. The invention can obviously improve the stability of the recombinant human nerve growth factor, in particular to the stability of the recombinant human nerve growth factor under low concentration by compounding trehalose, mannitol and polyvinyl alcohol with the recombinant human nerve growth factor.
The recombinant human nerve growth factor is used in the composition of the present invention in an amount of 0.005-1 parts 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 any one of the above-mentioned end values or combinations of the 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.
The trehalose is used in the composition of the invention in an amount of 20 to 70 parts by weight, for example, 40 to 70 parts by weight, 45 to 60 parts by weight.
The mannitol is used in the composition of the present invention in an amount of 5 to 30 parts by weight, for example, 5 to 20 parts by weight, 8 to 14 parts by weight.
The polyvinyl alcohol is used in the composition of the invention in an amount of 1 to 13 parts by weight, for example, 1 to 11 parts by weight and 5 to 9 parts by weight.
The invention also provides application of the composition containing the recombinant human nerve growth factor in preparing an ophthalmic preparation.
The ophthalmic preparation of the present invention can be used for treating keratopathy caused by reduced corneal sensitivity, corneal denervation or corneal damage, such as neurotrophic keratitis, trauma/surgery-related corneal damage/defect lesions, dry eye, etc.
The invention also provides an ophthalmic preparation, which comprises the composition containing the recombinant human nerve growth factor.
The ophthalmic formulation of the present invention has better stability than the prior art, thereby reducing the loss of active ingredient during manufacture, transportation and long-term storage of the ophthalmic formulation.
The ophthalmic preparation of the present invention has an osmotic pressure of 200 to 380mOsmol/kg, preferably 250 to 350mOsmol/kg, and more preferably 280 to 320mosm/kg.
The viscosity of the ophthalmic preparation of the present invention is 1-25mm 2/s, preferably 2-24mm 2/s.
The ophthalmic formulations of the present invention may also contain buffering agents to achieve a desired pH. Preferably, the ophthalmic formulation has a pH of 6.5-7.5, more preferably 7.0-7.4. The buffer is selected from at least one of phosphate, citrate and histidine.
The ophthalmic formulation of the present invention may further comprise an antioxidant compound, in particular selected from L-methionine and/or cysteine. Preferably, the antioxidant compound is present in an amount of 0.01-0.1mg/ml.
The ophthalmic preparation of the present invention may further comprise a high molecular polymer, specifically, polyethylene glycol, having a molecular weight of 4000-8000. Preferably, the content of the high molecular polymer is 5-25mg/ml.
The beneficial effects of the invention are as follows:
(1) The invention can obviously improve the stability of the recombinant human nerve growth factor, especially the stability of the recombinant human nerve growth factor with low concentration by compounding trehalose, mannitol and polyvinyl alcohol with the recombinant human nerve growth factor.
(2) The ophthalmic preparation provided by the invention can obviously restore cornea perception of a model rat, reduce cornea fluorescein sodium staining score, improve tear secretion and promote cornea nerve restoration, and has a good effect of repairing cornea epithelium and nerves.
(3) The ophthalmic preparation provided by the invention has good stability, so that the ophthalmic preparation has low storage requirement, can be stored for at least 7 days at 10-14 ℃, greatly reduces the use cost of patients, has more cost advantage compared with a public prescription, and can better meet the clinical requirements of patients with the eyes.
(4) The ophthalmic preparation (low concentration) has better light stability, the purity of the recombinant human nerve growth factor is higher than 90% when the light is irradiated for 10 days, the purity of the recombinant human nerve growth factor is higher than the release standard of products on the market, the purity of the recombinant human nerve growth factor is higher than 85% when the light is irradiated for 15 days, the release standard of the products on the market is met, and the quality of the ophthalmic preparation is better.
(5) The ophthalmic preparation has good stability, thereby reducing the loss of pharmacological active substances during the manufacture, transportation and long-term storage of the ophthalmic preparation and ensuring the treatment effect and the medication safety.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Before further describing embodiments of the invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is intended to be in the nature of specific embodiments and is not intended to be limiting of the scope of the invention.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, 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 belongs.
The invention does not limit the sources of the raw materials, if no special description exists, the adopted raw materials and auxiliary materials are all pharmaceutical grade commercial products, and the solvent is water for injection if no special description exists. Analytical methods in terms of the methods, the following methods were used in the examples of the present invention. The percentages stated in the invention are mass percentages unless otherwise indicated.
1. Experimental materials
The recombinant human nerve growth factor is recombinant human beta-nerve growth factor prepared by patent 201910390559.2 of Chongqing Kerun biological medicine Co. Commercially available recombinant human nerve growth factor products can also achieve similar technical effects;
Polyvinyl alcohol, purchased from Jiangsu alpha pharmaceutical industry groups;
l-methionine, available from Hubei eight peak pharmaceutical Co., ltd;
Disodium hydrogen phosphate dodecahydrate, available from the company of pharmaceutical excipients, hualiyi, et cetera;
Sodium dihydrogen phosphate monohydrate, available from the company of pharmaceutical excipients, inc. of Sichuan huali;
mannitol, purchased from Qingdao open moon seaweed group Co., ltd;
PEG6000, available from Hunan Erkang pharmaceutical Co., ltd;
trehalose dihydrate, available from japan corporation;
sodium hyaluronate, available from Shaanxi plus pharmaceutical excipients Inc.
2. RP-HPLC (reverse phase chromatography) method for evaluating purity and content of main peak
The measurement was carried out according to the pharmacopoeia of the people's republic of China (2015 edition, 0521 high performance liquid chromatography) and the detection was carried out by using a reversed phase chromatographic column. When the method is used for purity detection, the area normalization method is used for calculating the purity of the sample. When the method is used for content test, the recombinant human nerve growth factor of 20 mug/ml is used for external standard method quantification, and the peak area average value of the target protein at the concentration is 170. The samples above this concentration were diluted with phosphate buffer, the samples below this concentration were concentrated by ultrafiltration and 12000g centrifugation, all analyzed at 20. Mu.g/ml, and the chromatograms were recorded.
Content evaluation relative percentage (%) = (test sample peak area/170) ×100%.
1. Stability test of compositions containing recombinant human nerve growth factor
The formulations of the recombinant human nerve growth factor-containing compositions of examples 1-15 are shown in Table 1.
TABLE 1
The preparation method of the composition containing the recombinant human nerve growth factor comprises respectively weighing trehalose, mannitol and polyvinyl alcohol, dissolving in injectable water, adding the recombinant human nerve growth factor, mixing, adding phosphate buffer solution to adjust pH to 7.2, and filtering.
The effect of light on the stability of the composition at different storage times was investigated by RP-HPLC at a temperature of 12.+ -. 2 ℃ and a strong light of 5000.+ -. 500 Lux.
Wherein, the comparative composition 1 is recombinant human nerve growth factor 180 mug/ml, trehalose 50.4mg/ml, mannitol 13.1mg/ml, hydroxypropyl methylcellulose (HPMC, 4000 Pa.s) 1.1mg/ml. The preparation method is the same as the composition containing the recombinant human nerve growth factor.
Comparative composition 2 recombinant human nerve growth factor 180. Mu.g/ml, trehalose 50.4mg/ml, mannitol 13.1mg/ml, sodium hyaluronate 0.1mg/ml. The preparation method is the same as the composition containing the recombinant human nerve growth factor.
The results are shown in tables 2 and 3.
TABLE 2
As can be seen from Table 2, the recombinant human nerve growth factor-containing compositions of examples 1-15 of the present invention had a purity of greater than 90% in the compositions, a purity of greater than 90% in the comparative composition 1, and a purity of less than 90% in the comparative composition 2 after 7 days of illumination at 12.+ -. 2 ℃.
TABLE 3 Table 3
As can be seen from Table 3, the compositions of examples 1-15 of the present invention, which contained recombinant human nerve growth factor, all had a relative percentage of recombinant human nerve growth factor within the target concentration range of 80-120% after 7 days of illumination at 12.+ -. 2 ℃ whereas the comparative composition 1 had a relative percentage of recombinant human nerve growth factor of less than 80% after 3 days of illumination, and the comparative composition 2 had a relative percentage of recombinant human nerve growth factor of 61% after 1 day of illumination. The above results indicate that the recombinant human nerve growth factor-containing composition of the present invention has good photostability. The light stability of the recombinant human nerve growth factor can be improved by compounding trehalose, mannitol and polyvinyl alcohol with the recombinant human nerve growth factor.
As can be seen from tables 2 and 3, the compositions containing recombinant human nerve growth factor of examples 1 to 15 of the present invention satisfy the requirements of the purity and the target concentration range after 7 days of illumination at 12.+ -. 2 ℃ and the relative percentage of the recombinant human nerve growth factor in the composition, while the comparative composition 1 satisfies the requirements of the purity of the recombinant human nerve growth factor after 7 days of illumination at 12.+ -. 2 ℃ but the relative percentage of the recombinant human nerve growth factor is lower than 80% and fails to satisfy the requirements of the target concentration range. After 7 days of illumination at 12+ -2 ℃, the purity and relative percentage of recombinant human nerve growth factor of comparative composition 2 do not meet the requirements of purity and target concentration range. Therefore, compared with the comparative compositions 1 and 2, the composition containing the recombinant human nerve growth factor has better photostability, and the composition containing the recombinant human nerve growth factor with the concentration lower than 100 mug/ml still has better photostability.
To examine the effect of varying amounts of trehalose, mannitol, and polyvinyl alcohol in combination with recombinant human nerve growth factor on the recombinant human nerve growth factor-containing compositions, the following comparative examples 1-3 were prepared.
The formulations of the recombinant human nerve growth factor-containing compositions of comparative examples 1-3 are shown in Table 4.
TABLE 4 Table 4
The preparation method of the composition comprises respectively weighing other components except recombinant human nerve growth factor, dissolving in injectable water, adding recombinant human nerve growth factor, mixing, adding phosphate buffer solution to adjust pH to 7.2, and filtering.
The stability of the compositions of comparative examples 1-3 was tested with reference to the methods of the stability test described previously, and the results are shown in tables 5 and 6.
TABLE 5
As can be seen from Table 5, the purity of recombinant human nerve growth factor was already less than 89.5% in the composition of comparative example 1 when it was irradiated at 12.+ -. 2 ℃ for 7 days. The compositions of comparative example 2 and comparative example 3 containing recombinant human nerve growth factor were subjected to 7 days of light at 12.+ -. 2 ℃ and the purity of recombinant human nerve growth factor was still greater than 90%.
TABLE 6
As can be seen from Table 6, the composition of comparative example 1 containing recombinant human nerve growth factor had a relative percentage of recombinant human nerve growth factor at the lower limit of the target concentration range after 7 days of light at 12.+ -. 2 ℃ and the relative percentages of recombinant human nerve growth factor of comparative examples 2 and 3 were both lower than 80%.
As can be seen from tables 5 and 6, the compositions of comparative examples 1 to 3 containing recombinant human nerve growth factor could not meet the requirements of the purity and the target concentration range of recombinant human nerve growth factor at the same time in the light stability test.
In summary, the compositions of the present invention have better photostability, even with low concentrations of recombinant human nerve growth factor, thereby reducing the loss of pharmacologically active ingredients during manufacture, transport and long-term storage of ophthalmic formulations.
2. Evaluation of ophthalmic formulation performance
Taking the compositions of example 4, example 12 and example 15 as examples, respectively, the stability and efficacy of ophthalmic formulations containing low concentrations (100. Mu.g/ml, 10. Mu.g/ml and 5. Mu.g/ml) of recombinant human nerve growth factor were examined.
An ophthalmic formulation 1, comprising the recombinant human nerve growth factor composition of example 4, labeled as formulation 1, comprises recombinant human nerve growth factor 100 μg/ml, trehalose 50mg/ml, mannitol 14mg/ml, polyvinyl alcohol 9mg/ml, PEG6000 10mg/ml, L-methionine 0.01mg/ml, disodium hydrogen phosphate dodecahydrate 7.24mg/ml, and sodium dihydrogen phosphate monohydrate 1.08mg/ml.
An ophthalmic formulation 2, containing the recombinant human nerve growth factor of example 12, labeled as formulation 2, comprises recombinant human nerve growth factor 10 μg/ml, trehalose 70mg/ml, mannitol 20mg/ml, polyvinyl alcohol 11mg/ml, PEG6000 10mg/ml, L-methionine 0.01mg/ml, disodium hydrogen phosphate dodecahydrate 7.24mg/ml, and sodium dihydrogen phosphate monohydrate 1.08mg/ml.
An ophthalmic formulation 3, containing the recombinant human nerve growth factor composition of example 15, labeled as formulation 3, was prepared by the specific formulation of recombinant human nerve growth factor 5 μg/ml, trehalose 55mg/ml, mannitol 12mg/ml, polyvinyl alcohol 7mg/ml, PEG6000 10mg/ml, L-methionine 0.01mg/ml, disodium hydrogen phosphate dodecahydrate 7.24mg/ml, and sodium dihydrogen phosphate monohydrate 1.08mg/ml.
A comparative ophthalmic preparation comprises the comparative composition 1, and the specific formulation comprises 180 mug/ml of recombinant human nerve growth factor, 50.4mg/ml of trehalose, 13.1mg/ml of mannitol, 1.1mg/ml of HPMC (4000 mPas), 10.7mg/ml of PEG6000, 0.01mg/ml of L-methionine, 7.24mg/ml of disodium hydrogen phosphate dodecahydrate and 1.08mg/ml of sodium dihydrogen phosphate monohydrate.
2.1 Long term stability
The above ophthalmic preparations (2000 counts each, 2mL in size) were left for a long period of time at-20℃for 9 months, and the results of the evaluation of the samples by RP-HPLC are shown in Table 7.
TABLE 7
As can be seen from Table 7, the low concentration of recombinant human nerve growth factor-containing ophthalmic preparation of the present invention maintained the purity of the recombinant human nerve growth factor at a stable level when left at-20℃for 9 months, and was superior to the comparative ophthalmic preparation, satisfying the long-term storage requirement.
2.2 Acceleration stability
The above ophthalmic preparations (2000 counts each, 2mL in size) were subjected to accelerated stability studies at 2-8℃and the samples were evaluated by RP-HPLC, and the results are shown in Table 8.
TABLE 8
As can be seen from Table 8, the inventive ophthalmic preparation containing a low concentration of recombinant human nerve growth factor maintained the purity of the recombinant human nerve growth factor at a stable level when stored at 2-8℃for 4 months, and the purity of the recombinant human nerve growth factor was more than 90%, whereas the comparative ophthalmic preparation showed less than 90% when stored at 2-8℃for 4 months.
2.3 Light stability
The above ophthalmic preparations (2000 pieces each, 2ml in size) were stored at 12.+ -. 2 ℃ and irradiated with strong light (5000.+ -. 500 Lux) for 15 days, and the purity of the samples was evaluated by RP-HPLC, and the results are shown in Table 9.
TABLE 9
As shown in Table 9, the ophthalmic preparation containing the recombinant human nerve growth factor with low concentration has better light stability, the purity of the recombinant human nerve growth factor is more than 90% when the ophthalmic preparation is illuminated for 10 days, the purity of the recombinant human nerve growth factor is more than 85% when the ophthalmic preparation is illuminated for 15 days, and the purity of the recombinant human nerve growth factor is less than 90% when the ophthalmic preparation is illuminated for 10 days, and the purity of the recombinant human nerve growth factor is less than 85% when the ophthalmic preparation is illuminated for 15 days, so that the ophthalmic preparation does not meet the release standard of the marketed product.
Table 10
As can be seen from Table 10, the ophthalmic preparation of the present invention containing a low concentration of recombinant human nerve growth factor has a relative percentage of recombinant human nerve growth factor within the target concentration range when it is irradiated for 15 days, whereas the comparative ophthalmic preparation has a relative percentage of recombinant human nerve growth factor of less than 80% when it is irradiated for 3 days.
In summary, the ophthalmic formulation of the present invention containing a low concentration of recombinant human nerve growth factor has better photostability.
2.4 Evaluation of the Effect of neurotrophic keratitis
(One) test animals
SD rat (3 days old) with weight of 7-10g.
(II) test method
After the neonates were randomly grouped according to body weight, capsaicin (8 mg/ml) was injected subcutaneously in the nape of the neck a single time, and a cornea denervation animal model was established. After molding, the model group and the normal animal group are subjected to ophthalmic examination, and the molding is successful if the cornea perception sensitivity and the cornea sodium staining have obvious differences. Animals successfully molded are randomly divided into a model control group, an original preparation treatment group and a new preparation treatment group, and 16 normal mice are taken as normal control groups.
In this example, the daily effective dose of the composition was calculated as 0.1. Mu.g/kg (calculated as recombinant human nerve growth factor) for human, the weight of the adult was 60kg, and the equivalent daily dose of the mice was 1. Mu.g/kg (calculated as recombinant human nerve growth factor) when the conversion factor of the mice to the human dose was 10.0.
All animals received a daily dose of 10 μl/eye, except for the normal control group, which was given injection water, the original formulation treatment group was given the comparative ophthalmic formulation, and the new formulation treatment group was given the ophthalmic formulation of the prescription 1, and the formulation of the specific ophthalmic formulation was as follows:
The comparative ophthalmic preparation comprises 180 mug/mL of recombinant human nerve growth factor, 50.4mg/mL of trehalose, 13.1mg/mL of mannitol, 1.1mg/mL of HPMC (4000 mPas), 10.7mg/mL of PEG6000, 0.01mg/mL of L-methionine, 7.24mg/mL of disodium hydrogen phosphate dodecahydrate and 1.08mg/mL of sodium dihydrogen phosphate monohydrate.
Prescription 1 recombinant human nerve growth factor 100 mug/mL, trehalose 50mg/mL, mannitol 14mg/mL, polyvinyl alcohol 9mg/mL, PEG6000 10mg/mL, L-methionine 0.01mg/mL, disodium hydrogen phosphate dodecahydrate 7.24mg/mL and sodium dihydrogen phosphate monohydrate 1.08mg/mL.
After successful molding, ophthalmic examination was performed, designated Day1 (abbreviated as D1), with Day 4 administered (abbreviated as D5) and Day 8 administered (abbreviated as Day 9). Animal cornea nerve perception was measured by cornea perception sensitivity meter at Day1, day5 and Day9 (20:00 of the Day), after fluorescein sodium staining, slit lamp observation was performed to evaluate corneal epithelial defect, and at Day9, animal cornea was taken and subjected to gold chloride staining to evaluate recovery of cornea nerve.
The judgment standard is that the corneal fluorescein sodium staining score evaluates the corneal epithelial defect condition of animals, the cornea nerve perception is measured by a cornea perception sensitivity measuring instrument, and the recovery condition of cornea nerves is quantified by gold chloride cornea nerve counting.
The results are shown in tables 11-13 below.
TABLE 11 corneal perception sensitivity
As can be seen from Table 11, the D1 examination showed that the cornea sensitivity of the model control group, the original preparation treatment group and the freshly prepared treatment group were extremely significantly different (a P. Ltoreq.0.001) compared with the normal control group, and the molding was successful.
The cornea sensitivity of the original preparation treatment group and the cornea sensitivity of the new preparation treatment group are obviously different from that of the model control group after 4 days of administration (b P is less than or equal to 0.05). It was shown that the comparative ophthalmic formulation and the ophthalmic formulation of formula 1 have the effect of improving corneal sensitivity after 4 days of continuous administration.
The cornea sensitivity of the original preparation treatment group and the cornea sensitivity of the new preparation treatment group are extremely obviously different from that of the model control group after 8 days of administration (c P is less than or equal to 0.001). It was shown that comparing the ophthalmic formulation with the ophthalmic formulation of prescription 1 can further improve the corneal sensitivity after 8 days of continuous administration.
Table 12 corneal epithelial defect repair
As can be seen from Table 12, the D1 examination showed that the fluorescence staining scores of the model control group, the original preparation treatment group and the freshly prepared treatment group were extremely significantly different (a P. Ltoreq.0.001) compared with the normal control group, and the molding was successful.
4 Days after administration, the fluorescence staining scores of the original preparation treatment group and the new preparation treatment group are obviously different from that of the model control group (b P is less than or equal to 0.05). The comparative ophthalmic formulation and the ophthalmic formulation of formulation 1 were shown to have efficacy in repairing corneal epithelial defects after 4 days of continuous administration.
The fluorescence staining scores of the original preparation treatment group and the new preparation treatment group are extremely significantly different (c P is less than or equal to 0.001) compared with the model control group after 8 days of administration. It was shown that the comparison of the ophthalmic formulation with the ophthalmic formulation of prescription 1 further repaired the corneal epithelial defect after 8 days of continuous administration.
TABLE 13 corneal nerve recovery
| Group of | Test animal (Only) | Corneal nerve length (mean+ -SD) |
| Normal control group | 16 | 1240.99±171.27 |
| Model control group | 16 | 862.45±59.46a |
| Original preparation treatment group | 16 | 1140.60±78.41c |
| New formulation treatment group | 16 | 1130.62±79.87c |
As can be seen from Table 13, the D9 examination showed that the corneal nerve length of the model control group was significantly different (a P. Ltoreq.0.001) compared with that of the normal control group, and the modeling was successful.
After 8 days of administration, the corneal nerve lengths of the original preparation treatment group and the new preparation treatment group are obviously different from those of the model control group (c P is less than or equal to 0.001). The comparison of the ophthalmic formulation with the ophthalmic formulation of prescription 1 shows the efficacy of increasing the length of the corneal nerve in the model mice after 8 days of continuous administration.
In conclusion, the ophthalmic preparation of the prescription 1 can obviously improve the cornea perception sensitivity of the model mice, obviously reduce the cornea sodium fluorescein staining score of the model mice and obviously increase the cornea nerve length of the model mice after being continuously administered for 4 days and 8 days.
Claims (10)
1. The composition containing the recombinant human nerve growth factor is characterized by comprising, by weight, 0.005-1 part of the 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, which comprises the components of, 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, which comprises the following components, 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 a composition according to any one of claims 1 to 3 for the preparation of an ophthalmic formulation.
5. The use according to claim 4, wherein the ophthalmic formulation is for the treatment of keratopathy.
6. The use according to claim 5, wherein the keratopathy is caused by a decrease in corneal sensitivity, corneal denervation or corneal damage.
7. An ophthalmic formulation comprising the composition of any one of claims 1-3.
8. The ophthalmic formulation of claim 7, wherein the ophthalmic formulation has a pH of 6.5-7.5, an osmotic pressure of 200-380mOsmol/kg, and a viscosity of 1-25mm 2/s.
9. The ophthalmic formulation of claim 7, further comprising an antioxidant compound comprising L-methionine and/or cysteine, wherein the antioxidant compound is present in an amount of 0.01-0.1mg/ml.
10. The ophthalmic formulation of claim 7, further comprising a high molecular polymer, wherein the high molecular polymer is polyethylene glycol, has a molecular weight of 4000 to 8000, and is present in an amount of 5 to 25mg/ml.
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| CN202310835893.0A CN119258198A (en) | 2023-07-07 | 2023-07-07 | Composition containing recombinant human nerve growth factor and its application and ophthalmic preparation |
| PCT/CN2024/103736 WO2025011440A2 (en) | 2023-07-07 | 2024-07-05 | Ophthalmic preparation containing recombinant human β-nerve growth factor and use thereof |
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| CN101972224B (en) * | 2010-09-13 | 2012-01-04 | 舒泰神（北京）生物制药股份有限公司 | Eye in-situ gel |
| CN101972470B (en) * | 2010-09-13 | 2012-10-17 | 舒泰神（北京）生物制药股份有限公司 | An ophthalmic in situ gel |
| ES2893239T3 (en) * | 2015-04-21 | 2022-02-08 | Staidson Beijing Biopharmaceuticals Co Ltd | Composition of nerve growth factor and injection powder |
| DK3287140T3 (en) * | 2015-04-21 | 2021-07-19 | Beijing Staidson Medical Tech Co Ltd | Nerve growth factor composition and powder injection |
| ES2893952T3 (en) * | 2015-04-21 | 2022-02-10 | Staidson Beijing Biopharmaceuticals Co Ltd | Composition of nerve growth factor and powder for injection |
| CN107913246A (en) * | 2016-10-09 | 2018-04-17 | 刘力 | The medical composite for eye of local administration |
| IT201900022029A1 (en) * | 2019-11-25 | 2021-05-25 | Dompe Farm Spa | Ophthalmic formulations |
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